WO2011003905A1 - Biomarker for selecting patients and related methods - Google Patents

Biomarker for selecting patients and related methods Download PDF

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Publication number
WO2011003905A1
WO2011003905A1 PCT/EP2010/059635 EP2010059635W WO2011003905A1 WO 2011003905 A1 WO2011003905 A1 WO 2011003905A1 EP 2010059635 W EP2010059635 W EP 2010059635W WO 2011003905 A1 WO2011003905 A1 WO 2011003905A1
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WIPO (PCT)
Prior art keywords
patient
illo
ratio
immunogenic composition
ifny
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PCT/EP2010/059635
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French (fr)
Inventor
Bruce Acres
Benoît GRELLIER
Original Assignee
Transgene Sa
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Priority to CA2767458A priority Critical patent/CA2767458A1/en
Priority to AU2010270313A priority patent/AU2010270313B2/en
Application filed by Transgene Sa filed Critical Transgene Sa
Priority to RU2012103932/15A priority patent/RU2552292C2/en
Priority to CN201080031162.1A priority patent/CN102483405B/en
Priority to KR1020127000597A priority patent/KR20120089619A/en
Priority to SG2011088804A priority patent/SG176619A1/en
Priority to EP10739307.6A priority patent/EP2452194B1/en
Priority to ES10739307.6T priority patent/ES2558729T3/en
Priority to MX2012000509A priority patent/MX337287B/en
Priority to BR112012000522A priority patent/BR112012000522A2/en
Priority to DK10739307.6T priority patent/DK2452194T3/en
Priority to US13/383,189 priority patent/US9261512B2/en
Priority to NZ596325A priority patent/NZ596325A/en
Priority to JP2012518967A priority patent/JP5650212B2/en
Publication of WO2011003905A1 publication Critical patent/WO2011003905A1/en
Priority to IL216083A priority patent/IL216083A/en
Priority to ZA2012/00110A priority patent/ZA201200110B/en
Priority to HK12105900.4A priority patent/HK1165206A1/en

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1738—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5428—IL-10
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • 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 the field of immunology ana, in particular, to immunotherapy of a patient against diseases caused for example by infection cr cancers. More particularly, the invention relates to methods for predicting whether a patient is or is not susceptible to developing a prophylactic or therapeutic response, preferably immune response, after such immunotherapy. The present invention relates to methods and compositions for improving the survival rate of patients to be treated by an immunogenic composition, in particular a therapeutic vaccine.
  • Lave vaccines are typically attenuated non ⁇ pathogenic versions of an infectious agent that are capable of priming an immune response directed against a pathogenic version of the infectious agent.
  • the ideal viral vector In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen- specific antigens to the immune system. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis.
  • Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application (for a review on recombinant viral vaccines see for example Harrop and Carroll, 2006, Front Biosci., 11, 804-817 ; Yokoyama et al., 1997, J Vet Med Sci.,59, 311-322) .
  • IRMs xminune response modifiers
  • TLRs Toll-like receptors
  • Such compounds have been shown to stimulate a rapid release of certain T cell, dendritic ceil, or monocyte/macrophage- derived cytokines and are also capable of stimulating lymphocyte function, such as B cells to secrete antibodies which play an important role in the antiviral and antitumor activities of IRM compounds .
  • vaccination strategies have been proposed, most of them being based on a prime-boost vaccination regimen.
  • the immune system is first induced oy administering to the patient a priming composition and then boosted by administration of a boosting second composition
  • one treatment can be effective only in specific group of patients. It is thus desirable to provide to physicians tools and methods that will enable them to tailor optimal personalized patient therapies, i.e. to prescribe the right therapy to the right patient at right time, to provide a higher treatment success rate, to monitor the response to the treatment, to increase drug efficacy and safety, to eliminate the unnecessary treatment of patients for whom therapy is not appropriate, to spare the patient unnecessary toxicity and side effects, to reduce the cost to patients and insurers of unnecessary or dangerous ineffective medication, and tc improve patient quality of life, eventually making cancer a managed disease, with follow up assays as appropriate.
  • optimal personalized patient therapies i.e. to prescribe the right therapy to the right patient at right time, to provide a higher treatment success rate, to monitor the response to the treatment, to increase drug efficacy and safety, to eliminate the unnecessary treatment of patients for whom therapy is not appropriate, to spare the patient unnecessary toxicity and side effects, to reduce the cost to patients and insurers of unnecessary or dangerous ineffective medication, and tc improve patient quality of life, eventually making cancer a
  • Pharmacogenetics which consist in the study of individual response to drugs as a function of genetic differences. These responses relate to how a drug functions in any given individual, how it is metabolized, its toxicity and dosage requirements. With the human genome project, pharmacogenetics has expanded into pharmacogenomics . Pharmacogenomics goes beyond pharmacogenetics, with the potential to find uses from drug discovery and development, target discovery and validation, and clinical trials;
  • Metabolomics can also be applied to the field of predictive medicine. Unlike pharmacogenetics, which is limited to genetic factors, pharr ⁇ aco-metabolomics is able to predict an individual's response to a drug based not only on genetic factors, but also non-genetic factors, such as other drugs in the patient's body, the patient's current state of health, etc.
  • biomarker can be an indicator of normal biological processes, disease processes, or pharmacological responses to therapeutic intervention. Their role ranges from, stratifying the patient population in helping to identify responders versus non-responders to determining the efficacy of the therapeutic. Biomarkers can be a valuable tool m making better decisions that will reduce the cost for drug development and enabJ e therapies to reach the most appropriate patient population faster.
  • the invention provides rraterials and methods for predicting the efficacy of a treatment involving the administration of an immunogenic composition to a patient ⁇ i.e. immunotherapy treatment) using biological markers (biomarkers) that have been determined to be substantially reliable signature which correlates with the desired clinical outcome, lhe biomarkers are present in biological samples obtained from the patient, prior to the treatment with the said immunogenic composition.
  • biological markers biomarkers
  • the ability to predict the clinical outcome of a treatment, before its initiation, will enable clinicians and patients to identify ineffective therapy, make informed decisions regarding the course of treatment, including whether to abandon or to allow alternate therapy implementation.
  • the present Invention relates to the use of the interleukin-10 /J nterferon-gamma (ILl0/IFN ⁇ ) ratio as a biomarker for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition.
  • the interferon-gamma/ interleukin-10 (IFKy/ ILlO) ratio can be used as a biomarker according to the present invention, but in that case conclusions should be adapted.
  • the present Invention relates to a method for treating a patient for human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low IL10/IFN ⁇ ratio.
  • the present Invention thus relates to a method for treating a patient for human disease by administering an immunogenic composition, said method comprising the following steps :
  • the present Invention relates to a method for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition, said method comprising the steps of:
  • the present Invention relates to a metfio ⁇ for selecting a patient susceptible to developing prophylactic or therapeutic response, preferaoly immune response, by administration of an immunogenic composition, said method comprising the steps of:
  • the present Invention relates to a method for predicting whether a patient is or is not susceptible to respond positively to a treatment comprising administration of an immunogenic composition, saJ d method comprising che steps of:
  • IL10/IFN ⁇ ratio calculating IL10/IFN ⁇ ratio, wherein low IL10/IFN ⁇ ratio indicates that the patient is predicted to have an increased susceptibility to develop a prophylactic or Therapeutic response, preferably immune response.
  • the present Invention relates to a method for selecting a patient susceptible ic respond positively to a treatment comprising administration of an immunogenic compos.! tion, said method comprising the steps of:
  • the present Invention relates to an ex-vivo method for testing whether a patient will respond therapeutically to a method of treatment comprising administration of an immunogenic composition, wherein the testing method comprises the steps of:
  • the present Invention relates to an ex ⁇ vivo method for testing whether a patient will respond therapeutically to a method of treating cancer by administration of an immunogenic composition, wherein the testing method comprises the steps of:
  • the present Invention further relates to an ex vivo method of treatment comprising administration of an immunogenic composition, wherein the resting method comprises the step of measuring levels of ILlO and INFy in a biological sample, especially blood sample, from the patient, calculating ILlO/IFNf ⁇ ratio, wherein low ILlO/IFN ⁇ ratio indicates that the patient will develop a prophylactic or therapeutic response, in particular immune response, towards the immunogenic composition.
  • the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low IL!0/IFN ⁇ ratio,
  • the present Invention relates to a method for inducing an immune response io at ieasr one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients thai have low ILlO/IFN ⁇ ratio.
  • the present Invention relates to a method for inducing an Immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low ILlO/IFN ⁇ ratio and wherein said raised iiurrune response is innate immune response.
  • the innate immune response is body's inxtial immune defense against pathogens and is elicited by a variety of cells including antigen-presenting cells or "APCs". These cells express surface and cytoplasmic receptors that recognize molecules of foreign origin ⁇ e.g., bacterial and viral nucleic acids, proteins, carbohydrates).
  • the dendritic cells and macrophage Upon detecting these signals, the dendritic cells and macrophage elicit a defensive response that includes the release of cytokines (including interferons, TNF-alpha, and IL-12) and chemokines that attract cells such as immature dendritic cells, macrophage, NK ceils, and granulocytes, to the site of challenge.
  • cytokines including interferons, TNF-alpha, and IL-12
  • chemokines that attract cells such as immature dendritic cells, macrophage, NK ceils, and granulocytes
  • the present Invention thus relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps :
  • the present Invention relates to a method for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps : - selection of one patient in a patient population composed of patients that have low ILlO/IFN ⁇ ratio,
  • -he present Invention relates to a method for inducing an immune response ⁇ i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said raised immune response is innate immune response, said method comprising ⁇ .he following steps :
  • the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps :
  • the present Invention relates to a method for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps : measuring in the patient the levels of ILlO and
  • the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for creating human disease by administering an immunogenic composition wherein said raised immune response is innate immune response, said method comprising the following steps :
  • the terms “a” and “an” are used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced compounds or steps, unless the context dictates otherwise.
  • a cell includes a plurality of cells including a mixture thereof. More specifically, “at least one” and “one or more” means a number which Is one or greater than one, with a special preference for one, two or three.
  • patient refers to a vertebrate, particularly a member of the mammalian species and includes, but is not limited to, domestic animals, sport animals, primates including humans.
  • the term “treatment” or “treating” encompasses prophylaxis and/or therapy. Accordingly the immunogenic compositions or methods of the present invention are not limited to therapeutic applications and can be used in prophylaxis cries. This is covered by the term “co developing a prophylactic or therapeutic immune response” herein. “Prophylaxis” is not limited to preventing immediate diseases ⁇ e.g. infectious diseases), it further encompasses prevention of long term consequences of these infections such as cirrhosis or cancer.
  • an “effective amount” or a “sufficient amount” of an active compound is an amount sufficient to effect beneficial or desired results, including clinical results.
  • An effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” is an amount to effect beneficial clinical results, including, but not limited to, alleviation of one or more symptoms associated with viral infection as we] 1 as prevention of disease (e.g. prevention of one or more symptoms of infection).
  • a patient selected in a patient population composed of patients that have low ILlO/IFNy ratio should be understood as meaning a patient for who levels of interleukin-10 and interferon-gamma have beer, measured as disclosed herein, and who has low IL10/IFN ⁇ ratio.
  • the terms "a patient will respond therapeutically” or ⁇ a patient will respond positively to a treatment” mean that the said patient has an increase ot survival rate (see example section).
  • the method of the invention comprises an mxtial step consisting in measuring the interieukin-10 and interferon-gamma levels in biological samples from the patient before administration of the immunogenic composition.
  • the levels of interieukin-10 and interferon-gamma are measured in a biological sample obtained from the patient.
  • Biological samples include but are net limited to blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen.
  • the biological sample is blood, plasma or serum, in which case obtaining the samples from a patient is relatively simple and non-invasive procedure. Methods of obtaining blood or serum are well-known in the art are not part of the invention.
  • detecting and quantifying polypeptides including the instant biomarkers
  • methods include but are not limited to anribody-based methods, more specifically monoclonal antibodies-based methods.
  • the particular methods of detecting and quantifying the biomarkers are not important to the invention.
  • the materials and methods of the present invention may be used with Lurni nex technology (Lur ⁇ inex Corporation, Austin, Tex.) or enzyme-linked immunosorbant assays (ELISA, numerous KLISA kits are commercially available e.g. by CliniScience, Diacione, Biosource) .
  • the levels of interleukin-10 and interferon-gamma are determined by using antibodies.
  • said antibody (ies) is (are) specific of ILlO or ⁇ NF ⁇ .
  • said anc ⁇ bodies are monoclonal antibodies.
  • said antibodxe ⁇ are tagged for example by fluorescence, radiolabel, enzyme, biotin, or any other methods designed to render cells labelled with said antibodies detectable. These techniques are widely used and known in the art.
  • the method includes determining the levels of interleukin-10 and interferon-gamma in a patient prior administration of an immunogenic composition to the patient; calculating ILlO/IFNy ratio; comparing said IL10/IFM ⁇ ratio to a cut-off value; and predicting the efficacy of immunotherapy treatment based on the levels of ILlO/IFN ⁇ ratio compared to the cut-off value.
  • the said cut-off value is about 5, preferably about 4, more preferably about 3 and particularly about 2.
  • said cut-off value is about 3.7, and more preferably is 3.7.
  • the "Mow ILlO/IFNy ratio" according to r.he present invention is designating ILlO/IFNy ratios below about 5, preferably below about 4 and more preferably below about 3 and particularly blow about ?.
  • said the "low ILlO/IFNy ratio" according to the present invention is designating ILlO/IFNy ratios below about 3.7, and more preferably is below 3.7.
  • the levels of interleukin-10 and interferon-gamma are measured by Enzyme Linked Immune Sorbant Assay (ELISA) , by Luminex ⁇ analysis, by lab-on-chip systems, by radio-immune assay or other systems based on specific molecular recognition of ILlO and ItNy using antibodies or other specific molecules.
  • ELISA Enzyme Linked Immune Sorbant Assay
  • the terms "immunogenic conpositicn" "vaccine composition” , “vaccine” or similar terms can be used interchangeably and mean an agent suitable for stiraulating/ind ⁇ ciug/increasing a patient's immune system to ameliorate a current condition, e.g. to improve survival rate, or to protect against or to reduce present or future harm or infections (including viral, bacterial, parasitic infections), e.g. reduced tumour cell proliferation or survival, reduced pathogen replication or spread in a patient or a detectabiy reduced unwanted symptom (s) associated with a condition, extend patient survival.
  • a current condition e.g. to improve survival rate, or to protect against or to reduce present or future harm or infections (including viral, bacterial, parasitic infections), e.g. reduced tumour cell proliferation or survival, reduced pathogen replication or spread in a patient or a detectabiy reduced unwanted symptom (s) associated with a condition, extend patient survival.
  • Said immunogenic composition can contain at least (i) all or part of at least one targeted antigen and/or (ii) at least one recombinant vector expressing in vivo all or part of at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding all or part of at least one targeted antigen.
  • the immunogenic composition of the Invention comprises (iii) at least one immune response modifier, alone or in combination with (i) and/or (ii ) .
  • immune response modifiers include the CpG oligonucleotides (see US
  • the immunogenic comDosition comprises cells whicn stimulate a patient immune response to treat a disease, such as cancer.
  • Said cells can be antigen presenting cells, such as dendritic cells, combined with an antigenic composition (e.g. Provenge developed. by u i ',. .c- -;>) , tumor cells (e.g. GvAX developed, by Cell Genesis) or lymphocytes,
  • an antigenic composition e.g. Provenge developed. by u i ',. .c- -;>
  • tumor cells e.g. GvAX developed, by Cell Genesis
  • lymphocytes e.g. GvAX developed, by Cell Genesis
  • the term "antigen” refers to any substance, including complex antigen (e.g. tamour cells, virus infected cells, dendritic cells, etc%), that is capable of being the target of an immune response.
  • An antigen may be the target of, for example, a cell-mediated and/or humoral immune response raised by a patient.
  • the term "antigen” encompasses for example all or part of viral antigens, tumour-specific or tumour-related antigens, bacterial antigens, parasitic antigens, allergens and the like:
  • Viral antigens include for example antigens from hepatitis viruses A, B, C, D and E, HIV, herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, adenoviruses, coxsdkie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus, measles virus; some non-limiting examples of known viral antigens include the following : antigens derived from HIV-I such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vi£, vpr, vpu, rev or part and/or combinations thereof; antigens derived from human herpes viruses such as gH, gL gM gB gC gK
  • tumor-specific or -related antigens include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, renal cancer, malignant melanoma, laryngeal cancer, prostate cancer.
  • Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at low levels or at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded Dy mutant cellular genes, such as oncogenes (e.g., activated ras oncogene) , suppressor genes (e.g., mutant pb3) , fusion proteins resulting from internal deletions or chromosomal translocations.
  • oncogenes e.g., activated ras oncogene
  • suppressor genes e.g., mutant pb3
  • cancer antigens can be encoded by viral genes such as tnose carried on RNA and DNA tumor viruses.
  • t ⁇ mor-specific or -related antigens include MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV) , adenosine deaminase-binding protein (ADAbp) , cyciophilin b, Colorectal associated antigen (CRC) -C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-I and CAP-2, etv ⁇ , amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSATM2, and PSA-3, prostate- specific membrane antigen (PSMA) , T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-Al, MAGE-A
  • DPPIV
  • MUC-I MUC-I
  • HER2/neu HER2/neu
  • p21ras RCAS
  • aipha-fetoprotej n E-cadher in, alpha-catenin, beta- caienin and gamma-catenin, pl20ctn, gplOO . sup .
  • Pmelli7 FRAME, NY-E ⁇ O-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiorype, pl5, gp75, GM2 and GD2 gangliosides , viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PlA, ElBV- encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40) , SSX-I, SSX-4, SSX- 5, SCP-I and CT-7, and c-erbB ⁇ 2;
  • APC adenomatous polyposis coli protein
  • APC adenomatous polyposis coli protein
  • APC adenomatous polyposis coli protein
  • fodrin Connexin 37
  • Ig-idiorype pl5
  • gp75 GM
  • - bacterial antigens includes for example antigens from Mycobacteria causing TB and leprosy, pneumocci, aerooic grarn negative bacilli, mycoplasma, staphy Ioccocal infections, streptococcal infections, salrr.onellae, chlamydiae, neisseriae;
  • antigens includes for exampJ e anzigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis;
  • allergens refer to a substance that can induce an allergic or asthmatic response in a susceptible patient.
  • the list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin) .
  • natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Canine (Canis familiaris) ; Dermatophagoides (e.g. Dermatophagoides farinae) ; Felis (Felis domesticus) ; Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum) ; Cryptomeria
  • Cupress ⁇ s e.g. C ⁇ pressus sempervirens
  • Juniperus sabinoides Juniperus virginiana, Juniperus communis and Juniperus ashei
  • Thuya e.g. Thuya orientalis
  • Chamaecyparis e.g. Chamaecyparis obtusa
  • Periplaneta e.g. Periplaneta americana
  • Agropyron e.g. Agropyron repens
  • Secale e.g. Secale cereale
  • Triticum e.g. Triticum aestivum
  • Dactylis e.g. Dactylis glomerata
  • Festuca ⁇ e.g.
  • Festuca elatior ; Poa (e.g. Poa pratensis or Poa compressa) ; Avena (e.g. Avena sativa) ; Holcus (e.g. Holcus lanatus); Anthoxcintbum (e.g. Anthoxanthum odoratum) ; Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phie ⁇ m (e.g. Phle ⁇ m pratense) ; Phalaris (e.g. Phalaris ar ⁇ ndinacea) ; Paspalum (e.g. Paspalum noratum) ; Sorghum (e.g. Sorgnum halepensis) ; and 3romus (e.g. 3romus ir.ermis).
  • Avena e.g. Avena sativa
  • Holcus e.g
  • said anc igen is encoded by an heterologous nucleotide sequence and is expressed in vivo by a recombinant vector.
  • the heterologous nucleotide sequence of the present invention encodes one or more of ail or part of the following an t igens HBV-PreSl PreS2 and Surface env proteins, core and polHIV- gpl20 gp40,gpl60, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef; HPV-El, E2, E3, E4, E5, E6, El, E8, Ll, L2 (see for example WO 90/10459, WO 98/04705, WO 99/03885); HCV env protein El or E2, core protein, NS2, NS3, NS4a f NS4b, NS5a, N35b, p7 (see for example WO2004111082 , WO2005051420 ) ; Muc-1 (see for example US 5,861,381; US6, 054 , 438 ;
  • the immunogenic composition contains at least two antigens, or an heterologous nucleotide sequence encoding at least two antigens, or at least two heterologous nucleotide sequences encoding at least two antigens, or any combination thereof.
  • said heterologous nucleotide sequence of the present invention encodes all or part of HPV antigen (s) selected in the group consisting of E6 early coding region of HPV, E7 early coding region of HPV and derivates or combination thereof.
  • HPV antigen encoded by the recombinant vector according to the invention is selected in the group consisting of an HPV ⁇ 6 polypeptide, an HPV E7 polypeptide or both an HPV E6 polypeptide and an HPV E7 polypeptide.
  • the 5 present invention encompasses the use of any HPV E6 polypeptide which binding to p53 is altered or at least significantly reduced and/or the use of any HPV E7 polypeptide which binding to Rb is altered, or at lea ⁇ t significantly reduced ⁇ Munger et al., 1989, EMBO J. 8, 4099-
  • HPV-16 E6 variant which is suitable for the purpose of the present invention is deleted of one or more amino acid residues
  • invention is deleted of one or more amino acid residues located from approximately position 21 to approximately position 26 (+1 representing the first amino acid of the native HPV-16 E7 polypeptide, with a special preference for the complete deletion of residues 21 to 26
  • HPV-I 6 early polypeptide (s) in use in the invention is/are further modified so as to improve MHC class 1 and/or MHC class ⁇ I presentation, and/or to stimulate anti-HPV immunity.
  • HPV E6 and ⁇ 7 polypeptides are nuclear proteins ana
  • membrane presentation permits to improve their therapeutic efficacy (see for example WG99/03885) .
  • Membrane anchorage can be easily achieved by incorporating in the HPV early polypeptide a membrane-anchoring sequence and if the native polypeptide lacks it a secretory sequence (i.e. a signal peptide).
  • Tney usually comprise 15 uo 35 essentially hydrophobic amino acids which are then removed by a specific ER-Iocated en ⁇ opepnidase ⁇ o give the mature polypeptide.
  • Membrane- ancncring sequences are usually highly hydrophobic in nature and serves to anchor the polypeptides m the cell membrane
  • membrane-anchoring and secretory sequences which can be used in the context of the present invention is vast. They may be obtained from any membrane- anchored and/or secreted polypeptide comprising it (e.g. cellular or viral polypeptides) such as the rabies glycoprotein, of the HIV virus envelope glycoprotein or of the measles virus F protein or may be synthetic.
  • the membrane anchoring and/or secretory sequences inserted in each of the early HPV-16 polypeptides used according to the invention may have a common or different origin.
  • the preferred site of insertion of the secretory sequence is the N-terrninus downstream of the codon for initiation of translation and thar of the membrane-anchoring sequence is the C-terminus, for example immediately upstream of the stop codon.
  • HPV E6 polypeptide in use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the measles F protein.
  • the HPV E7 polypeptide in use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the rabies glycoprotein.
  • the therapeutic efficacy of the recombinant vector can also be improved by using one or more nucleic acid encoding 5 3 miTiunoporentiator polypeptide (s ).
  • a polypeptide such as calreticulin (Cheng et ai., 2001, J. Clin, Invest. 108, 669-678), Mycobacterium tuberculosis heat snock procein 70 (HSP70) (Chen et al . , 2000, Cancer Res. 60, 10 1035-1042), ubiquitin ⁇ Rodriguez et al., 1997, J. Virol.
  • 15 recombinant vector according to the invention comprises a nucleic acid encoding one or more early polypeptide (s) as above defined, and more particularly HPV-16 and/or HPV-18 early E6 and/or E7 polypeptides.
  • 20 sai ⁇ heterologous nucleotide sequence of the present invention encodes all or part of MUC 1 antigen or derlvates thereof.
  • said heterologous nucleotide sequence of the present inventdon is said heterologous nucleotide sequence of the present inventdon
  • heterologous nucleotide sequence of the present invention encodes one or more fusion protein wherein
  • the configuration is not native m the sense that at least one of the NS polypeptides appears in an order which i s distinct from that of the native configuration.
  • the native configuration would be H53-NS4A-NS5B with NS3 at the N-terminus and NS5B at the C-terminus.
  • a non-native configuration can be NS5B-NS3-NS4A, NS5B-NS4A-NS3, NS4A-NS3-NS5B, NS4A-NS5B-NS3 or NS3-NS5B-NS4A.
  • the fusion protein according to the invention comprises ai least one of the followings:
  • each of the NS polypeptides can be independently native or modified.
  • the NS4A polypeptide included in the NS4/- ⁇ -NG3 portion can be native whereas the NS3 polypeptide comprises at i east one of the modifications described below.
  • r.ne nucleic acid molecule in use m the invention may be optimized for providing high level expression of the targeted antigen (e.g. HPV early polypeptide ( s )) in a particular host ceil or organism, e.g. a hurran host cell or organism.
  • codon optimisation is performed by replacing one or more "native" (e.g. HPV) codon corresponding to a codon infrequently used in the mammalian host cell by one or more codon encoding the same amino acid which is more frequently used. This can be achieved by conventional mutagenesis or by chemical synthetic techniques
  • the term "recombinant vector” refers tc viral as well as non viral vectors, including extrachromosomal (e.g. episome) , multicopy and integrating vectors (i.e. for being incorporated into the host chromosomes) .
  • extrachromosomal e.g. episome
  • multicopy and integrating vectors i.e. for being incorporated into the host chromosomes
  • vectors for use in gene therapy i.e. which are capable of delivering the nucleic acid to a host organism
  • expression vectors for use in various expression systems are particularly important in the context of the invention.
  • Suitable non viral vectors include plasmids such as pREP4, pCEP4 ( Invitrogene) , pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76, 12735-12746).
  • Suitable viral vectors may be derived from a variety of different viruses (e.g. retrovirus, adenovirus, AAV, poxvirus, herpes virus, measle virus, foamy virus and the like) .
  • the term "viral vector” encompasses vector DNA/RNA as well as viral particles generated thereof.
  • ViraJ vectors can be i ep] ication-competent , or can be genetically disabled so as to be replication-defective or replication-impaired.
  • replication-competent encompasses replication-selective and conditionally-replicative viral vectors which are engineered to replicate better or selectively in specific host cells (e.g. tumoral cells) .
  • the recombinant vector in use in the invention is a recombinant adenoviral vector (for a review, see “Adenoviral vectors for gene therapy", 2002, Ed D. Curlel and J. Douglas, Academic Press) . It can be derived from a variety of human or animal sources and any serotype can be employed from the adenovirus serotypes 1 through 51. Particularly preferred are human adenoviruses 2 (Ad2), 5
  • Ad5 Ad5
  • Ad6 Ad6
  • 11 AdIl
  • 24 Ad24
  • 35 Ad35
  • the adenoviral vector in use in the present invention can be replication-competent.
  • Numerous examples of replication-competent adenoviral vectors are readily available to those skill in the art (see, for example,
  • they can be engineered from a wild-type adenovirus genome by deletion in the ElA CR2 domain (see for example WO00/24408) and/or by replacement of the native El and/or E4 promoters with tissue, tumor or cell status-specific promoters (see for example " JS 5,998,20s, WO99/25860, US 5,698,443, WO00/46355,
  • the adenoviral vector in use in the invention is replication-defective (see for example WO94/28152; Lusky et al . , 1998, J. Virol 72, 2022-2032).
  • Preferred replication-defective adenoviral vectors are El- defective (see for example US 6,136,594 and US 6,013,638), with an Ei deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of the human adenovirus type 5 disclosed in the GeneBank under the accession number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280-285).
  • the cloning capacity can further be improved by deleting additional portion (s) of the adenoviral genome (all or part 5 of the non. essential E3 region or of other essential E2, KA regions) .
  • Insertion of a nacieic aci ⁇ in any location of the adenoviral vecror can be performed through homologous recombination as described in Charrier et al. (1996, J. Virol. 70, 4805-4810) .
  • ⁇ ,he nucleic acid encoding 10 the HPV- 16 E6 polypeptide can be inserted in replacement of the El region and the nucleic aci ⁇ encoding the FPV-16 E7 polypeptide in replacement: of the E3 region or vice versa.
  • the vector in use in the invention is a poxviral vector (see for example Cox et
  • suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266
  • the vector may also be obtained from any other member of the poxviridae, in particular fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); canarypox (e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev Biol Stand., 84, 159-
  • fowlpox e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163
  • canarypox e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev Biol Stand., 84, 159-
  • said virus can be a replication competent poxvirus, especially replication competent vaccinia virus .
  • replication competent poxvirus especially replication competent vaccinia virus .
  • examples of these virus are provided m WO9531105 (e.g. products JX594, VV TK- GMCSF or JX963, VV TK- VGF- GMCSF), WO0073479, WO2009/06b547, WO2009/065546.
  • the nucleic acid encoding she antigen of the Invention is preferably inserted in a nonessential locus of the poxviral genome, in order that L"he recombinant poxvirus remains viable and infectious.
  • Nonessential regions are non- coding intergenic regions or any gene for which inactj vation or deletion does not significantly impair viral growth, replication or infection.
  • the antigen- encoding nucleic acid is preferably inserted in the thymidine 5 kinase gene (tic) (Hruby et al., 1983, Proc. Natl. Acad. Sci USA 80, 3411-3415 ; Weir et al., 1983, J. Virol. 46, 530- 537).
  • tic thymidine 5 kinase gene
  • other insertion sites are also appropriate, e.g. in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189-4198), in the KlL locus, in the u gene (Zhou et al.,
  • the antigen-encoding nucleic acid can be inserted in any one of the identified deletions I TO VII as
  • 25 nucleic acid is preferably introduced in the intergenic region situated between ORFs 7 and 9 (see for example EP 314
  • said recombinant vector is a recombinant plasmid DNA or a recombinant viral 30 vector.
  • said recombinant viral vector is a recombinant vaccinia vector.
  • said recombinant vaccinia vector is a recombinant MVA vector .
  • the antigen-encoding nucleic acid in use in the invention is m a form suitable for its expression in a host cell or organism, whicn means that the nucleic acid sequence encoding the antigen are placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism-
  • regulatory sequence refers to any sequence that allows, conirio ⁇ tes or modulates the expression of a nucleic acid m a given host cell, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA ⁇ into the host cell.
  • the nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell.
  • the gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to whicn it is operatively linked.
  • the gene expression sequence may, for example, be a mammalian or viiai promoter, such as a constitutive or inducible promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters.
  • HPRT hypoxanthine phosphoribosyl transferase
  • adenosine deaminase adenosine deaminase
  • pyruvate kinase pyruvate kinase
  • b-actin promoter b-actin promoter
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV) , simian virus (e.g., SV40), papilloma virus, adenovirus, human Immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus .
  • CMV cytomegalovirus
  • simian virus e.g., SV40
  • papilloma virus e.g., SV40
  • HSV40 human Immunodeficiency virus
  • HSV40 human Immunodeficiency virus
  • Rous sarcoma virus Rous sarcoma virus
  • cytomegalovirus cytomegalovirus
  • LTR long terminal
  • Inducible promoters are expressed in the presence of an inducing agent.
  • the metal lothionein promoter is induced to promote transcription and translation in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill In the art.
  • the gene expression sequence shall include, as necessary, 5' non- transcribing and 5' non-translating sequences Involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • Preferred promoters for use in a poxviral vector include without limitation vaccinia promoters 7.5K, H5R, TK, p23, pll and KIT.., chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechnlques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158).
  • the promoter is of special importance and the present invention encompasses the use of constitutive promoters which direct expression of the nucleic acid in many types of host cells and those which direct expression only in certain host cells or in response to specific events or exogenous factors (e.g. by temperature, nutrient additive , hormone or other ligand) .
  • Suitable promoters are widely described in literature and one may cite more specifically viral promoters such a ⁇ RSV, SV40, CMV and MLP promoters.
  • Preferred promoters for use in a poxviral vector include without limitation vaccinia promoters 7.5K, H5R, TK, p28, pll and KIL, chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described In Chakrabarti et al. (1997, Bi otechn Lques 23, 1094-1097), Hammond et al. (1997, J, Virologies! Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 351-158).
  • the regulatory elements controlling the expression of the nucLeic acid molecule of the invention may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine- Dalgamo sequences, etc. ) into the host cell or organism.
  • transcription e.g. polyA transcription termination sequences
  • mRNA transport e.g. nuclear localization signal sequences
  • processing e.g. splicing signals
  • stability e.g. introns and non-coding 5' and 3' sequences
  • translation e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine- Dalgamo sequences,
  • the recombinant vector in use in the present invention can farther comprise at least one nucleic acid encoding at least one cytokine.
  • Suitable cytokines include without limitation interleukins (e.g. IL ⁇ 2, IL- 7, IL- 15, IL-18, IL-21) and interferons (e.g. 15 "1 Ny, INF ⁇ ) , with a special preference for mterieukin IL-2.
  • the recombinant vaccine of the invention comprises a cytokine-expressing nucleic acid
  • said nucleic acid may be carried by tne recombinant vector encoding the one or more antigen (s) or by an independent recombinant vector which can De of the same or a different origin.
  • the recombinant vector in use in the present invention is encoding all or part of the MUCi antigen and at least one cytokines above listed, and preferably an ⁇ nterleukin, especially IL2,
  • the recombinant vector in use in the present invention is an MVA encoding ail or part of the MUCi antigen and at least one cytokines above listed, and preferably an inr.erleuk.in, especially 11,2.
  • Infectious viral particles comprising the above- described recombinant viral vector can be produced by routine process.
  • Rn exemplary process comprises the steps of:
  • Cells appropriate for propagating adenoviral vectors are for example 293 cells, PERC6 cells, HER96 cells, or cells as disclosed in WO 94/28152, WO 97/00326, US 6,127,175.
  • Cells appropriate for propagating poxvirus vectors are avian cells, ana most preferably primary chicken embryo fibroblasts (C ⁇ E" ⁇ prepared from chicken embryos obtained from fertilized eggs.
  • the infectious viral particles may be recovered frcm the culture supernatant or from the cells after lysis (e.g. by chemical means, freezing/thawing, osmotic shock, mecanic shock, sonication and the like) .
  • the viral particles can be isolated by consecutive rounds of plaque purification and then purified using the techniques of the art (chromatographic methods, ultracentrifugation on caesium chloride or sucrose gradient).
  • the method or use for treating a pat Lenr. for human disease human disease according io the Invention can be carried out in the selected patients in conjunction with one or more conventional therapeutic modalities (e.g. radiation, chemotherapy and/or surgery ⁇ .
  • one or more conventional therapeutic modalities e.g. radiation, chemotherapy and/or surgery ⁇ .
  • the use of multiple therapeutic approaches provides the selected patient with a broader based intervention.
  • the method or use for treating a patient for human disease human disease according to the Invention can be preceded or followed by a surgical intervention. In another embodiment, it can be preceded or followed by radiotherapy (e.g. gamma radiation).
  • the method or use of the invention is associated to chemotherapy with one or more drugs (e.g. drugs which are conventionally used for treating or preventing viral infections, virus-associated pathologic conditions, cancer, and the like).
  • drugs e.g. drugs which are conventionally used for treating or preventing viral infections, virus-associated pathologic conditions, cancer, and the like.
  • the present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
  • the present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
  • the administration of said chemotherapeutic agent is done before administration of said immunogenic composition.
  • the administration of said chemotherapeutic agent is done after administration of said Immunogenic composition.
  • the administration of said chemothera sufficientlyic agent is done concomitantly with administ rar 1 on of said immunogenic composition.
  • said chemotherapeutic agent is cisplatin and/or Gemcitabine, or similar.
  • the present Invention further concerns a method of improving cytotoxic effectiveness of cytotoxic drugs (i.e. chemotherapeutic agent ⁇ or radiotherapy which comprises co- treating a patient selected in a patient population composed of patients that have low ILlO/IFN ⁇ ratio with an immunogenic composition according to the Invention.
  • cytotoxic drugs i.e. chemotherapeutic agent ⁇ or radiotherapy
  • the method or use of immunogenic composition of the invention is carried out according to a prime boost therapeutic modality which comprises sequential administration of: one or more primer compos It " i on (s) and one or more booster composition (s ) .
  • the priming and the boosting compositions use different vehicles which comprise or encode at least an antigenic domain in common.
  • the priming immunogenic composition is initially administered to ⁇ he host organism and the boosting immunogenic composition is subsequently administered to the same host organism after a period varying from one day to tv/elve months.
  • the method of the invention may comprise one to ten sequential administrations of the priming composition followed by one to ten sequential administrations of the boosting composition. Desirably, injection intervals are a matter of one week to six months.
  • the priming and boosting compositions can be administered at the same site or at alternative sites by the same route or by different routes of administration.
  • the desired clinical benefit is independent of a demonstrable immune response to the vaccine.
  • the Invention relates to a method as above described wherein said human disease is cancer.
  • said cancer is for example breast cancer, colon cancer, kidney cancer, rectal cancer, lung cancer, cancer of the head and neck, renal cancer, malignant melanoma., laryngeal cancer, ovarian cancer, vicai cancer, prostate cancer, nori Small cell Lung Cancer, haematological cancecs, gastric cancers, myeloma .
  • the Invention relates to a method as above described wherein said human disease is Infectious disease.
  • said infectious disease is a viral induced disease, such as for example disease induced by HTV, HCV, HBV, HPV, and the like.
  • an immunogenic composition comprising ail or part of a targeted antigen for the manufacture of a medicament for treat inq a patient for human disease in a particular patient; population wherein the patients of said population have low ILl 0/IF 1 NY ratio.
  • an immunogenic composition for the manufacture of a medicament for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFN ⁇ ratio.
  • an immunogenic composition for the manufacture of a medicament for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFN ⁇ ratio.
  • an immunogenic composition for the manufacture of a medicament for inducing an immune response to a targeted antigen (j . e. the raised immune response) m a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFN ⁇ ratio.
  • an immunogenic composition for the manufacture of a medicament for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have J ow ILlO/IFN ⁇ ratio and wherein said raised immune response is innate immune response.
  • said "raised immune response" in said patient population is directed towards a tumour-specific or -related antigens and/or viral anrigen.
  • said "raised immune response” in said patient population is directed towards distinct antigens.
  • said "raised immune response” in said patient population is directed towards all or part of MUCl antigen.
  • said "raised immune response” in said patient population is T ceil immune response, and preferably CD8+ (Cytotoxic T Lymphocytes) immune response.
  • said "raised immune response" in said patient: population is a non specific immune response or an immune response to disease associated antigens not contained in the vaccine formulation or an immune response to disease associated antigens which is not measureable by current techniques.
  • said "raised immune response” in said patient population is a stimulation of the innate immune response .
  • the ability to induce or stimulate ar. immune response upon administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays which are standard in the arr..
  • assays which are standard in the arr..
  • Measurement of cellular immunity can be performed by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T ⁇ cells (e.g.
  • IL-IO or IFN gamma-producing cells by ELIspot
  • determination of the activation status of immune effector cells e.g. T cell proliferation assays by a classical [ 3 Hl thymidine uptake
  • assaying for antigen- specific T lymphocytes in a sensitized patient e.g. pepfi ⁇ e- specific lysis in a cytotoxicity assay
  • detection of antigen specific T ceils e.g. tetramers.
  • the ability to stimulate a humoral response may be determined by antibody binding and/or competit Lon in binding (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press) .
  • the method of the invention can also be further validated in animal models challenged with an appropriate tumor-inducing agent (e.g. MUCl-express: ng murine tumor ceils cells) co determine antitumor activity, reflecting an induction or an enhancement of an anti-antigen immune response.
  • an appropriate tumor-inducing agent e.g. MUCl-express: ng murine tumor ceils cells
  • the present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an immunogenic composition, said method comprising the following steps :
  • the present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an .immunogenic composition, said method comprising the following steps :
  • the present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an immunogenic composition and chemotherapeutic agent (see above) , said method comprising the following steps :
  • the present Invention relates to the use of ILlO/IFN ⁇ ratio as a biomarker for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition.
  • the present Invention relates to the use of ILlO/IFN ⁇ ratio as a biomarker for predicting whether a patient is or is nor su.sceptib] e "co developing prophylactic or therapeutic immune response by administration ot an immunogenic composition, wherein low ILlO/IE " Ny ratio indicates tnar. the patient is predicted to hcve an increased t-usceptibility zo develop a prophylactic or therapeutic response, preferably immune response,
  • the present Invention relates to the use of the ILlO/IFN ⁇ ratio as a biomarker for predicting whether a patient is or is not susceptible to survive longer after administration of an immunogenic composition, wherein low ILlO/IFN ⁇ ratio indicates that the patient is predicted to have a longer survival rate compared to treated patients who have higher ILlO/IFN ⁇ ratio.
  • the present invention relates to an ex- vivo method for predicting whether a patient is or is not susceptible to survive longer after administration of an immunogenic composition, wherein the testing method comprises the step of measuring levels of TLIO and INFy in a biological sample (e.g. blood or plasma or serum) from che patient, calculating ILlO/IFN ⁇ ratio, wherein low ILlO/TFN ⁇ ratio indicates chat the patient will have a longer survival race.
  • a biological sample e.g. blood or plasma or serum
  • Trius tne Invention further concerns the use of the ILlO/IFN ⁇ ratio as a biomarker for predicting whether a patient which is undergoing chemotherapeutic treatment with a cheinotherapeutic agent is or is not susceptible to developing prophylactic or therapeutic immune response (e.g. to survive longer) after administration of an immunogenic composition.
  • the Invention further concerns the use of the IL10/IFN ⁇ ratio as a biomarker for predicting whether a patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent is or is not susceptible to developing prophylactic or therapeutic immune response (e.g. to survive longer) after administration of an immunogenic composition, wherein low IT 10/IE 1 N/ ratio indicates that the patient Is predicted to have an increased susceptibility to develop a prophylactic or therapeutic immune response.
  • Tne present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotnerapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
  • kits i.e. companion test
  • the kit of parts, or kits may include reagents for collecting and or measuring serum levels of levels of ILlO and IFNy. Such reagents may include antibodies.
  • the kits may further include equipment for collecting an ⁇ /or processing biological samples.
  • the kits are also likely to contain instructions for use, cut-off values (see above) and/or instructions for their determination, and instructions for interpreting the data obtained from the use of the kits .
  • the said kit of parts, or kits may further include an immunogenic composition as above disclosed, and/or as disclosed in the Example section below.
  • the invention further provides computer programs and/or algorithms for monitoring clinical trial, levels of ILlO and IFN ⁇ and ILlO and IFN ⁇ ratio, determining whether such ratio are above or below a threshold level, and/or recommending treatment to improve a patient's response to an immunotherapy treatment.
  • the computer programs or algorithms may be provided aJ ong with necessary hardware, e.g., in the form of a kit or apparatus, which may also accept biological samples and measure the relative levels of ILlO and IFN ⁇ present therein and calculate ILlO and TFN ⁇ ratio.
  • the above- described computer programs and/or apparatus are likely to be provided to physicians or clinical laboratories with appropriate instructions and reagents, including antibodies. Use of levels of ILlO and INF ⁇ in the generation of an algorithm for recommending modification to a treatment to improve patient' s response io immunotherapy treatment by immunogenic composition.
  • Fig-ure 1 Survival curves describing vaccine immunotherapy in lung cancer: patients with a ratio of plasma IL-10/I FNg ⁇ or > 3.7, prior to treatment
  • Figure 2 Survival curves describing chemotherapy in lung cancer: patients with a ratio of plasma IL ⁇ 10/IFNg ⁇ or > 3.7 prior to treatment.
  • Example 1 The immunogenic composition, noted vaccine TG4010, was used to treat non-small cell lung cancer (NSCLC) patients in combination with standard chemotherapy.
  • NSCLC non-small cell lung cancer
  • TG4010 is a recombinant Modified Virus Ankara (MVA) expressing both IL2 and the tumor-associated antigen MUCl (see Rochlitz et al., 2003, J. Gene Med. r 5, 690-699).
  • MVA Modified Virus Ankara
  • PFS progression-free survival
  • Blood samples were taken prior to treatment on day 1 (first day of treatment) and were shipped immediately to a central immunology lab where plasma was isolated and stored frozen until analysis.
  • Plasma samples were assessed, at a central laboratory for cytokines, using a Luminex mutli-analyte profiling system.

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Abstract

The present invention concerns biomarkers and use thereof for determining whether a subject is or is not susceptible to developing a prophylactic or therapeutic immune response after such treatment.

Description

BIOMARKER FOR SELECTING PATIENTS AND REIATED METHODS
The present invention relates to the field of immunology ana, in particular, to immunotherapy of a patient against diseases caused for example by infection cr cancers. More particularly, the invention relates to methods for predicting whether a patient is or is not susceptible to developing a prophylactic or therapeutic response, preferably immune response, after such immunotherapy. The present invention relates to methods and compositions for improving the survival rate of patients to be treated by an immunogenic composition, in particular a therapeutic vaccine.
Traditional vaccination techniques involving the introduction into an animal system of an antigen (e.g. peptides, proteins) which can induce an immune response, and thereby protect said animal against infection for example, have been known for many years. These techniques have further included the development of both live and inactivated vaccines. Lave vaccines are typically attenuated non¬ pathogenic versions of an infectious agent that are capable of priming an immune response directed against a pathogenic version of the infectious agent.
Numerous research groups have also investigated the use of vaccines cts a potential therapeutic modal.ity for various cancer types. This specific type of vaccine strategy is generally referred to as immunotherapy.
In recent years there have been advances in the development of recombinant vaccines, especially recombinant live vaccines, in which foreign antigens of interest are encoded and expressed from a vector. Among them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigaced for their ability to express proteins from foreign pathogens or tumoral tissue, and :o induce specific immunological responses against these antigens in vivo. Generally, these qene-bαsed vaccines can stimulate potent numoral and cellular immune responses and VJ rai vectors may be an effective strategy for both the delivery of antigen-encoαxng genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen- specific antigens to the immune system. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application (for a review on recombinant viral vaccines see for example Harrop and Carroll, 2006, Front Biosci., 11, 804-817 ; Yokoyama et al., 1997, J Vet Med Sci.,59, 311-322) .
Following the observation in the early 1990 's that piasmid DNA vectors coαid di reefJy transfect animal cells in vjvo, significant research efforts have also been undertaken to develop vaccination techniques based upon the use of DNA pl&smids to induce immune response, by direct introduction into animals of DNA which encodes for antigens. Such techniques which are widely referred as DNA vaccination have now been used to elicit protective immune responses in large number of disease models. For a review on DNA vaccines, see Reyes-Sandoval and Erti, 2001 (Current Molecular Medicine, 1, 217-243) . A general problem in vaccine field however has been the identification of a means of inducing a sufficiently strong immune response in vaccinated individuals to protect and/or treat against infection and disease, and "hereby to extend the survival of patient having fatal disease, for example, cancer .
Therefore there has Joeen for example major effort in recent years, to discover new drug corrpounds that act cy stimulating certain key aspects of the immune system which will serve to increase the immune response induced by vaccines. Most of these compounds, referred as xminune response modifiers (IRMs) or adjuvants, appear to act through basic immune system mechanisms via Toll-like receptors (TLRs) to induce various important cytokines biosynthesis (e.g., interferons, interleukins, tumor necrosis factor, etc. see for example Schiller et al., 2006, Exp Dermatol., 15, 331-341). Such compounds have been shown to stimulate a rapid release of certain T cell, dendritic ceil, or monocyte/macrophage- derived cytokines and are also capable of stimulating lymphocyte function, such as B cells to secrete antibodies which play an important role in the antiviral and antitumor activities of IRM compounds .
Alternatively, vaccination strategies have been proposed, most of them being based on a prime-boost vaccination regimen. According to these "prime-boost" vaccination protocols, the immune system is first induced oy administering to the patient a priming composition and then boosted by administration of a boosting second composition
(see for example EP1411974 or US20030191076) .
Moreover, it has been shown in the health care context that one treatment can be effective only in specific group of patients. It is thus desirable to provide to physicians tools and methods that will enable them to tailor optimal personalized patient therapies, i.e. to prescribe the right therapy to the right patient at right time, to provide a higher treatment success rate, to monitor the response to the treatment, to increase drug efficacy and safety, to eliminate the unnecessary treatment of patients for whom therapy is not appropriate, to spare the patient unnecessary toxicity and side effects, to reduce the cost to patients and insurers of unnecessary or dangerous ineffective medication, and tc improve patient quality of life, eventually making cancer a managed disease, with follow up assays as appropriate.
With these regards, literature proposes various tools and methods, such as for example :
Pharmacogenetics, which consist in the study of individual response to drugs as a function of genetic differences. These responses relate to how a drug functions in any given individual, how it is metabolized, its toxicity and dosage requirements. With the human genome project, pharmacogenetics has expanded into pharmacogenomics . Pharmacogenomics goes beyond pharmacogenetics, with the potential to find uses from drug discovery and development, target discovery and validation, and clinical trials;
Metabolomics can also be applied to the field of predictive medicine. Unlike pharmacogenetics, which is limited to genetic factors, pharrαaco-metabolomics is able to predict an individual's response to a drug based not only on genetic factors, but also non-genetic factors, such as other drugs in the patient's body, the patient's current state of health, etc.
The role of biornarkers is becoming increasingly important in the clinical development of therapeutics. A biomarker can be an indicator of normal biological processes, disease processes, or pharmacological responses to therapeutic intervention. Their role ranges from, stratifying the patient population in helping to identify responders versus non-responders to determining the efficacy of the therapeutic. Biomarkers can be a valuable tool m making better decisions that will reduce the cost for drug development and enabJ e therapies to reach the most appropriate patient population faster.
The invention provides rraterials and methods for predicting the efficacy of a treatment involving the administration of an immunogenic composition to a patient {i.e. immunotherapy treatment) using biological markers (biomarkers) that have been determined to be substantially reliable signature which correlates with the desired clinical outcome, lhe biomarkers are present in biological samples obtained from the patient, prior to the treatment with the said immunogenic composition. The ability to predict the clinical outcome of a treatment, before its initiation, will enable clinicians and patients to identify ineffective therapy, make informed decisions regarding the course of treatment, including whether to abandon or to allow alternate therapy implementation.
The Applicant has now identified new tool and vaccination strategy. More specifically, the present Invention relates to the use of the interleukin-10 /J nterferon-gamma (ILl0/IFNγ) ratio as a biomarker for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition. Alternatively, the interferon-gamma/ interleukin-10 (IFKy/ ILlO) ratio can be used as a biomarker according to the present invention, but in that case conclusions should be adapted.
Van den Boogaardt et al, 2006 {Transplantation, 82, 844-848) have shown that the interferon-gamma/interleukin-10 (IFNγ/IL10) ratio is a valuable tool to discriminate between non-rejecting and rejecting patients for renal transplantations .
Jamal et al . , 2007, (Tuberculosis, 87, 279-287) have shown that in pulmonary and extra pulmonary tuberculosis, there is a direct relationship between inter feron- gamπia/interleuJ<in~10 {IFNy/ILlO) ratio ana disease severity ranking .
Similarly, Gomes-Silva et a], 2007 , (Clinical and Experimental Immunology, 149, 440-444) indicate that high IFNY and low ILlO are associated with severity of mucosal leishmaniasis .
According to a first embodiment,, the present Invention relates to a method for treating a patient for human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low IL10/IFNγ ratio.
The present Invention thus relates to a method for treating a patient for human disease by administering an immunogenic composition, said method comprising the following steps :
- selection of one patient in a patient population composed of patients that have low ILlO/ IFNγ ratio,
- administering to sad d selected patient the said immunogenic composition.
According to another embodiment, the present Invention relates to a method for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition, said method comprising the steps of:
- obtaining a blood sample from the patient; - measuring levels of ILlO and IFNγ in the said blood sample, and
- calculating ILlO/IFNy ratio, wherein low ILlO/IFNy ratio indicates that the patient is predicted to have an increased susceptibility to develop a prophylactic or Therapeutic response, preferably immune response.
According to another embodiment, the present Invention relates to a metfioα for selecting a patient susceptible to developing prophylactic or therapeutic response, preferaoly immune response, by administration of an immunogenic composition, said method comprising the steps of:
obtaining a blood sample from the patient;
- measuring levels of ILlO and IFNγ in the said blood sample, and
calculating ILlO/IFNγ ratio, wherein low IL10/IFNγ ratio indicates that the patient has an increased susceptibility to developing a prophylactic or therapeutic response, preferably immune response. According to another embodiment, the present Invention relates to a method for predicting whether a patient is or is not susceptible to respond positively to a treatment comprising administration of an immunogenic composition, saJ d method comprising che steps of:
- obtaining a blood sample from the patient;
- measuring levels of ILlO and IFNγ in the said blood sample, and
calculating IL10/IFNγ ratio, wherein low IL10/IFNγ ratio indicates that the patient is predicted to have an increased susceptibility to develop a prophylactic or Therapeutic response, preferably immune response.
According to another embodiment, the present Invention relates to a method for selecting a patient susceptible ic respond positively to a treatment comprising administration of an immunogenic compos.! tion, said method comprising the steps of:
obtaining a blood sample from trie patient;
- measuring levels of ILlO and IFNγ in the said blood sample, and
calculating ILlO /I FNY ratio, wherein low ILlO/IFNy ratio indicates that the patient has an increased susceptibility to developing a prophylactic or therapeutic response, preferably immune response. According to another embodiment, the present Invention relates to an ex-vivo method for testing whether a patient will respond therapeutically to a method of treatment comprising administration of an immunogenic composition, wherein the testing method comprises the steps of:
- obtaining a blood sample from the patient;
- measuring levels of ILlO and IFNγ in the said blood sample, and calculating ILlO/lFNy ratio, wherein Jow IL10/IFNγ ratio indicates that the patient will develop a prophylactic or therapeutic response, preferably immune response, towards trie immunogenic composition.
According to another embodiment, the present Invention relates to an ex~vivo method for testing whether a patient will respond therapeutically to a method of treating cancer by administration of an immunogenic composition, wherein the testing method comprises the steps of:
obtaining a blood sample from the patient;
- measuring levels of ILlO and IFNγ in the said blood sample, and
calculating IL10/IFNγ ratio, wherein low ILlO/IFNγ ratio indicates chat. ύhe patient will respond therapeutically to the method of treating cancer.
The present Invention further relates to an ex vivo method of treatment comprising administration of an immunogenic composition, wherein the resting method comprises the step of measuring levels of ILlO and INFy in a biological sample, especially blood sample, from the patient, calculating ILlO/IFNfγ ratio, wherein low ILlO/IFNγ ratio indicates that the patient will develop a prophylactic or therapeutic response, in particular immune response, towards the immunogenic composition.
According to another embodiment, the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low IL!0/IFNγ ratio,
According to another embodiment, the present Invention relates to a method for inducing an immune response io at ieasr one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients thai have low ILlO/IFNγ ratio.
According to another embodiment, the present Invention relates to a method for inducing an Immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said patient is selected in a patient population composed of patients that have low ILlO/IFNγ ratio and wherein said raised iiurrune response is innate immune response. The innate immune response is body's inxtial immune defense against pathogens and is elicited by a variety of cells including antigen-presenting cells or "APCs". These cells express surface and cytoplasmic receptors that recognize molecules of foreign origin {e.g., bacterial and viral nucleic acids, proteins, carbohydrates). Upon detecting these signals, the dendritic cells and macrophage elicit a defensive response that includes the release of cytokines (including interferons, TNF-alpha, and IL-12) and chemokines that attract cells such as immature dendritic cells, macrophage, NK ceils, and granulocytes, to the site of challenge. The innate immune response thus confers non-specific protection while the body is generating the adaptive response.
The present Invention thus relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps :
- selection of one patient in a patient population composed of patients tnat have low ILlO/ItNy ratio,
- administering to said selected patient the said immunogenic composition.
According to another embodiment, the present Invention relates to a method for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps : - selection of one patient in a patient population composed of patients that have low ILlO/IFNγ ratio,
- administering to said selected patient the said immunogenic compos ii ion.
According to another embodiment, -he present Invention relates to a method for inducing an immune response {i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition wherein said raised immune response is innate immune response, said method comprising τ.he following steps :
- selection of patient in a patient population composed of patients that have low IL10/IFNγ ratxo,
- administering to said selected patients the said immunogenic composition.
According to another embodiment, the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps :
- measuring in the patient the levels of ILlO and IFNγf
calculating IL10/IFKγ ratio, and
- administering to the patient the said immunogenic compositior- if said patient has low ILlO/IFNγ ratio. According to another embodiment, the present Invention relates to a method for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease by administering an immunogenic composition, said method comprising the following steps : measuring in the patient the levels of ILlO and
IFNy,
calculating TLlO /IFNy ratio, and
- administering to the patient the said immunogenic composition if said patient has low ILlO/I FNy ratio.
According to another embodiment, the present Invention relates to a method for inducing an immune response (i.e. the raised immune response) in a patient for creating human disease by administering an immunogenic composition wherein said raised immune response is innate immune response, said method comprising the following steps :
measuring in the patient the levels of ILlO and IFNy calculating ILlO/IFNγ ratio, and
- administering to the patient the said immunogenic composition if said patient has low ILlO/IFNγ ratio.
As used herein throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced compounds or steps, unless the context dictates otherwise. For example, the term "a cell" includes a plurality of cells including a mixture thereof. More specifically, "at least one" and "one or more" means a number which Is one or greater than one, with a special preference for one, two or three.
The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term" .
The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5%. For sake of clarity, it is added that "about x" is including the x specific value.
The terms "patient", "subject" refer to a vertebrate, particularly a member of the mammalian species and includes, but is not limited to, domestic animals, sport animals, primates including humans.
As used herein, the term "treatment" or "treating" encompasses prophylaxis and/or therapy. Accordingly the immunogenic compositions or methods of the present invention are not limited to therapeutic applications and can be used in prophylaxis cries. This is covered by the term "co developing a prophylactic or therapeutic immune response" herein. "Prophylaxis" is not limited to preventing immediate diseases {e.g. infectious diseases), it further encompasses prevention of long term consequences of these infections such as cirrhosis or cancer.
An "effective amount" or a "sufficient amount" of an active compound is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations. A "therapeutically effective amount" is an amount to effect beneficial clinical results, including, but not limited to, alleviation of one or more symptoms associated with viral infection as we] 1 as prevention of disease (e.g. prevention of one or more symptoms of infection).
The terms "a patient selected in a patient population composed of patients that have low ILlO/IFNy ratio" should be understood as meaning a patient for who levels of interleukin-10 and interferon-gamma have beer, measured as disclosed herein, and who has low IL10/IFNγ ratio. When the number of patient tested is above 1, the said patients form a patient population. According to special embodiments, the terms "a patient will respond therapeutically" or Λλa patient will respond positively to a treatment" mean that the said patient has an increase ot survival rate (see example section).
Tn a preferred embodiment of the invention, the method of the invention comprises an mxtial step consisting in measuring the interieukin-10 and interferon-gamma levels in biological samples from the patient before administration of the immunogenic composition.
According to the present invention, the levels of interieukin-10 and interferon-gamma are measured in a biological sample obtained from the patient. Biological samples include but are net limited to blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen. In a preferred embodiment, the biological sample is blood, plasma or serum, in which case obtaining the samples from a patient is relatively simple and non-invasive procedure. Methods of obtaining blood or serum are well-known in the art are not part of the invention.
In addition, numerous methods for detecting and quantifying polypeptides, including the instant biomarkers, are known. Such methods include but are not limited to anribody-based methods, more specifically monoclonal antibodies-based methods. The particular methods of detecting and quantifying the biomarkers are not important to the invention. For example the materials and methods of the present invention may be used with Lurni nex technology (Lurαinex Corporation, Austin, Tex.) or enzyme-linked immunosorbant assays (ELISA, numerous KLISA kits are commercially available e.g. by CliniScience, Diacione, Biosource) . According to one embodiment of the Invention, the levels of interleukin-10 and interferon-gamma are determined by using antibodies.
According to one specific embodiment of the Invention, said antibody (ies) is (are) specific of ILlO or ΣNFγ.
According to one specific embodiment of the Invention, said anc ~ bodies are monoclonal antibodies.
According to one specific embodiment of the Invention, said antibodxeε are tagged for example by fluorescence, radiolabel, enzyme, biotin, or any other methods designed to render cells labelled with said antibodies detectable. These techniques are widely used and known in the art.
In related aspects the method includes determining the levels of interleukin-10 and interferon-gamma in a patient prior administration of an immunogenic composition to the patient; calculating ILlO/IFNy ratio; comparing said IL10/IFMγ ratio to a cut-off value; and predicting the efficacy of immunotherapy treatment based on the levels of ILlO/IFNγ ratio compared to the cut-off value.
According to special embodiment, the said cut-off value is about 5, preferably about 4, more preferably about 3 and particularly about 2. According to one preferred embodiment, said cut-off value is about 3.7, and more preferably is 3.7. /According to prefer red embodiment, the "Mow ILlO/IFNy ratio" according to r.he present invention is designating ILlO/IFNy ratios below about 5, preferably below about 4 and more preferably below about 3 and particularly blow about ?.. According to one preferred embodiment, said the "low ILlO/IFNy ratio" according to the present invention is designating ILlO/IFNy ratios below about 3.7, and more preferably is below 3.7. According to preferred embodiment, the levels of interleukin-10 and interferon-gamma are measured by Enzyme Linked Immune Sorbant Assay (ELISA) , by Luminex © analysis, by lab-on-chip systems, by radio-immune assay or other systems based on specific molecular recognition of ILlO and ItNy using antibodies or other specific molecules.
As useα herein, the terms "immunogenic conpositicn" "vaccine composition" , "vaccine" or similar terms can be used interchangeably and mean an agent suitable for stiraulating/indαciug/increasing a patient's immune system to ameliorate a current condition, e.g. to improve survival rate, or to protect against or to reduce present or future harm or infections (including viral, bacterial, parasitic infections), e.g. reduced tumour cell proliferation or survival, reduced pathogen replication or spread in a patient or a detectabiy reduced unwanted symptom (s) associated with a condition, extend patient survival. Said immunogenic composition can contain at least (i) all or part of at least one targeted antigen and/or (ii) at least one recombinant vector expressing in vivo all or part of at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding all or part of at least one targeted antigen. According to an alternate embodiment, the immunogenic composition of the Invention comprises (iii) at least one immune response modifier, alone or in combination with (i) and/or (ii ) . Examples of such immune response modifiers (IRMs), include the CpG oligonucleotides (see US
6,194,388; US2006094683; WO 2004039829 for example), j ipopoJ ysaccnarides, polyinosic:polycyt idylic acid complexes
(Kadowaki, et al., 2001, J. Immunol. 366, 2291-2295), and polypeptides and proteins known to induce cytokine production from dendritic cells and/or monocyte/macrophages. Other examples of such immune response modifiers (IRMs) are small organic molecule such as imidazoquinolinamines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, imidazonaphthyridine amines, oxazoloσuinoline amines, thiazoloquinoline amines and 1,2-bridged imidazoquinoline amines (see for example US 4,689,338; US 5,389,640; US 6,110,929; and US 6,331,539), In another emDodiment, the immunogenic comDosition comprises cells whicn stimulate a patient immune response to treat a disease, such as cancer. Said cells can be antigen presenting cells, such as dendritic cells, combined with an antigenic composition (e.g. Provenge developed. by u i ',.
Figure imgf000018_0001
.c- -;>) , tumor cells (e.g. GvAX developed, by Cell Genesis) or lymphocytes,
As used herein, the term "antigen" refers to any substance, including complex antigen (e.g. tamour cells, virus infected cells, dendritic cells, etc...), that is capable of being the target of an immune response. An antigen may be the target of, for example, a cell-mediated and/or humoral immune response raised by a patient. The term "antigen" encompasses for example all or part of viral antigens, tumour-specific or tumour-related antigens, bacterial antigens, parasitic antigens, allergens and the like:
Viral antigens include for example antigens from hepatitis viruses A, B, C, D and E, HIV, herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, adenoviruses, coxsdkie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus, measles virus; some non-limiting examples of known viral antigens include the following : antigens derived from HIV-I such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vi£, vpr, vpu, rev or part and/or combinations thereof; antigens derived from human herpes viruses such as gH, gL gM gB gC gK gE or gD or part and/or combinations thereof or Immediate Early protein such asICP27, ICP47, ICP4, ICP36 from HSVl or KSV2 ; antigens derived from cytomegalovirus, especially human cytomegalovirus such as gB or derivatives thereof ; antigens derived from Epstein Barr virus such as gp350 or derivatives thereof; antigens derived from Varicella Zoster Virus such as gpl, 11, 111 and IE63; antigens derived from a hepatitis virus pucn as hepatitis B , hepatitis C or hepatitis E virus antigen (e.g. env protein El oc E2, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p/, or pQrt and/or combinations thereof of HCV) ; anr.igens derived from human papilloma viruses (for example HPV6, 11, 16, 18 , e.g. Ll, L2, El, E2, E3, E4, E5, E6, E7, or parr and/or combinations thereof) ; antigens derived from other viral pathogens, such as Respiratory Syncytial virus (e.g. F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, flaviviruses (e. g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenza virus cells (e.g. HA, NP, NA, or M proteins, or part and/or combinations thereof) ;
tumor-specific or -related antigens include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, renal cancer, malignant melanoma, laryngeal cancer, prostate cancer. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at low levels or at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded Dy mutant cellular genes, such as oncogenes (e.g., activated ras oncogene) , suppressor genes (e.g., mutant pb3) , fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as tnose carried on RNA and DNA tumor viruses. Some nor-limiting examples of tαmor-specific or -related antigens include MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV) , adenosine deaminase-binding protein (ADAbp) , cyciophilin b, Colorectal associated antigen (CRC) -C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-I and CAP-2, etvβ, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSA™2, and PSA-3, prostate- specific membrane antigen (PSMA) , T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4 , MAGE-Δ5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE- A9, MAGE-AlO, MAGE-AIl, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE- Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE- 1, GAGE-2, GAGE-3, GAGE-4, GAGE- 5, GAGE- 6, GAGE-7, GΛGE-8, GAGE-9), BAGE, RAGE, LAGE-I, NAG, GnT-V, MUM-], CDK4, tyrosinase, p53, MUC family (e.g. MUC-I), HER2/neu, p21ras, RCAS], aipha-fetoprotej n, E-cadher in, alpha-catenin, beta- caienin and gamma-catenin, pl20ctn, gplOO . sup . Pmelli7 , FRAME, NY-EΞO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiorype, pl5, gp75, GM2 and GD2 gangliosides , viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PlA, ElBV- encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40) , SSX-I, SSX-4, SSX- 5, SCP-I and CT-7, and c-erbB~2;
- bacterial antigens includes for example antigens from Mycobacteria causing TB and leprosy, pneumocci, aerooic grarn negative bacilli, mycoplasma, staphy Ioccocal infections, streptococcal infections, salrr.onellae, chlamydiae, neisseriae;
other antigens includes for exampJ e anzigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis;
allergens refer to a substance that can induce an allergic or asthmatic response in a susceptible patient. The list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin) . Examples of natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Canine (Canis familiaris) ; Dermatophagoides (e.g. Dermatophagoides farinae) ; Felis (Felis domesticus) ; Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum) ; Cryptomeria
(Cryptomeria japonica) ; Alternaria (Alternaria alternata) ;
Alder; Alnus {Alnus gultinoasa) ; Betula (Betuia verrucosa) ;
Quercus (Quercus alba) ; Olea (Olea europa) ; Artemisia
(Artemisia vulgaris}; P±antago (e.g. Plantago lanceolaca) ; Parietaria (e.g. Parietaria officinalis or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g.
Apis multif lorum) ; Cupressαs (e.g. Cαpressus sempervirens,
Cupressus aπzonica and Cupressαs macrocarpa) ; Juniperuε
(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei) ; Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa) ; Periplaneta (e.g. Periplaneta americana) ; Agropyron (e.g. Agropyron repens ) ; Secale (e.g. Secale cereale) ; Triticum (e.g. Triticum aestivum) ; Dactylis (e.g. Dactylis glomerata) ; Festuca {e.g. Festuca elatior) ; Poa (e.g. Poa pratensis or Poa compressa) ; Avena (e.g. Avena sativa) ; Holcus (e.g. Holcus lanatus); Anthoxcintbum (e.g. Anthoxanthum odoratum) ; Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phieυm (e.g. Phleαm pratense) ; Phalaris (e.g. Phalaris arαndinacea) ; Paspalum (e.g. Paspalum noratum) ; Sorghum (e.g. Sorgnum halepensis) ; and 3romus (e.g. 3romus ir.ermis).
According to one special embodiment, said anc igen is encoded by an heterologous nucleotide sequence and is expressed in vivo by a recombinant vector.
In a particularly preferred embodiment the heterologous nucleotide sequence of the present invention, encodes one or more of ail or part of the following antigens HBV-PreSl PreS2 and Surface env proteins, core and polHIV- gpl20 gp40,gpl60, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef; HPV-El, E2, E3, E4, E5, E6, El, E8, Ll, L2 (see for example WO 90/10459, WO 98/04705, WO 99/03885); HCV env protein El or E2, core protein, NS2, NS3, NS4af NS4b, NS5a, N35b, p7 (see for example WO2004111082 , WO2005051420 ) ; Muc-1 (see for example US 5,861,381; US6, 054 , 438 ; WO98/04727; WO98/37095) .
According to variants of the invention, the immunogenic composition contains at least two antigens, or an heterologous nucleotide sequence encoding at least two antigens, or at least two heterologous nucleotide sequences encoding at least two antigens, or any combination thereof.
According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes all or part of HPV antigen (s) selected in the group consisting of E6 early coding region of HPV, E7 early coding region of HPV and derivates or combination thereof. The HPV antigen encoded by the recombinant vector according to the invention is selected in the group consisting of an HPV Ξ6 polypeptide, an HPV E7 polypeptide or both an HPV E6 polypeptide and an HPV E7 polypeptide. The 5 present invention encompasses the use of any HPV E6 polypeptide which binding to p53 is altered or at least significantly reduced and/or the use of any HPV E7 polypeptide which binding to Rb is altered, or at leaεt significantly reduced {Munger et al., 1989, EMBO J. 8, 4099-
10 4105; Crook ez al., 1991, Cell 67, 547-556; Heck et al . , 1992, Proc. Natl. Acad. Sci . USA 89, 4442-4446; Phelps ΘL al., 1992, J. Virol. 66, 2148-2427). A non-oncogenic HPV-16 E6 variant which is suitable for the purpose of the present invention is deleted of one or more amino acid residues
15 located from approximately position 118 to approximately position 122 (+1 representing the first methionine residue of the native HPV-16 E6 polypeptide) , with a special preference for the complete deletion of residues 118 to 122 (CPEEK) . A non-oncogenic HPV-16 E7 variant which is suitable for the
20 purpose of the present: invention is deleted of one or more amino acid residues located from approximately position 21 to approximately position 26 (+1 representing the first amino acid of the native HPV-16 E7 polypeptide, with a special preference for the complete deletion of residues 21 to 26
25 (DLYCYE) . According to a preferred embodiment, tne one or more HPV-I 6 early polypeptide (s) in use in the invention is/are further modified so as to improve MHC class 1 and/or MHC class ΪI presentation, and/or to stimulate anti-HPV immunity. HPV E6 and Ξ7 polypeptides are nuclear proteins ana
30 iz. has been previously shown that membrane presentation permits to improve their therapeutic efficacy (see for example WG99/03885) . Thus, it may be advisable to modify at least one of the HPV early polypeptide (s) so as to be anchored to the cell membrane. Membrane anchorage can be easily achieved by incorporating in the HPV early polypeptide a membrane-anchoring sequence and if the native polypeptide lacks it a secretory sequence (i.e. a signal peptide).
Membrane-anchoring and secretory sequences are known in the art. Briefly, secretory sequences are present at the N-
Lerminus of the membrane presented or secreted polypeptides and initiate their passage into the endoplasmic reticulum
(ER). Tney usually comprise 15 uo 35 essentially hydrophobic amino acids which are then removed by a specific ER-Iocated enαopepnidase τo give the mature polypeptide. Membrane- ancncring sequences are usually highly hydrophobic in nature and serves to anchor the polypeptides m the cell membrane
(see for example Branden and Tooze, 1991, in Introduction to
Prouein Structure p. 202-214, NY Garland) .
The choice of the membrane-anchoring and secretory sequences which can be used in the context of the present invention is vast. They may be obtained from any membrane- anchored and/or secreted polypeptide comprising it (e.g. cellular or viral polypeptides) such as the rabies glycoprotein, of the HIV virus envelope glycoprotein or of the measles virus F protein or may be synthetic. The membrane anchoring and/or secretory sequences inserted in each of the early HPV-16 polypeptides used according to the invention may have a common or different origin. The preferred site of insertion of the secretory sequence is the N-terrninus downstream of the codon for initiation of translation and thar of the membrane-anchoring sequence is the C-terminus, for example immediately upstream of the stop codon.
The HPV E6 polypeptide in use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the measles F protein.
Optionally or in combination, the HPV E7 polypeptide in use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the rabies glycoprotein.
The therapeutic efficacy of the recombinant vector can also be improved by using one or more nucleic acid encoding 5 3 miTiunoporentiator polypeptide (s ). For example,- it may be advantageous to link the HPV early polypeptide ( s ) to a polypeptide such as calreticulin (Cheng et ai., 2001, J. Clin, Invest. 108, 669-678), Mycobacterium tuberculosis heat snock procein 70 (HSP70) (Chen et al . , 2000, Cancer Res. 60, 10 1035-1042), ubiquitin {Rodriguez et al., 1997, J. Virol. 71, 8497-8503) or the translocation domain of a bacterial toxin such as Pseυdomonas aeruginosa exotoxin A (ETA(dΙIl)) (Hung et al., 2001 Cancer Res. 61, 3698-3703).
According to another and preferred embodiment, the
15 recombinant vector according to the invention comprises a nucleic acid encoding one or more early polypeptide (s) as above defined, and more particularly HPV-16 and/or HPV-18 early E6 and/or E7 polypeptides.
According to another special and preferred embodiment, 20 saiα heterologous nucleotide sequence of the present invention, encodes all or part of MUC 1 antigen or derlvates thereof.
According to another special embodiment, said heterologous nucleotide sequence of the present inventdon,
25 encodes one or more of all or part of the followings: HCV env protein El or E2, core proreiπ, NS2, NS3, NG4a, NS4b, NS5a, NSBb, p7 or derivates thereof. According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes one or more fusion protein wherein
30 the configuration is not native m the sense that at least one of the NS polypeptides appears in an order which i s distinct from that of the native configuration. Thus, if the fusion protein comprises a NS3 polypeptide, a NS4A polypeptide and a NS5B polypeptide, the native configuration would be H53-NS4A-NS5B with NS3 at the N-terminus and NS5B at the C-terminus. In contrast, a non-native configuration can be NS5B-NS3-NS4A, NS5B-NS4A-NS3, NS4A-NS3-NS5B, NS4A-NS5B-NS3 or NS3-NS5B-NS4A. In particular, the fusion protein according to the invention comprises ai least one of the followings:
o A NS4A polypeptide fused directly or through a linker to the N-tecmmαs oi a NS3 polypeptide;
o A NS3 polypeptide fused directly or through a linker to the N-terrrαnus of a NS5B polypeptide;
o A NS4B polypeptide fused αirect Ly or tnrough a linker to the N-terminus of a NS5B polypeptide;
o A NS4A polypeptide fused directly or through a linker to the N-terminus of a NS3 polypeptide which is fused directly or through a linker to the N- terrainus of a NS4B polypeptide; and/or
o A NS3 polypeptide fused directly or through a linker to the N-terminus of a KS4B polypeptide which is fused directly or through a linker to the N~terminus of a NS5B polypeptide.
In such specific portions of the fusion protein of the invention, each of the NS polypeptides can be independently native or modified. For example, the NS4A polypeptide included in the NS4/-^-NG3 portion can be native whereas the NS3 polypeptide comprises at i east one of the modifications described below.
If needed, r.ne nucleic acid molecule in use m the invention may be optimized for providing high level expression of the targeted antigen (e.g. HPV early polypeptide ( s )) in a particular host ceil or organism, e.g. a hurran host cell or organism. Typically, codon optimisation is performed by replacing one or more "native" (e.g. HPV) codon corresponding to a codon infrequently used in the mammalian host cell by one or more codon encoding the same amino acid which is more frequently used. This can be achieved by conventional mutagenesis or by chemical synthetic techniques
(e.g. resulting in a synthetic nucleic acid) . It is not necessary to replace ail native codons corresponding to infrequently used codons since increased expression can be achieved even with partial replacement. Moreover, some deviations from strict adherence to optimised codon usage may be made to accommodate the introduction of restriction site (s) .
As used herein, the term "recombinant vector" refers tc viral as well as non viral vectors, including extrachromosomal (e.g. episome) , multicopy and integrating vectors (i.e. for being incorporated into the host chromosomes) . Particularly important in the context of the invention are vectors for use in gene therapy (i.e. which are capable of delivering the nucleic acid to a host organism) as well as expression vectors for use in various expression systems. Suitable non viral vectors include plasmids such as pREP4, pCEP4 ( Invitrogene) , pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76, 12735-12746). Suitable viral vectors may be derived from a variety of different viruses (e.g. retrovirus, adenovirus, AAV, poxvirus, herpes virus, measle virus, foamy virus and the like) . As used herein, the term "viral vector" encompasses vector DNA/RNA as well as viral particles generated thereof. ViraJ vectors can be i ep] ication-competent , or can be genetically disabled so as to be replication-defective or replication-impaired. The term "replication-competent" as used herein encompasses replication-selective and conditionally-replicative viral vectors which are engineered to replicate better or selectively in specific host cells (e.g. tumoral cells) .
In one aspect, the recombinant vector in use in the invention is a recombinant adenoviral vector (for a review, see "Adenoviral vectors for gene therapy", 2002, Ed D. Curlel and J. Douglas, Academic Press) . It can be derived from a variety of human or animal sources and any serotype can be employed from the adenovirus serotypes 1 through 51. Particularly preferred are human adenoviruses 2 (Ad2), 5
(Ad5), 6 (Ad6), 11 (AdIl), 24 (Ad24) and 35 (Ad35). Such adenovirus are available from the American Type Culture
Collection (ATCC, Pockville, Md. ) , and have been the patient, of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them (see for example US 6,133,028; US 6,110,735; WO
02/40665; WO 00/505'/3; EP 1016711; Vogels et al., 2003, J.
Virol. 77, 8263-8271) .
The adenoviral vector in use in the present invention can be replication-competent. Numerous examples of replication-competent adenoviral vectors are readily available to those skill in the art (see, for example,
Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-
2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727). For example, they can be engineered from a wild-type adenovirus genome by deletion in the ElA CR2 domain (see for example WO00/24408) and/or by replacement of the native El and/or E4 promoters with tissue, tumor or cell status-specific promoters (see for example "JS 5,998,20s, WO99/25860, US 5,698,443, WO00/46355,
WO00/15820 and WO01/36650) .
Alternatively, the adenoviral vector in use in the invention is replication-defective (see for example WO94/28152; Lusky et al . , 1998, J. Virol 72, 2022-2032). Preferred replication-defective adenoviral vectors are El- defective (see for example US 6,136,594 and US 6,013,638), with an Ei deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of the human adenovirus type 5 disclosed in the GeneBank under the accession number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280-285). The cloning capacity can further be improved by deleting additional portion (s) of the adenoviral genome (all or part 5 of the non. essential E3 region or of other essential E2, KA regions) . Insertion of a nacieic aciα in any location of the adenoviral vecror can be performed through homologous recombination as described in Charrier et al. (1996, J. Virol. 70, 4805-4810) . For example, τ,he nucleic acid encoding 10 the HPV- 16 E6 polypeptide can be inserted in replacement of the El region and the nucleic aciα encoding the FPV-16 E7 polypeptide in replacement: of the E3 region or vice versa.
In another and preferred aspect, the vector in use in the invention is a poxviral vector (see for example Cox et
15 al. in "Viruses in Human Gene Therapy" Ed J. M. Hos, Carolina Academic Press) . According to another preferred embodiment it is selected in the group consisting of vaccinia virus, suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266
20 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), the Wyeth strain and the highly attenuated attenuated virus derived thereof including MVA (for review see Mayr, A,, et al . , 1975, Infection 3, 6-14) and derivates thereof (such as MVA vaccinia strain 5^5 (ECACC V00120707 - "JS 6,913,752),
25 NYVAC (see WO 92/15672 - Cartaglia et al . , 1992, Virology, 188, 2T"?"232). Determination of the complete sequence of the MVA genome and comparison with the Copenhagen VV genome has allowed the precise identification of the seven deletions (I to VII) which occurred in the MVA genome (Antoine et al.,
30 1998, Virology 244, 365-396), any of which can be used to insert the antigen-encoding nucleic acid. The vector may also be obtained from any other member of the poxviridae, in particular fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); canarypox (e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev Biol Stand., 84, 159-
163); pigeonpox; swinepox and the like. By way of example, persons skilled in the arc may refer to WO 92 15672
(incorporated by reference) which describes the production of expression vectors based on poxviruses capable of expressing such heterologous nucleoside sequence, especially nucleotide sequence encoding antigen.
According to special embodiment, said virus can be a replication competent poxvirus, especially replication competent vaccinia virus . Examples of these virus are provided m WO9531105 (e.g. products JX594, VV TK- GMCSF or JX963, VV TK- VGF- GMCSF), WO0073479, WO2009/06b547, WO2009/065546.
The basic technique for inserting the nucleic acid and associated regulatory elements required for expression in a poxviral genome is described in numerous documents accessible to the man skilled in the art (Paul et al., 2002, Cancer gene
Ther. 9, 470-477; Piccini et al . , 1987, Methods of Enzymology
153, 545-563 ; US 4,769,330 ; US 4,772,848 ; US 4,603,112 ; DS 5,100,587 and US 5,179,993). Usually, one proceed through homologous recombination between overlapping sequences (i.e. desired insertion site) present both in the viral genome and a plasmid carrying the nucleic acid to insert.
The nucleic acid encoding she antigen of the Invention is preferably inserted in a nonessential locus of the poxviral genome, in order that L"he recombinant poxvirus remains viable and infectious. Nonessential regions are non- coding intergenic regions or any gene for which inactj vation or deletion does not significantly impair viral growth, replication or infection. One may also envisage insertion in an essential viral locus provided that the defective function is supplied in trans during production of viral particles, for example by using an helper cell line carrying the complementing sequences corresponding to those deleted in the poxviral genome.
When using the Copenhagen vaccinia virus, the antigen- encoding nucleic acid is preferably inserted in the thymidine 5 kinase gene (tic) (Hruby et al., 1983, Proc. Natl. Acad. Sci USA 80, 3411-3415 ; Weir et al., 1983, J. Virol. 46, 530- 537). However, other insertion sites are also appropriate, e.g. in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189-4198), in the KlL locus, in the u gene (Zhou et al.,
10 1990, J. Gen. Virol. 71, 2185-2190) or at the Left end of the vaccinia virus genome where a variety of spontaneous or engineered deletions have been reported in the literature (Altenburger et al., 1989, Archives Virol. 105, 15-27 ; Moss et al. 1981, J. Virol. 40, 387-395 ; Panicali et al., 1981,
15 J. Virol. 37, 1000-1010 ; Perkus et al, 1989, J. Virol. 63, 3829-3836 ; Perkus et al, 1990, Virol. 179, 276-286 ; Perkus et al, 1991, Virol. 180, 406-410).
When using MVA, the antigen-encoding nucleic acid can be inserted in any one of the identified deletions I TO VII as
20 well as in the D4R locus, but insertion in deletion II or III is preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-
1038 ; Sutter et al., 1994, Vaccine 12, 1032-1040).
When using fowlpox virus, although insertion within the thymidine kinase gene may be considered, the antigen-encoding
25 nucleic acid is preferably introduced in the intergenic region situated between ORFs 7 and 9 (see for example EP 314
569 and US 5,180, 675) .
According to one special embodiment, said recombinant vector is a recombinant plasmid DNA or a recombinant viral 30 vector.
According to another special embodiment, said recombinant viral vector is a recombinant vaccinia vector. According to another specicil embodiment , said recombinant vaccinia vector is a recombinant MVA vector .
Preferably, the antigen-encoding nucleic acid in use in the invention is m a form suitable for its expression in a host cell or organism, whicn means that the nucleic acid sequence encoding the antigen are placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism- As used herein, the term "regulatory sequence" refers to any sequence that allows, conirioαtes or modulates the expression of a nucleic acid m a given host cell, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA} into the host cell. It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on factors such as the host cell, the vector and the level of expression desired. The nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to whicn it is operatively linked. The gene expression sequence may, for example, be a mammalian or viiai promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV) , simian virus (e.g., SV40), papilloma virus, adenovirus, human Immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus . Other constitutive promoters are known to rhose of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metal lothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill In the art. In general, the gene expression sequence shall include, as necessary, 5' non- transcribing and 5' non-translating sequences Involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired. Preferred promoters for use in a poxviral vector (see below) include without limitation vaccinia promoters 7.5K, H5R, TK, p23, pll and KIT.., chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechnlques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158).
The promoter is of special importance and the present invention encompasses the use of constitutive promoters which direct expression of the nucleic acid in many types of host cells and those which direct expression only in certain host cells or in response to specific events or exogenous factors (e.g. by temperature, nutrient additive , hormone or other ligand) . Suitable promoters are widely described in literature and one may cite more specifically viral promoters such a≤ RSV, SV40, CMV and MLP promoters. Preferred promoters for use in a poxviral vector include without limitation vaccinia promoters 7.5K, H5R, TK, p28, pll and KIL, chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described In Chakrabarti et al. (1997, Bi otechn Lques 23, 1094-1097), Hammond et al. (1997, J, Virologies! Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 351-158).
Those skilled m tne art will appreciate that the regulatory elements controlling the expression of the nucLeic acid molecule of the invention may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine- Dalgamo sequences, etc. ) into the host cell or organism.
Alternatively, the recombinant vector in use in the present invention can farther comprise at least one nucleic acid encoding at least one cytokine. Suitable cytokines include without limitation interleukins (e.g. IL~2, IL- 7, IL- 15, IL-18, IL-21) and interferons (e.g. 15"1Ny, INFα) , with a special preference for mterieukin IL-2. When the recombinant vaccine of the invention comprises a cytokine-expressing nucleic acid, said nucleic acid may be carried by tne recombinant vector encoding the one or more antigen (s) or by an independent recombinant vector which can De of the same or a different origin. According to one most preferred embodiment, the recombinant vector in use in the present invention is encoding all or part of the MUCi antigen and at least one cytokines above listed, and preferably an ±nterleukin, especially IL2, Preferably the recombinant vector in use in the present invention is an MVA encoding ail or part of the MUCi antigen and at least one cytokines above listed, and preferably an inr.erleuk.in, especially 11,2.
Infectious viral particles comprising the above- described recombinant viral vector can be produced by routine process. Rn exemplary process comprises the steps of:
a. introducing the viral vector into a suitable cell line,
b. culturing said cell line under suitable conditions so as to allow the production of said infectious viral particle,
c. recovering the produced infectious viral particle from the culture of said cell line, and
d. optionally purifying said recovered infectious viral particle.
Cells appropriate for propagating adenoviral vectors are for example 293 cells, PERC6 cells, HER96 cells, or cells as disclosed in WO 94/28152, WO 97/00326, US 6,127,175.
Cells appropriate for propagating poxvirus vectors are avian cells, ana most preferably primary chicken embryo fibroblasts (CΞE"} prepared from chicken embryos obtained from fertilized eggs.
The infectious viral particles may be recovered frcm the culture supernatant or from the cells after lysis (e.g. by chemical means, freezing/thawing, osmotic shock, mecanic shock, sonication and the like) . The viral particles can be isolated by consecutive rounds of plaque purification and then purified using the techniques of the art (chromatographic methods, ultracentrifugation on caesium chloride or sucrose gradient).
If desired, the method or use for treating a pat Lenr. for human disease human disease according io the Invention (i.e. by administering an immunogenic composition comprising at least one antigen) can be carried out in the selected patients in conjunction with one or more conventional therapeutic modalities (e.g. radiation, chemotherapy and/or surgery} . The use of multiple therapeutic approaches provides the selected patient with a broader based intervention. Jn one embodiment, the method or use for treating a patient for human disease human disease according to the Invention can be preceded or followed by a surgical intervention. In another embodiment, it can be preceded or followed by radiotherapy (e.g. gamma radiation). Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters which can be used (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field) . In still another embodiment, the method or use of the invention is associated to chemotherapy with one or more drugs (e.g. drugs which are conventionally used for treating or preventing viral infections, virus-associated pathologic conditions, cancer, and the like).
The present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
- selection of patient in a patient population composed of patients that have low ILlO/IFNy ratio, - administering to said selected patients an immunogenic composition according to the Invention and a chemotherapeutic agent.
The present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
measuring in biological sample (e.g. blood or plasma or serum) from the patient trie levels of ILlO and IFNy,
calculating ILiO/IFNγ ratio, and
- administering to the patient the said immunogenic composition if said patient has low ILlO/IFNy ratio according to the Invention.
According to one embodiment, the administration of said chemotherapeutic agent is done before administration of said immunogenic composition.
According to another embodiment, the administration of said chemotherapeutic agent is done after administration of said Immunogenic composition.
According to another embodiment, the administration of said chemotherapeuxic agent is done concomitantly with administ rar 1 on of said immunogenic composition.
According to one embodiment, said chemotherapeutic agent is cisplatin and/or Gemcitabine, or similar.
The present Invention further concerns a method of improving cytotoxic effectiveness of cytotoxic drugs (i.e. chemotherapeutic agent} or radiotherapy which comprises co- treating a patient selected in a patient population composed of patients that have low ILlO/IFNγ ratio with an immunogenic composition according to the Invention. In another embodiment, the method or use of immunogenic composition of the invention is carried out according to a prime boost therapeutic modality which comprises sequential administration of: one or more primer compos It"i on (s) and one or more booster composition (s ) . Typically, the priming and the boosting compositions use different vehicles which comprise or encode at least an antigenic domain in common. The priming immunogenic composition is initially administered to τhe host organism and the boosting immunogenic composition is subsequently administered to the same host organism after a period varying from one day to tv/elve months. The method of the invention may comprise one to ten sequential administrations of the priming composition followed by one to ten sequential administrations of the boosting composition. Desirably, injection intervals are a matter of one week to six months. Moreover, the priming and boosting compositions can be administered at the same site or at alternative sites by the same route or by different routes of administration.
According to a special embodiment, the desired clinical benefit is independent of a demonstrable immune response to the vaccine.
According to one special embodiment, the Invention relates to a method as above described wherein said human disease is cancer.
According to a preferred embodiment, said cancer is for example breast cancer, colon cancer, kidney cancer, rectal cancer, lung cancer, cancer of the head and neck, renal cancer, malignant melanoma., laryngeal cancer, ovarian cancer, ceivicai cancer, prostate cancer, nori Small cell Lung Cancer, haematological cancecs, gastric cancers, myeloma .
According to one special embodiment, the Invention relates to a method as above described wherein said human disease is Infectious disease. According to a preferred embodiment, said infectious disease is a viral induced disease, such as for example disease induced by HTV, HCV, HBV, HPV, and the like.
In a further embodiment there is provided the use of an immunogenic composition comprising ail or part of a targeted antigen for the manufacture of a medicament for treat inq a patient for human disease in a particular patient; population wherein the patients of said population have low ILl 0/IF1NY ratio.
In a further embodiment there is provided the use of an immunogenic composition for the manufacture of a medicament for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFNγ ratio.
In another embodiment there is provided the use of an immunogenic composition for the manufacture of a medicament for inducing an immune response to at least one antigen (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFNγ ratio.
In another embodiment there is provided the use of an immunogenic composition for the manufacture of a medicament for inducing an immune response to a targeted antigen (j . e. the raised immune response) m a patient for treating human disease in a particular patient population wherein the patients of said population have low ILlO/IFNγ ratio.
In another embodiment there is provided the use of an immunogenic composition for the manufacture of a medicament for inducing an immune response (i.e. the raised immune response) in a patient for treating human disease in a particular patient population wherein the patients of said population have J ow ILlO/IFNγ ratio and wherein said raised immune response is innate immune response.
According to one special embodiment, said "raised immune response" in said patient population is directed towards a tumour-specific or -related antigens and/or viral anrigen. According to one eirhoctiment, said "raised immune response" in said patient population is directed towards distinct antigens. According ~o one special embodiment, said "raised immune response" in said patient population is directed towards all or part of MUCl antigen. According to another special embodiment, said "raised immune response" in said patient population is T ceil immune response, and preferably CD8+ (Cytotoxic T Lymphocytes) immune response. According to another special embodiment, said "raised immune response" in said patient: population is a non specific immune response or an immune response to disease associated antigens not contained in the vaccine formulation or an immune response to disease associated antigens which is not measureable by current techniques. According to another special embodiment, said "raised immune response" in said patient population is a stimulation of the innate immune response .
The ability to induce or stimulate ar. immune response upon administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays which are standard in the arr.. For a general description of techniques available to evaluate the onset and activation of an immune response, see for example Coligan et al. (1992 and 1994, Current Protocols in Immunology ; ed J Wiley & Sons Ino, National Institute of Health). Measurement of cellular immunity can be performed by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T~~cells (e.g. quantification of IL-IO or IFN gamma-producing cells by ELIspot) , by determination of the activation status of immune effector cells (e.g. T cell proliferation assays by a classical [3Hl thymidine uptake) , by assaying for antigen- specific T lymphocytes in a sensitized patient (e.g. pepfiαe- specific lysis in a cytotoxicity assay) or by detection of antigen specific T ceils by fluorescent MHC and/or peptide multimers (e.g. tetramers). The ability to stimulate a humoral response may be determined by antibody binding and/or competit Lon in binding (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press) . The method of the invention can also be further validated in animal models challenged with an appropriate tumor-inducing agent (e.g. MUCl-express: ng murine tumor ceils cells) co determine antitumor activity, reflecting an induction or an enhancement of an anti-antigen immune response.
Thus the present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an immunogenic composition, said method comprising the following steps :
- selection of patient in a patient population composed of patients that have low ILlO/IFNγ ratio,
- administering to said selected patients the said immunogenic composition.
The present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an .immunogenic composition, said method comprising the following steps :
- measuring in the patient the levels of ILlO and IFNY,
calculating ILlO/IFNγ ratio, and - administering to the patient the said immunogenic composition if said patient has low ILlO/IFNγ ratio.
The present invention further concerns a method for extending the survival rate of a patient treated for human disease, for example cancer, by administering an immunogenic composition and chemotherapeutic agent (see above) , said method comprising the following steps :
measuring in the patient the levels of XLlO and
IFNY,
- calculating IL10/lFNγ ratio, and
- admJ nistermg to the patient the said immunogenic composition and chemotherapeutic agent if said patient has low ILlO/IFNγ ratio.
According to another embodiment, the present Invention relates to the use of ILlO/IFNγ ratio as a biomarker for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response, preferably immune response, by administration of an immunogenic composition.
More specifically, the present Invention relates to the use of ILlO/IFNγ ratio as a biomarker for predicting whether a patient is or is nor su.sceptib] e "co developing prophylactic or therapeutic immune response by administration ot an immunogenic composition, wherein low ILlO/IE"Ny ratio indicates tnar. the patient is predicted to hcve an increased t-usceptibility zo develop a prophylactic or therapeutic response, preferably immune response,
Tn other words, the present Invention relates to the use of the ILlO/IFNγ ratio as a biomarker for predicting whether a patient is or is not susceptible to survive longer after administration of an immunogenic composition, wherein low ILlO/IFNγ ratio indicates that the patient is predicted to have a longer survival rate compared to treated patients who have higher ILlO/IFNγ ratio.
In other words, the present invention relates to an ex- vivo method for predicting whether a patient is or is not susceptible to survive longer after administration of an immunogenic composition, wherein the testing method comprises the step of measuring levels of TLIO and INFy in a biological sample (e.g. blood or plasma or serum) from che patient, calculating ILlO/IFNγ ratio, wherein low ILlO/TFNγ ratio indicates chat the patient will have a longer survival race.
Trius tne Invention further concerns the use of the ILlO/IFNγ ratio as a biomarker for predicting whether a patient which is undergoing chemotherapeutic treatment with a cheinotherapeutic agent is or is not susceptible to developing prophylactic or therapeutic immune response (e.g. to survive longer) after administration of an immunogenic composition.
Thus the Invention further concerns the use of the IL10/IFNγ ratio as a biomarker for predicting whether a patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent is or is not susceptible to developing prophylactic or therapeutic immune response (e.g. to survive longer) after administration of an immunogenic composition, wherein low IT 10/IE1N/ ratio indicates that the patient Is predicted to have an increased susceptibility to develop a prophylactic or therapeutic immune response.
Tne present Invention thus relates to a method for improving the treatment of a cancer patient which is undergoing chemotnerapeutic treatment with a chemotherapeutic agent, said method comprising the following steps :
- measuring in the patient the levels of ILlC) and IFNy,
calculating ILl0/IFNγ ratio, and - administering to the patient the said immunogenic composition and chemotherapeutic agent if said patient has low ILl07TFNy ratio according to the Invention .
The invention also provides kits (i.e. companion test) which include parts for practicing the methods described herein and that will be apparent from the examples provided herein. The kit of parts, or kits, may include reagents for collecting and or measuring serum levels of levels of ILlO and IFNy. Such reagents may include antibodies. The kits may further include equipment for collecting anα/or processing biological samples. The kits are also likely to contain instructions for use, cut-off values (see above) and/or instructions for their determination, and instructions for interpreting the data obtained from the use of the kits .
According to one special embodiment, the said kit of parts, or kits, may further include an immunogenic composition as above disclosed, and/or as disclosed in the Example section below.
The invention further provides computer programs and/or algorithms for monitoring clinical trial, levels of ILlO and IFNγ and ILlO and IFNγ ratio, determining whether such ratio are above or below a threshold level, and/or recommending treatment to improve a patient's response to an immunotherapy treatment. The computer programs or algorithms may be provided aJ ong with necessary hardware, e.g., in the form of a kit or apparatus, which may also accept biological samples and measure the relative levels of ILlO and IFNγ present therein and calculate ILlO and TFNγ ratio. The above- described computer programs and/or apparatus are likely to be provided to physicians or clinical laboratories with appropriate instructions and reagents, including antibodies. Use of levels of ILlO and INFγ in the generation of an algorithm for recommending modification to a treatment to improve patient' s response io immunotherapy treatment by immunogenic composition.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced in a different way from what is specifically described herein.
All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication or entry were specifically and individually indicated to be incorporated by reference.
Examples
Fig-ure 1: Survival curves describing vaccine immunotherapy in lung cancer: patients with a ratio of plasma IL-10/I FNg < or > 3.7, prior to treatment
95 Group 1 : Vaccine (i.e. immunogenic composition)
+ chemotnerapy in patients with a low ratio of plasma IL- 10/IFNγ. Low plasma IL-10/IFNγ ratio defined as < 3.7. 40 patients. Median survival= 21.2 months
- - -- -- - Group 2 : Vaccine + chemotherapy in patients with high ratio of plasma ΪL-10/IFNγ. High ratio of plasma IL-10/IFNγ defined as > 3.7. 21 patients. Median survival= 5.6 months Significant difference, by log rank: p = 0.007
0 Complete + Censored
Figure 2: Survival curves describing chemotherapy in lung cancer: patients with a ratio of plasma IL~10/IFNg < or > 3.7 prior to treatment.
Group 1 : Chemotherapy in patients with a low ratio of plasma IL-10/IFNγ. Low ratio of plasma IL-10/IFNγ defined as < 3.7. 57 patients. Median survival= 10.8 months
~ - - - - Group 2 : Chemotherapy in patients with high ratio of plasma ΪL-10/IFNγ. High ratio of plasma IL-10/IFNγ defined as > 3.7. 11 patients. Median survival= 8.4 months
Not Significantly different, by log rank: p = 0.92
0 Complete ■+ Censored
Among patients with a pre-treatment ratio of ILlO/IFNg of < 3.7, those treated with TG4010 + chemotherapy survived significantly longer (median survival = 21.2 months) than did patients treated with chemotherapy alone (median survival = 10.8 months) (p~0.03 by log rank test) . Median survival among patients independent of plasma IL-10/IFNγ ratio is not significantly different: 10.7 months for patients treated with TG4010 and chemotherapy; and 10.3 months for patients treated with chemotherapy alone.
Example 1 : The immunogenic composition, noted vaccine TG4010, was used to treat non-small cell lung cancer (NSCLC) patients in combination with standard chemotherapy.
TG4010 is a recombinant Modified Virus Ankara (MVA) expressing both IL2 and the tumor-associated antigen MUCl (see Rochlitz et al., 2003, J. Gene Med.r 5, 690-699). One hundred and forty eight patients were randomized to receive :
chemotherapy (Cisplatin 75mg/mz on di and Gemcάrabine 1250mg/m? on day 1 and day 8 every 3 weeks for up to 6 cycles) either alone (Study Arm 2) or
chemotherapy together with TG4010 (Study Arm
1) .
Tumors were evaluated (WHO criteria) every 6 weeks. Endpoints were progression-free survival (PFS) at 6 months and overall survival with intent to treat analysis.
Blood samples were taken prior to treatment on day 1 (first day of treatment) and were shipped immediately to a central immunology lab where plasma was isolated and stored frozen until analysis.
Plasma samples were assessed, at a central laboratory for cytokines, using a Luminex mutli-analyte profiling system.
Figure 1 shows that patients [Arm 1 (TG4010 + chemotherapy) ] with a ratio of plasma concentrations of Interleukin-10 / Interferon gamma of < 3.7 prior to therapy survive longer (median survival = 21.2 months) than do patients with a ratio of plasma concentrations of Interleuκin-10 / Interferon gamma > 3.7 prior to therapy (median survival of 5.6 months.
The data in Figure 2 demonstrates that the effect of selecting parlents based on the ratio of plasma concentrations of Interleukin-10 / Interferon gamma < 3.7 pricx- to therapy is restricted to patients receiving the vaccine in rhat Figure 2 shows that pat ients with a ratio of plasma concentrations of InterleukIn-10 / Interferon gamma < or > 3.7 prior to therapy with TG4010 and have the same survival expectancy.

Claims

1 - An ex-vivo method for testing whether a patient will respond therapeutically to a method of treatment comprising administration of an immunogenic composition, wherein the testing method comprises the steps of:
obtaining a biological sample from the patient; - measuring levels of ILlO and IFNy in said biological sample;
calculating ILlO/ IFNγ ratio; and comparing said ILlO/ IFNy ratio with a threshold level, wherein low ILlO/ IFNy ratio indicates that the patient will develop a prophylactic or therapeutic response towards the immunogenic composition with low ILlO/ IFNy ratio being below 5.
2 - The method of claim 1 wherein said low ILlO/ IFNy ratio being below 4.
3 - The method of claims 1 or 2 wherein said method of treatment is a method of treating cancer.
4 - The method of any of the preceding claims, wherein said ILlO/ IFNy ratio is measured with Luminex technology or enzyme-linked immunosorbant assays
5 - The method of any of claims, wherein said ILlO/ IFNy ratio is determined by using antibodies specific for ILlO and INFy, respectively.
6 - The method of any of the preceding claims, wherein said biological sample is selected in the group consisting of total blood sample, plasma or serum. 7 - The method of any of the preceding claims, wherein said immunogenic composition contains at least one recombinant vector expressing in vivo all or part of at least one heterologous nucleotide sequence.
8 - The method of claim 7, wherein said recombinant vector is a viral vector.
9 - The method of any of claims 7 and 8, wherein said viral vector is replication-competent.
10 - The method of any of claims 7 and 8, wherein said viral vector is replication-defective.
11 - The method of any of claims 7 to 10, wherein said viral vector is adenoviral .
12 - The method of any of claims 7 to 10, wherein said viral vector is vaccinia vector .
13 - The method of any of claims 7 to 10, wherein said viral vector is MVA vector .
14 - The method of any of the preceding claims, wherein said patient Is a patient treated with chemotherapeutic agent.
15 - Use of ILlO/ IFNy ratio as a biomarker for predicting whether a patient is or is not susceptible to developing prophylactic or therapeutic response by administration of an immunogenic composition.
16 - A kit for testing whether a patient will respond therapeutically to a method of treatment comprising administration of an immunogenic composition wherein the kit comprises :
- antibodies for determining the levels of ILlO and IFNy in a biological sample from the patient; and
- instructions for Interpreting the data obtained saying that a low ILlO/ IFNγ ratio indicates that the patient will develop a prophylactic or therapeutic response towards the immunogenic composition wherein low ratio is ratio of less than about 5.
17 - Ex -vivo method for predicting whether a patient is or is not susceptible to survive longer after administration of an immunogenic composition, wherein the testing method comprises the step of:
obtaining a biological sample from the patient;
measuring levels of ILlO and IFNy in said biological sample;
calculating ILlO/ IFNy ratio; and
comparing said ILlO/ IFNy ratio with a threshold level, wherein low ILlO/ IFNy ratio indicates that the patient will have a longer survival rate with low ILlO/ IFNy ratio being below 5.
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