WO2014191222A1 - Predictive biomarker for cancer therapy - Google Patents

Predictive biomarker for cancer therapy Download PDF

Info

Publication number
WO2014191222A1
WO2014191222A1 PCT/EP2014/059995 EP2014059995W WO2014191222A1 WO 2014191222 A1 WO2014191222 A1 WO 2014191222A1 EP 2014059995 W EP2014059995 W EP 2014059995W WO 2014191222 A1 WO2014191222 A1 WO 2014191222A1
Authority
WO
WIPO (PCT)
Prior art keywords
agonist
dna
cells
tlr
activated
Prior art date
Application number
PCT/EP2014/059995
Other languages
French (fr)
Inventor
Matthias Schroff
Manuel Schmidt
Kerstin KAPP
Burghardt Wittig
Original Assignee
Mologen Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to BR112015029852A priority Critical patent/BR112015029852A2/en
Priority to RU2015150739A priority patent/RU2672358C2/en
Priority to MX2015016305A priority patent/MX365512B/en
Priority to CA2907980A priority patent/CA2907980A1/en
Priority to PL14725114T priority patent/PL3004875T3/en
Priority to KR1020157029934A priority patent/KR101937442B1/en
Priority to DK14725114.4T priority patent/DK3004875T3/en
Priority to AU2014273400A priority patent/AU2014273400B2/en
Application filed by Mologen Ag filed Critical Mologen Ag
Priority to CN201480024805.8A priority patent/CN105247365B/en
Priority to US14/891,869 priority patent/US10006032B2/en
Priority to JP2016515715A priority patent/JP6441320B2/en
Priority to ES14725114T priority patent/ES2795929T3/en
Priority to EP14725114.4A priority patent/EP3004875B1/en
Publication of WO2014191222A1 publication Critical patent/WO2014191222A1/en
Priority to ZA2015/06813A priority patent/ZA201506813B/en
Priority to IL242551A priority patent/IL242551A/en
Priority to HK16104519.6A priority patent/HK1216779A1/en

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • 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 generally to the identification of cancer patients as to whether they will respond to specific therapies. More particularly the invention relates to a method and DNA construct for identifying responders to a therapy involving immune activating DNA constructs, as well as to a DNA construct intended to treat cancer patients individually characterized as potential responders prior to the start of a treatment with said construct.
  • Biomarkers are substances found in blood or other body fluids, in tissues or as receptors on cells that signal the presence or absence of a condition or disease, like cancer for instance. Biomarkers can be differentiated into predictive, prognostic and pharmacodynamics biomarkers.
  • Predictive biomarkers are used to assess the probability that a patient will respond to or benefit from a particular treatment.
  • Prognostic biomarkers allow for the classification of tumours with regard to their aggressive potential to guide a decision of whom to treat or how aggressively to treat.
  • Pharmacodynamic biomarkers measure the near-term treatment effects of a drug on the tumour and might be used to guide dose selection during clinical development of a new anti-cancer drug.
  • Targeted therapies are treatments that work on a molecular level to stop a cancer from growing or spreading.
  • therapies are usually related to the use of very aggressive drugs, there is a need for tools allowing to predict whether a patient will respond to a targeted therapy or not.
  • Another object of the invention is the immune activating DNA itself, which can be used in such a method, i.e. to treat a patient identified as responder prior to the start of treatment
  • the present invention provides a method for predicting or monitoring whether a patient suffering from cancer or an autoimmune disease will respond or responds to treatment with a TLR-9 agonist by determining the frequency of activated natural killer T (NKT) cells. It is preferred to determine the cluster of differentiation or cluster of designation (CD) molecules CD3+/CD56+/CD69+ for immunophenotyping of activated NKT cells. The ratio of activated NKT cells of the whole NKT cell population may also be determined in order to assess the probability whether a patient will respond to a treatment with a TLR-9 agonist.
  • NKT natural killer T
  • the TLR-9 agonist is a DNA construct comprising at least one sequence motif NVCGNV, wherein N ! N 2 and N 3 N 4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
  • a responder to the treatment with the TLR-9 agonist shall have a frequency of at least 3 % of activated NKT cells of the whole NKT cell population.
  • an induction therapy with a non-DNA drug may be performed.
  • Such a therapy comprises chemotherapy with or without angiogenesis inhibitor or the use of antibodies.
  • a further object of the present disclosure is a TLR-9 agonist for use in a method of treatment of cancer, characterised in that the patient has elevated levels of activated NKT cells.
  • the used TLR-9 agonist comprises at least a DNA construct comprising at least one sequence motif N 1 N 2 CGN 3 N 4 , wherein N J N 2 and N 3 N 4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
  • the agonist may further be characterised by N J N 2 being an element selected from the group comprising GT, GG, GA, AT or AA, N 3 N 4 being an element selected from the group comprising CT or TT.
  • the agonist may comprise DNA that is a linear open-chained DNA construct com- prising single or double-stranded DNA or is a linear double-stranded DNA construct, which comprises at least one end with a single stranded loop. Instead of using loops to protect the ends of the linear double strands at least one L-DNA nucleotide may be used as part of the DNA double strand.
  • the sequence motif N ! N 2 CGN 3 N 4 shall be located within a single-stranded and/or a double-stranded region of the DNA sequence.
  • At least one nucleotide of a DNA TLR-9 agonist is modified with a functional group selected from the group com- prising carboxyl, amine, amide, aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehyde groups.
  • the agonist comprising a DNA construct can be linked to a compound selected from the group comprising peptides, proteins, carbohydrates, antibodies, lipids, micelles, vesicles, synthetic molecules, polymers, micro projectiles, metal particles, nanoparticles, or a solid phase.
  • the agonist comprising a DNA construct can be part of a pharmaceutical composition, wherein the pharmaceutical composition can be a vaccine.
  • Another object of the present disclosure is a kit for predicting or monitoring whether a patient suffering from cancer or auto immune disease will respond or responds to treatment with a TLR-9 agonist comprising means for detecting and quantifying the frequency of activated NKT cells of the whole NKT cell population.
  • the TLR-9 agonist may comprise a DNA construct comprising at least one sequence motif N 1 N 2 CGN 3 N 4 , wherein N J N 2 and N 3 N 4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
  • Figure 1 ROC curves in verum patients for activated NKT cells.
  • Figure 2 Activated NKT cells versus sensitivity and specificity.
  • Figure 3 a: Kaplan-Meier plot in biomarker positive patients for verum and placebo; b: Kaplan-Meier curve for the biomarker negative patients in both the verum and placebo arm.
  • Figure 4 a: Kaplan-Meier plot of biomarker positive patients versus bi- omarker negative patients in the verum arm; b: Kaplan-Meier plot of the placebo group for biomarker positive and negative patients.
  • MGN1703 designates a DNA construct of a covalently closed partially self-complementary DNA chain having a double stranded stem and single stranded terminal loops bearing unmethylated CG motifs.
  • a "stem” shall be understood as a DNA double strand formed by base pairing either within the same DNA molecule (which is then partially self-complementary) or within different DNA molecules (which are partially or completely complementary).
  • Intramolecular base pairing designates base pairing within the same molecules, and base pairing between different DNA molecules is termed as intermolecular base-pairing.
  • a "loop" within the meaning of the present disclosure shall be understood as an unpaired, single-stranded region either within or at the end of a stem structure.
  • a “hairpin” is a distinct combination of a stem and a loop, which occurs when two self-complementary re- gions of the same DNA molecule hybridize to form a stem with an unpaired loop.
  • a "dumbbell-shape” describes a linear DNA construct with hairpins at both ends flanking a stem region.
  • a “linear DNA construct” within the context of the present disclosure describes a linear dumbbell-shaped DNA construct comprising single stranded loops at both ends of a double stranded DNA stem.
  • Immunomodulation refers to immune activation and immunosuppression.
  • Immune activation means preferentially that effector cells of the immune system are activated in order to proliferate, migrate, differentiate or become active in any other form.
  • B cell proliferation for instance can be induced without co-stimulatory signals by immune activating DNA molecules, which normally require a co-stimulatory signal from helper T-cells.
  • Immunosuppression on the other hand shall be understood as reducing the activation or efficacy of the immune system.
  • Immunosuppression is generally deliberately induced to prevent for instance the rejection of a transplanted organ, to treat graft-versus-host disease after a bone marrow transplant, or for the treatment of autoimmune diseases such as, for ex- ample, rheumatoid arthritis or Crohn's disease.
  • immunomodulation may also refer to influencing the nature or the character of an immune reaction, either by affecting an immune reaction, which is still developing or maturing or by modulating the character of an established immune reaction.
  • the term conveyorvaccination used in this disclosure refers to the administration of antigenic material (a vaccine) to produce immunity to a disease.
  • Vaccines can prevent or ameliorate the effects of infection by many pathogens such as viruses, fungi, protozoan parasites, bacteria but also of allergic diseases and asthma, as well as of tumors.
  • Vaccines typically con- tain one or more adjuvants, e.g. immune activating nucleic acids, used to boost the immune response.
  • Vaccination is generally considered to be the most effective and cost-effective method of preventing infectious and other diseases.
  • the material administered can, for example, be live but weakened forms of path- ogens (bacteria or viruses), killed or inactivated forms of these pathogens, purified material such as proteins, nucleic acids encoding antigens, or cells such as tumor cells or dendritic cells.
  • DNA vaccination has recently been developed. DNA vaccination works by insertion (and expression, triggering immune system recognition) of DNA encoding antigens into human or animal cells. Some cells of the immune system that recognize the proteins ex- pressed will mount an attack against these proteins and against cells expressing them.
  • DNA vaccines One advantage of DNA vaccines is that they are very easy to produce and store.
  • DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types.
  • Vaccination can be used as a prophylactic approach, leading to immunity against the antigen in the vaccinated, healthy individual upon exposure to the antigen.
  • a therapeutic vaccination can cause an improved response of the immune system of the vaccinated, diseased individual, by guiding the immune system of the individual towards the an- tigens. Both prophylactic and therapeutic vaccination can be applied to humans as well as animals.
  • cancer comprises cancerous diseases or a tumor being treated or pre- vented that is selected from the group comprising, but not limited to, mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarial carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and/or liver metastases.
  • Autoimmune diseases comprise rheumatoid arthritis, Crohn's disease, systemic lupus (SLE), autoimmune thyroiditis, Hashimoto's thyroiditis, multiple sclerosis, Graves' disease, myasthenia gravis, celiac disease and Addison's disease.
  • TLRs toll-like receptors
  • TLRs are part of the innate immune system.
  • TLRs are a family of specialized immune receptors that induce protective immune responses when they detect highly conserved pathogen-related molecular patterns, such as proteins, lipid structures, sacharidic structures, and certain nucleic acids.
  • Synthetic agonists for several TLRs, including TLR-3, TLR-4, TLR-7, TLR-8, and TLR-9, have been or are being developed for the treatment of cancer, generally with the intention to activate the immune system in the presence of tumours.
  • TLR-9 recognizes the presence of unmethylated CG-containing DNA sequences, which are typically found in bacteria, but practically never in human genomic DNA.
  • dumbbell-shaped DNA molecules are surprisingly suitable for the induction of an immune response.
  • a dumbbell-shape DNA construct having unmethylated CG motifs in the single stranded terminal loops was used.
  • monocytes activated monocytes 1 , activated monocytes 2, B-Cells, activated B-cells, T-cells, activated T-cells, natural killer (NK)-cells, activated NK-cells, NKT-cells, activated NKT- cells, plasmacytoid dendritic cells (pDCs), activated pDCs, myeloid dendritic cells (mDCs), and activated mDCs.
  • monocytes activated monocytes 1 , activated monocytes 2, B-Cells, activated B-cells, T-cells, activated T-cells, natural killer (NK)-cells, activated NK-cells, NKT-cells, activated NKT- cells, plasmacytoid dendritic cells (pDCs), activated pDCs, myeloid dendritic cells (mDCs), and activated mDCs.
  • Table 2 summarizes the context of cell types, CDs and the determined frequencies.
  • Table 2 Relation of cell types, CDs and determined frequencies
  • NK-cells CD3- / CD56+ all PBMCs activated NK-cells CD3- / CD56+ / CD69+ all natural killer cells
  • NKT cells CD3+ / CD56+ all PBMCs activated NKT cells CD3+ / CD56+ / CD69+ all NKT cells plasmacytoid dendritic cells Linl- / CD 123+ / HLA- all PBMCs
  • a receiver operating characteristic (ROC) curve shows the performance of a binary classifier system as its discrimination threshold is varied. ROC curves are created by plotting the fraction of true positives out of the positives versus the frac- tion of false positives out of the negatives at various threshold settings. The true positives are also designated as sensitivity and the false positives is one minus the specificity or true negative rate.
  • ROC analysis is used in medicine, radiology, biometrics, and other areas for many decades and is increasingly used in machine learning and data mining research.
  • biomarkers it can be used to study the study whether a potential biomarker can have clinical validity, i.e., whether it can be used for predictive purposes.
  • a successful diagnostic test or biomarker will result in a curve that bends above the diagonal while an unsuccessful test will mirror the diagonal, or fall below it.
  • a ROC curve provides information whether a diagnostic test is successful or not.
  • ROC curves in verum patients for activated NKT cells were established (figure 1). The area under the curve was determined to be 0.71 , which is a clear indication of the reliability of the biomarker.
  • the Youden-index which can be used to determine an optimum cut-off value for the test's readout, shows an optimum at cut-off at 3.08% activated NKT cells.
  • Figure 2 shows activated NKT cells versus sensitivity and specificity.
  • FIG. 3a shows a Kaplan-Meier plot in biomarker positive patients for verum and placebo (solid line: patients treated with MGN1703; dotted line: patients treated with placebo). It is obvious that the survival probability within the biomarker positive group is surprisingly related to the application of the TLR-9 agonist.
  • Figure 3b shows a Kaplan-Meier curve for the biomarker negative patients in both the verum and placebo arm. It is obvious that the time of progression free survival is clearly shorter within this verum group as compared to the biomarker positive verum patients (comp. fig. 3a).
  • Figure 4a shows a Kaplan-Meier plot of biomarker positive patients versus biomarker negative patients, only from the verum treatment arm (solid line: biomarker positive patients; dotted line: biomarker negative patients).
  • solid line biomarker positive patients
  • dotted line biomarker negative patients
  • Figure 4b shows a Kaplan-Meier plot of the placebo group for biomarker positive and negative patients (solid line: biomarker positive patients; dotted line: biomarker negative patients). It is obvious that both groups show a more or less identical progression free survival as they were only treated with placebo. Clearly, the biomarker is suitable for assessing whether a patient is a responder to the treatment with a TLR-9 agonist or not. Further, the placebo group shows that the biomarker is not related to effects caused by the overall health status of a patient.
  • the present invention provides new predictive biomarker for responder to a cancer treatment with a TLR-9 agonist, especially a covalently closed partially self-complementary DNA chain having a double stranded stem an single stranded terminal loops bearing un- methylated CG motifs.
  • the determination of the frequencies of activated NKT cells (CD3+/CD56+/Cd69+) at baseline allows assessing the probability whether a patient is a re- sponder to treatment with the DNA construct or not.
  • the patients with responder-like characteristics behave the same way as non-responders did, showing that the prolonged progression-free survival time of the biomarker positive verum patients is in fact due to the applicability of the biomarkers for the selected therapy, not just a better overall health or any non-specific effect.
  • FACS Fluorescence activated cell sorting
  • Cells were stained with the following combination of monoclonal antibodies: Anti- Lineage marker-FITC, (antibody cocktail containing antibodies directed against CD3, CD 14, CD16, CD19, CD20, CD56); Anti-CD 123 -PE; Anti-HLA-DR-PerCP; Anti-CD40-APC; PDC were gated as: lineage negative, HLA-DR positive, CD123 positive cells. Within the PDC population CD40 was used as activation marker.
  • Anti- Lineage marker-FITC antibody cocktail containing antibodies directed against CD3, CD 14, CD16, CD19, CD20, CD56
  • Anti-CD 123 -PE Anti-HLA-DR-PerCP
  • Anti-CD40-APC Anti-CD40-APC
  • PDC were gated as: lineage negative, HLA-DR positive, CD123 positive cells.
  • CD40 was used as activation marker.
  • NK cells were gated as: CD3 negative, CD56 positive cells
  • NK-T cells were gated as: CD3 positive, CD56 positive cells
  • T cells were gated as: CD3 positive, CD56 negative [0071] CD69 was used as activation marker for all 3 populations. [0072] CD86 expression of myeloid dendritic cells (MDC):
  • Cells were stained with the following combination of monoclonal antibodies: Anti- Lineage marker-FITC, (antibody cocktail containing antibodies directed against CD3, CD 14, CD16, CD19, CD20, CD56); Anti-CDl lc-PE; Anti-HLA-DR-PerCP ; Anti-CD86-APC
  • MDC were gated as: lineage negative, HLA-DR positive, CD1 lc positive cells.
  • CD86 Within the MDC population CD86 was used as activation marker.
  • CD86 expression of B cells and monocytes CD 169 expression of monocytes
  • B cells were gated as CD 19 positive cells. Within the B cell population CD86 was used as activation marker.
  • Monocytes were gated as CD 14 positive cells. Within the monocyte population CD86 and CD 169 were used as activation markers.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Physiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)

Abstract

The present invention relates generally to the identification of patients suffering from cancer whether they will respond to specific therapies. More particularly the invention relates to a method and means for identifying responder to a therapy TLR-9 agonists.

Description

PREDICTIVE BIOMARKER FOR CANCER THERAPY
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the identification of cancer patients as to whether they will respond to specific therapies. More particularly the invention relates to a method and DNA construct for identifying responders to a therapy involving immune activating DNA constructs, as well as to a DNA construct intended to treat cancer patients individually characterized as potential responders prior to the start of a treatment with said construct.
BACKGROUND OF THE INVENTION
[0002] Biomarkers are substances found in blood or other body fluids, in tissues or as receptors on cells that signal the presence or absence of a condition or disease, like cancer for instance. Biomarkers can be differentiated into predictive, prognostic and pharmacodynamics biomarkers.
[0003] Predictive biomarkers are used to assess the probability that a patient will respond to or benefit from a particular treatment. Prognostic biomarkers allow for the classification of tumours with regard to their aggressive potential to guide a decision of whom to treat or how aggressively to treat. Pharmacodynamic biomarkers measure the near-term treatment effects of a drug on the tumour and might be used to guide dose selection during clinical development of a new anti-cancer drug.
[0004] Targeted therapies are treatments that work on a molecular level to stop a cancer from growing or spreading. In order to avoid unnecessary approaches, because such therapies are usually related to the use of very aggressive drugs, there is a need for tools allowing to predict whether a patient will respond to a targeted therapy or not.
[0005] With regards to the fact that therapies become more and more target specific, the role of biomarkers will increase. They will help to individualise therapeutic approaches and to open the way to individualised oncology. Even the possibility to distinguish a responder to a specific therapy from a non-responder will help to prevent that patients will be subject to unnecessary treatments. [0006] A commonly known and well-approved treatment of cancer is chemotherapy. One major disadvantage of chemotherapy is the use of very aggressive pharmaceuticals causing severe side effects. In order to minimize these side effects many attempts have been undertaken to minimize dosage of chemotherapeutics like for instance combining chemotherapy with immune activating agents. One approach is the use of immune activating DNA.
[0007] So-called unmethylated CG sequences have been shown to activate the immune system very effectively (Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM; CpG motifs in bacterial DNA trigger direct B-cell activation; Nature 1995 Apr 6 374:6522 546-9). Those sequences are derived from bacteria. EP 1 196 178 discloses a covalently closed circular DNA with partially self-complementary sequences resulting in a DNA construct having a double stranded stem with single stranded loops at both ends comprising the unmethylated CG motifs.
[0008] The combination of the dumbbell- shaped DNA constructs of EP 1 196 178 with chemotherapeutics to treat cancerous diseases has been proposed by EP 1 776 124. Patients who were treated with the DNA constructs of EP 1 196 178 received subsequently chemo- therapeutic. It turned out that the amount of chemotherapeutic could be reduced by the disclosed combination. But this document does not disclose information about tools for identifying whether a patient will respond to the application of the combination therapy at all.
[0009] It is an object of the present invention to provide a method for predicting whether a cancer patient will respond to the treatment with immune activating DNA. Another object of the invention is the immune activating DNA itself, which can be used in such a method, i.e. to treat a patient identified as responder prior to the start of treatment
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides a method for predicting or monitoring whether a patient suffering from cancer or an autoimmune disease will respond or responds to treatment with a TLR-9 agonist by determining the frequency of activated natural killer T (NKT) cells. It is preferred to determine the cluster of differentiation or cluster of designation (CD) molecules CD3+/CD56+/CD69+ for immunophenotyping of activated NKT cells. The ratio of activated NKT cells of the whole NKT cell population may also be determined in order to assess the probability whether a patient will respond to a treatment with a TLR-9 agonist.
[0011] It is intended that the TLR-9 agonist is a DNA construct comprising at least one sequence motif NVCGNV, wherein N!N2 and N3N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
[0012] A responder to the treatment with the TLR-9 agonist shall have a frequency of at least 3 % of activated NKT cells of the whole NKT cell population.
[0013] It is further intended that previously an induction therapy with a non-DNA drug may be performed. Such a therapy comprises chemotherapy with or without angiogenesis inhibitor or the use of antibodies.
[0014] A further object of the present disclosure is a TLR-9 agonist for use in a method of treatment of cancer, characterised in that the patient has elevated levels of activated NKT cells.
[0015] It is intended that the used TLR-9 agonist comprises at least a DNA construct comprising at least one sequence motif N1N2CGN3N4, wherein NJN2 and N3N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine The agonist may further be characterised by NJN2 being an element selected from the group comprising GT, GG, GA, AT or AA, N3N4 being an element selected from the group comprising CT or TT.
[0016] The agonist may comprise DNA that is a linear open-chained DNA construct com- prising single or double-stranded DNA or is a linear double-stranded DNA construct, which comprises at least one end with a single stranded loop. Instead of using loops to protect the ends of the linear double strands at least one L-DNA nucleotide may be used as part of the DNA double strand. [0017] The sequence motif N!N2CGN3N4 shall be located within a single-stranded and/or a double-stranded region of the DNA sequence. It is further intended that at least one nucleotide of a DNA TLR-9 agonist is modified with a functional group selected from the group com- prising carboxyl, amine, amide, aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehyde groups.
[0018] The agonist comprising a DNA construct can be linked to a compound selected from the group comprising peptides, proteins, carbohydrates, antibodies, lipids, micelles, vesicles, synthetic molecules, polymers, micro projectiles, metal particles, nanoparticles, or a solid phase.
[0019] It is further intended that the agonist comprising a DNA construct can be part of a pharmaceutical composition, wherein the pharmaceutical composition can be a vaccine.
[0020] Another object of the present disclosure is a kit for predicting or monitoring whether a patient suffering from cancer or auto immune disease will respond or responds to treatment with a TLR-9 agonist comprising means for detecting and quantifying the frequency of activated NKT cells of the whole NKT cell population.
[0021] It is intended for the kit that the TLR-9 agonist may comprise a DNA construct comprising at least one sequence motif N1N2CGN3N4, wherein NJN2 and N3N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
BRIEF SUMMARY OF THE FIGURES
[0022] Figure 1 : ROC curves in verum patients for activated NKT cells.
[0023] Figure 2: Activated NKT cells versus sensitivity and specificity.
[0024] Figure 3: a: Kaplan-Meier plot in biomarker positive patients for verum and placebo; b: Kaplan-Meier curve for the biomarker negative patients in both the verum and placebo arm. [0025] Figure 4: a: Kaplan-Meier plot of biomarker positive patients versus bi- omarker negative patients in the verum arm; b: Kaplan-Meier plot of the placebo group for biomarker positive and negative patients. DETAILLED DESCRIPTION OF THE INVENTION
[0026] Within the meaning of the present invention MGN1703 designates a DNA construct of a covalently closed partially self-complementary DNA chain having a double stranded stem and single stranded terminal loops bearing unmethylated CG motifs. [0027] A "stem" according to the present disclosure shall be understood as a DNA double strand formed by base pairing either within the same DNA molecule (which is then partially self-complementary) or within different DNA molecules (which are partially or completely complementary). Intramolecular base pairing designates base pairing within the same molecules, and base pairing between different DNA molecules is termed as intermolecular base-pairing.
[0028] A "loop" within the meaning of the present disclosure shall be understood as an unpaired, single-stranded region either within or at the end of a stem structure. A "hairpin" is a distinct combination of a stem and a loop, which occurs when two self-complementary re- gions of the same DNA molecule hybridize to form a stem with an unpaired loop.
[0029] A "dumbbell-shape" describes a linear DNA construct with hairpins at both ends flanking a stem region. Thus, a "linear DNA construct" within the context of the present disclosure describes a linear dumbbell-shaped DNA construct comprising single stranded loops at both ends of a double stranded DNA stem.
[0030] Immunomodulation according to the present disclosure refers to immune activation and immunosuppression. Immune activation means preferentially that effector cells of the immune system are activated in order to proliferate, migrate, differentiate or become active in any other form. B cell proliferation for instance can be induced without co-stimulatory signals by immune activating DNA molecules, which normally require a co-stimulatory signal from helper T-cells. [0031] Immunosuppression on the other hand shall be understood as reducing the activation or efficacy of the immune system. Immunosuppression is generally deliberately induced to prevent for instance the rejection of a transplanted organ, to treat graft-versus-host disease after a bone marrow transplant, or for the treatment of autoimmune diseases such as, for ex- ample, rheumatoid arthritis or Crohn's disease.
[0032] In this context, immunomodulation may also refer to influencing the nature or the character of an immune reaction, either by affecting an immune reaction, which is still developing or maturing or by modulating the character of an established immune reaction.
[0033] The term„vaccination" used in this disclosure refers to the administration of antigenic material (a vaccine) to produce immunity to a disease. Vaccines can prevent or ameliorate the effects of infection by many pathogens such as viruses, fungi, protozoan parasites, bacteria but also of allergic diseases and asthma, as well as of tumors. Vaccines typically con- tain one or more adjuvants, e.g. immune activating nucleic acids, used to boost the immune response. Vaccination is generally considered to be the most effective and cost-effective method of preventing infectious and other diseases.
[0034] The material administered can, for example, be live but weakened forms of path- ogens (bacteria or viruses), killed or inactivated forms of these pathogens, purified material such as proteins, nucleic acids encoding antigens, or cells such as tumor cells or dendritic cells. In particular, DNA vaccination has recently been developed. DNA vaccination works by insertion (and expression, triggering immune system recognition) of DNA encoding antigens into human or animal cells. Some cells of the immune system that recognize the proteins ex- pressed will mount an attack against these proteins and against cells expressing them. One advantage of DNA vaccines is that they are very easy to produce and store. In addition, DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types. [0035] Vaccination can be used as a prophylactic approach, leading to immunity against the antigen in the vaccinated, healthy individual upon exposure to the antigen. Alternatively, a therapeutic vaccination can cause an improved response of the immune system of the vaccinated, diseased individual, by guiding the immune system of the individual towards the an- tigens. Both prophylactic and therapeutic vaccination can be applied to humans as well as animals.
[0036] The term "cancer" comprises cancerous diseases or a tumor being treated or pre- vented that is selected from the group comprising, but not limited to, mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarial carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and/or liver metastases.
[0037] Autoimmune diseases according to the present disclosure comprise rheumatoid arthritis, Crohn's disease, systemic lupus (SLE), autoimmune thyroiditis, Hashimoto's thyroiditis, multiple sclerosis, Graves' disease, myasthenia gravis, celiac disease and Addison's disease.
[0038] During experiments with immune activating dumbbell-shaped DNA constructs with unmethylated CG sequences in the single-stranded terminal loops it turned out that elevated frequencies of certain cell types of the immune activation and effector pathway were related to a successful therapy with the DNA constructs.
[0039] In vertebrates, the so-called "toll-like receptors" (TLRs) are part of the innate immune system. TLRs are a family of specialized immune receptors that induce protective immune responses when they detect highly conserved pathogen-related molecular patterns, such as proteins, lipid structures, sacharidic structures, and certain nucleic acids. Synthetic agonists for several TLRs, including TLR-3, TLR-4, TLR-7, TLR-8, and TLR-9, have been or are being developed for the treatment of cancer, generally with the intention to activate the immune system in the presence of tumours. TLR-9 recognizes the presence of unmethylated CG-containing DNA sequences, which are typically found in bacteria, but practically never in human genomic DNA. Thus, unmethylated CG-containing DNA sequences have been de- signed as artificial TLR-9 agonists. The effect of such unmethylated CG-containing DNA constructs depends on their interaction with TLR-9, and DNA-protein interaction depends on the conformation of both DNA and protein. Experimental data demonstrate that dumbbell- shaped DNA molecules are surprisingly suitable for the induction of an immune response. [0040] In order to identify a tool for predicting whether a cancer patient will respond to the application of a TLR-9 agonist, a dumbbell-shape DNA construct having unmethylated CG motifs in the single stranded terminal loops was used.
[0041] In total, 46 patients suffering from metastatic colorectal cancer who had previously been treated for 4.5 to 6 month in a standard first-line combination therapy with or without a human monoclonal antibody inhibiting vascular endothelial growth factor A were selected for the study. After a treatment-free interval of ca. 1-6 weeks, a randomization of the patients was performed, so that 32 patients received 60 mg per dose of the DNA construct MGN1703 and 14 patients received a placebo, each patient twice weekly by subcutaneous injection.
[0042] After 12 weeks of treatment, all patients were examined for tumour-progression. Based on the presence or absence of tumour progression, patients were divided into two groups, designated as "PFS groups". Patients whose tumour had not progressed were designated as progression- free patients and labelled "PFS1", whiles patients with tumour progression were labelled as "PFS0". Obviously, a lack of tumour progression (PFS1) indicates a possible response to the treatment, while tumour growth (PFS2) indicates a lack of response. Treatment was continued for each patient until tumour progression was found.
[0043] Table 1 summarizes the results. It is obvious that nearly all progression-free patients received the DNA construct.
[0044] Table 1 : Designation of patients
Treatment arms Group designation
PFSO PFS1 Total
MGN1703 19 13 32
Placebo 13 1 14
Total 32 14 46
[0045] Prior to the first application of MGN1703 or placebo (at baseline) blood samples of all patients were collected. The distribution of the following immunovariables, e.g. part of whole PBMC population and relation of activated and non-activated sub populations was determined. A correlation of the respective PFS group designation after 12 weeks and all im- munovariables at baseline was investigated The following immuno variables were determined: monocytes , activated monocytes 1 , activated monocytes 2, B-Cells, activated B-cells, T-cells, activated T-cells, natural killer (NK)-cells, activated NK-cells, NKT-cells, activated NKT- cells, plasmacytoid dendritic cells (pDCs), activated pDCs, myeloid dendritic cells (mDCs), and activated mDCs. Table 2 summarizes the context of cell types, CDs and the determined frequencies.
[0046] Table 2: Relation of cell types, CDs and determined frequencies
Cell Type Identified by Frequency
as percentage of monocytes CD 14+ all PBMCs activated monocytes 1 CD14+ / CD86+ all monocytes activated monocytes 2 CD14+ / CD169+ all monocytes
B-cells CD 19+ all PBMCs activated B-cells CD19+ / CD86+ all B-cells
T-cells CD3+ / CD56- all PBMCs activated T-cells CD3+ / CD56- / CD69+ all T-cells
natural killer (NK-)cells CD3- / CD56+ all PBMCs activated NK-cells CD3- / CD56+ / CD69+ all natural killer cells
NKT cells CD3+ / CD56+ all PBMCs activated NKT cells CD3+ / CD56+ / CD69+ all NKT cells plasmacytoid dendritic cells Linl- / CD 123+ / HLA- all PBMCs
(pDCs) DR+
activated pDCs Linl- / CD 123+ / HLA- all pDCs
DR+ / CD40+
myeloid dendritic cells Linl- / CDl lc+ / HLA- all PBMCs
(mDCs) DR+
activated mDCs Linl- / CDl lc+ / HLA- all mDCs
DR+ / CD86+
[0047] To assess whether one of the immunovariables may serve as a proper biomarker, a so-called Cox regression was calculated for each immunovariable. A Cox regression allows estimating the effect of parameter(s) without any consideration of the hazard function. The resulting hazard ratio from such a calculation should be below 1 and related to a significant p value being below 0.05. Otherwise the observed effect would not be related to the applied TLR-9 agonist. Those criteria applied only to activated NKT cells having a hazard ratio of about 0.933 and a p value of 0.0309. [0048] Surprisingly, the percentage of activated NKT cells could be used to predict treatment success within the verum group. The relationship between the percentage of activated NKT cells and the PFS group status was studied using advanced, well-established statistical analyses within the verum group.
[0049] A receiver operating characteristic (ROC) curve, or simply ROC curve, shows the performance of a binary classifier system as its discrimination threshold is varied. ROC curves are created by plotting the fraction of true positives out of the positives versus the frac- tion of false positives out of the negatives at various threshold settings. The true positives are also designated as sensitivity and the false positives is one minus the specificity or true negative rate.
[0050] ROC analysis is used in medicine, radiology, biometrics, and other areas for many decades and is increasingly used in machine learning and data mining research. In biomarkers, it can be used to study the study whether a potential biomarker can have clinical validity, i.e., whether it can be used for predictive purposes. A successful diagnostic test or biomarker will result in a curve that bends above the diagonal while an unsuccessful test will mirror the diagonal, or fall below it. Thus, a ROC curve provides information whether a diagnostic test is successful or not.
[0051] ROC curves in verum patients for activated NKT cells were established (figure 1). The area under the curve was determined to be 0.71 , which is a clear indication of the reliability of the biomarker. The Youden-index, which can be used to determine an optimum cut-off value for the test's readout, shows an optimum at cut-off at 3.08% activated NKT cells. Figure 2 shows activated NKT cells versus sensitivity and specificity.
[0052] All patients were now sorted into groups depending on their level of activated NKT cells. Patients with cell levels above the cut-off of 3.08% activated NKT cells were designated as biomarker positive, while patients with levels below the cut-off were labelled biomarker negative. [0053] Figure 3a shows a Kaplan-Meier plot in biomarker positive patients for verum and placebo (solid line: patients treated with MGN1703; dotted line: patients treated with placebo). It is obvious that the survival probability within the biomarker positive group is surprisingly related to the application of the TLR-9 agonist.
[0054] Figure 3b shows a Kaplan-Meier curve for the biomarker negative patients in both the verum and placebo arm. It is obvious that the time of progression free survival is clearly shorter within this verum group as compared to the biomarker positive verum patients (comp. fig. 3a).
[0055] Figure 4a shows a Kaplan-Meier plot of biomarker positive patients versus biomarker negative patients, only from the verum treatment arm (solid line: biomarker positive patients; dotted line: biomarker negative patients). Clearly, the biomarker positive patients have a significant advantage in survival probability compared to the biomarker negative pa- tients, even when both groups received the verum treatment MGN 1703.
[0056] Figure 4b shows a Kaplan-Meier plot of the placebo group for biomarker positive and negative patients (solid line: biomarker positive patients; dotted line: biomarker negative patients). It is obvious that both groups show a more or less identical progression free survival as they were only treated with placebo. Clearly, the biomarker is suitable for assessing whether a patient is a responder to the treatment with a TLR-9 agonist or not. Further, the placebo group shows that the biomarker is not related to effects caused by the overall health status of a patient. [0057] The present invention provides new predictive biomarker for responder to a cancer treatment with a TLR-9 agonist, especially a covalently closed partially self-complementary DNA chain having a double stranded stem an single stranded terminal loops bearing un- methylated CG motifs. The determination of the frequencies of activated NKT cells (CD3+/CD56+/Cd69+) at baseline allows assessing the probability whether a patient is a re- sponder to treatment with the DNA construct or not. Importantly, in the placebo arm, the patients with responder-like characteristics, behave the same way as non-responders did, showing that the prolonged progression-free survival time of the biomarker positive verum patients is in fact due to the applicability of the biomarkers for the selected therapy, not just a better overall health or any non-specific effect.
[0058] MATERIAL AND METHODS
[0059] Sample handling
[0060] Whole blood (10 mL) for FACS was collected in Streck Cyto-Chex® BCT tubes. Within 2 hours after sampling the blood samples were shipped to the analytical laboratory. According to established Protocol, samples were stored at room temperature. The frequency and activation status of plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC), monocytes, natural killer (NK) cells, NKT cells, B cells, T cells and other cell populations were evaluated.
[0061] Analytical Methods
[0062] Fluorescence activated cell sorting (FACS) were performed according to established principles. Whole blood samples were stained with fluorescencelabeled antibodies and incubated. Phenotypical analysis of the immune cells was performed with a FACScalibur (Becton Dickinson) flow cytometer. Frequencies of the respective analyzed cell populations were documented for each sample of the patients. [0063] Analysis of human PBMC for the activation of specific cell populations
[0064] CD40 expression of plasmacytoid dendritic cells (pDC)
[0065] Cells were stained with the following combination of monoclonal antibodies: Anti- Lineage marker-FITC, (antibody cocktail containing antibodies directed against CD3, CD 14, CD16, CD19, CD20, CD56); Anti-CD 123 -PE; Anti-HLA-DR-PerCP; Anti-CD40-APC; PDC were gated as: lineage negative, HLA-DR positive, CD123 positive cells. Within the PDC population CD40 was used as activation marker.
[0066] Activation of NK-, NK-T and T cells using the activation marker CD69
[0067] Cells were stained with the following combination of monoclonal antibodies: Anti CD3-FITC; Anti CD56-PE; Anti CD69-APC
[0068] NK cells were gated as: CD3 negative, CD56 positive cells
[0069] NK-T cells were gated as: CD3 positive, CD56 positive cells
[0070] T cells were gated as: CD3 positive, CD56 negative [0071] CD69 was used as activation marker for all 3 populations. [0072] CD86 expression of myeloid dendritic cells (MDC):
[0073] Cells were stained with the following combination of monoclonal antibodies: Anti- Lineage marker-FITC, (antibody cocktail containing antibodies directed against CD3, CD 14, CD16, CD19, CD20, CD56); Anti-CDl lc-PE; Anti-HLA-DR-PerCP ; Anti-CD86-APC
[0074] MDC were gated as: lineage negative, HLA-DR positive, CD1 lc positive cells.
[0075] Within the MDC population CD86 was used as activation marker.
[0076] CD86 expression of B cells and monocytes; CD 169 expression of monocytes
[0077] Cells were stained with the following combination of monoclonal antibodies: Anti CD14-FITC; Anti CD169-PE; Anti CD19-PerCP; Anti CD86-APC
[0078] B cells were gated as CD 19 positive cells. Within the B cell population CD86 was used as activation marker.
[0079] Monocytes were gated as CD 14 positive cells. Within the monocyte population CD86 and CD 169 were used as activation markers.

Claims

Claims
A method for predicting or monitoring whether a patient suffering from cancer or an autoimmune disease will respond or responds to treatment with a TLR-9 agonist by determining the frequency of activated natural killer T (NKT) cells.
The method of claim 1, wherein the TLR-9 agonist is a DNA construct comprising at least one sequence motif NVCGNV, wherein N!N2 and N3N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine
The method of claim 1 to 2, wherein the ratio of activated NKT cells of the whole NKT cell population is determined.
The method of any one of claims 1 to 3, wherein a responder to the treatment with the TLR-9 agonist has a frequency of at least 3 % of activated NKT cells of the whole NKT cell population.
The method of any one of claims 1 to 4, wherein previously an induction therapy with a non-DNA drug was performed.
A TLR-9 agonist for use in a method of treatment of cancer characterised in that the patient has elevated levels of activated NKT cells.
The agonist of claim 6, comprising at least a DNA construct comprising at least one sequence motif NVCGNV, wherein N!N2 and N3N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine .
8. The agonist of claim 7, wherein NJN2 is an element selected from the group comprising GT, GG, GA, AT or AA, N3N4 is an element selected from the group comprising CT or TT.
9. The agonist of claim 7 or 8, wherein the DNA is a linear open-chained DNA construct comprising single or double-stranded DNA or is a linear double-stranded DNA construct, which comprises at least one end with a single stranded loop.
10. The agonist of any one of claims 7 to 9, wherein the sequence motif N!N2CGN3N4 is located within a single- stranded and/or a double-stranded region of the DNA sequence.
11. The agonist of any one of claims 1 to 8, comprising at least one L-DNA nucleotide.
12. The agonist of any one of claims 7 to 11, wherein at least one nucleotide is modified with a functional group selected from the group comprising carboxyl, amine, amide, aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehyde groups.
13. The agonist of any one of claims 7 to 11, wherein the DNA construct is linked to a compound selected from the group comprising peptides, proteins, carbohydrates, antibodies, lipids, micelles, vesicles, synthetic molecules, polymers, micro projectiles, metal particles, nanoparticles, or a solid phase.
14. The agonist of any one of claims 7 to 12, wherein the DNA construct is part of a pharmaceutical composition.
15. The agonist of claim 13, wherein the pharmaceutical composition is a vaccine.
16. A kit for predicting or monitoring whether a patient suffering from cancer or auto immune disease will respond or responds to treatment with a TLR-9 agonist comprising means for detecting and quantifying the frequency of activated NKT cells of the whole NKT cell population.
17. The kit of claim 15, wherein the TLR-9 agonist comprises a DNA construct comprising at least one sequence motif
Figure imgf000017_0001
wherein N'N2 and N N4 is any combination of A, C, T, and G, and C is deoxycytidine, G is deoxyguanosine, A is deoxy- adenosine and T is deoxythymidine.
PCT/EP2014/059995 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy WO2014191222A1 (en)

Priority Applications (16)

Application Number Priority Date Filing Date Title
CN201480024805.8A CN105247365B (en) 2013-05-30 2014-05-15 Predictability biomarker use for cancer treatment
RU2015150739A RU2672358C2 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
US14/891,869 US10006032B2 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
PL14725114T PL3004875T3 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
KR1020157029934A KR101937442B1 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
DK14725114.4T DK3004875T3 (en) 2013-05-30 2014-05-15 PREDICTIVE BIOMARKER FOR CANCER THERAPY
AU2014273400A AU2014273400B2 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
BR112015029852A BR112015029852A2 (en) 2013-05-30 2014-05-15 method for predicting or monitoring whether a patient suffering from cancer or an autoimmune disease will respond or respond to treatment with a tlr-9 agonist, tlr-9 agonist for use in a cancer treatment method, and a kit to predict or monitor if a patient suffering from cancer or autoimmune disease will respond or respond to treatment with a tlr-9 agonist
MX2015016305A MX365512B (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy.
CA2907980A CA2907980A1 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
JP2016515715A JP6441320B2 (en) 2013-05-30 2014-05-15 Predictive biomarkers for cancer treatment
ES14725114T ES2795929T3 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
EP14725114.4A EP3004875B1 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy
ZA2015/06813A ZA201506813B (en) 2013-05-30 2015-09-15 Predictive biomarker for cancer therapy
IL242551A IL242551A (en) 2013-05-30 2015-11-11 Predictive biomarker for cancer therapy
HK16104519.6A HK1216779A1 (en) 2013-05-30 2016-04-20 Predictive biomarker for cancer therapy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1309657.3 2013-05-30
GB1309657.3A GB2514591A (en) 2013-05-30 2013-05-30 Predictive biomarker for cancer therapy

Publications (1)

Publication Number Publication Date
WO2014191222A1 true WO2014191222A1 (en) 2014-12-04

Family

ID=48805478

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/059995 WO2014191222A1 (en) 2013-05-30 2014-05-15 Predictive biomarker for cancer therapy

Country Status (18)

Country Link
US (1) US10006032B2 (en)
EP (1) EP3004875B1 (en)
JP (1) JP6441320B2 (en)
KR (1) KR101937442B1 (en)
CN (2) CN105247365B (en)
AU (1) AU2014273400B2 (en)
BR (1) BR112015029852A2 (en)
CA (1) CA2907980A1 (en)
DK (1) DK3004875T3 (en)
ES (1) ES2795929T3 (en)
GB (1) GB2514591A (en)
HK (1) HK1216779A1 (en)
IL (1) IL242551A (en)
MX (1) MX365512B (en)
PL (1) PL3004875T3 (en)
RU (1) RU2672358C2 (en)
WO (1) WO2014191222A1 (en)
ZA (1) ZA201506813B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3392345A1 (en) 2017-04-22 2018-10-24 Mologen AG Biomarker for small cell lung cancer therapy
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)
US11578331B2 (en) 2015-09-09 2023-02-14 Gilead Sciences, Inc. Combination comprising immunostimulatory oligonucleotides
US11583581B2 (en) 2015-09-21 2023-02-21 Gilead Sciences, Inc. Methods of treating a retroviral infection

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102290205B1 (en) 2014-06-04 2021-08-20 엑시큐어, 인크. Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
CN111278509A (en) * 2017-08-31 2020-06-12 莫洛根股份公司 TLR-9 agonists for modulating tumor microenvironment

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010055340A1 (en) * 2008-11-12 2010-05-20 Ludwig Institute For Cancer Research Use of inkt or tlr agonists for protecting against or treating a disease such as acute infection or cancer

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0314682D0 (en) * 2003-06-24 2003-07-30 Isis Innovation Materials and methods relating to the modulation of T cell response to soluble antigen
WO2006015560A1 (en) * 2004-08-09 2006-02-16 Mologen Ag Immunomodulating agent used in conjunction with chemotherapy
EP1922545A1 (en) * 2005-09-08 2008-05-21 Mologen AG Functional in vitro immunoassay
EP2079305A4 (en) * 2006-12-12 2010-01-27 Idera Pharmaceuticals Inc Synthetic agonists of tlr9
EP2112930B1 (en) * 2007-02-21 2017-01-11 Vaccinex, Inc. Modulation of nkt cell activity with antigen-loaded cdid molecules
CN102864151B (en) * 2007-08-01 2015-07-08 艾德拉药物股份有限公司 Novel synthetic agonists of tlr9
CN101821412A (en) * 2007-08-15 2010-09-01 艾德拉药物股份有限公司 TOLL sample receptor modulators
DK2219672T3 (en) * 2007-11-09 2016-05-17 Peregrine Pharmaceuticals Inc The anti-VEGF antibody compositions and methods
EP2073009A1 (en) * 2007-12-19 2009-06-24 Cell Med Research GMBH Method for T, NK and NKT cells
AU2009241645B2 (en) 2008-05-02 2014-07-17 Blink Therapeutics Limited Products and methods for stimulating an immune response
WO2009138494A2 (en) * 2008-05-15 2009-11-19 Ablynx N.V. Amino acid sequences directed against toll-like receptors and polypeptides comprising the same for the treatment of diseases related to toll-like receptors
JP5705836B2 (en) * 2009-05-29 2015-04-22 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Modulators for HER2 signaling in gastric cancer patients expressing HER2
MX340363B (en) * 2010-11-19 2016-07-06 Idera Pharmaceuticals Inc Immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response.
GB201021867D0 (en) * 2010-12-23 2011-02-02 Mologen Ag Non-coding immunomodulatory DNA construct
PT2744826T (en) * 2011-08-16 2022-05-19 Morphosys Ag Combination therapy with an anti - cd19 antibody and a purine analog
EP2682750A1 (en) * 2012-07-02 2014-01-08 Sotio a.s. In vitro method for the diagnosis and surveillance of cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010055340A1 (en) * 2008-11-12 2010-05-20 Ludwig Institute For Cancer Research Use of inkt or tlr agonists for protecting against or treating a disease such as acute infection or cancer

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "A novel iNKT-mediated vaccine maneuver overcomes PD-1-mediated T cell exhaustion in cancer -- Varghese et al. 188 (1001): 165.13 -- The Journal of Immunology", 1 January 2012 (2012-01-01), XP055131252, Retrieved from the Internet <URL:http://www.jimmunol.org/cgi/content/meeting_abstract/188/1_MeetingAbstracts/165.13> [retrieved on 20140723] *
ANONYMOUS: "Maintenance with the TLR-9 agonist MGN1703 versus placebo in patients with advanced colorectal carcincoma (mCRC): A randomized phase II trial (IMPACT). -- Riera-Knorrenschild et al. 31 (15): 3643 -- ASCO Meeting Abstracts", 20 May 2013 (2013-05-20), XP055131313, Retrieved from the Internet <URL:http://meeting.ascopubs.org/cgi/content/abstract/31/15_suppl/3643> [retrieved on 20140723] *
H.-J. SCHMOLL ET AL: "O-0012 * UPDATED RESULTS OF THE RANDOMIZED PHASE 2 IMPACT TRIAL: MAINTENANCE WITH TLR-9 AGONIST MGN1703 VS PLACEBO IN PATIENTS WITH METASTATIC COLORECTAL CARCINOMA (MCRC)", ANNALS OF ONCOLOGY, vol. 24, no. suppl 4, 1 June 2013 (2013-06-01), pages iv16 - iv17, XP055131314, ISSN: 0923-7534, DOI: 10.1093/annonc/mdt201.12 *
HANS-JOACHIM SCHMOLL ET AL: "Maintenance treatment with the immunomodulator MGN1703, a Toll-like receptor 9 (TLR9) agonist, in patients with metastatic colorectal carcinoma and disease control after chemotherapy: a randomised, double-blind, placebo-controlled trial", JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, 10 May 2014 (2014-05-10), XP055131369, ISSN: 0171-5216, DOI: 10.1007/s00432-014-1682-7 *
J. VAS ET AL: "Regulatory Roles for NKT Cell Ligands in Environmentally Induced Autoimmunity", THE JOURNAL OF IMMUNOLOGY, vol. 181, no. 10, 3 November 2008 (2008-11-03), pages 6779 - 6788, XP055131246, ISSN: 0022-1767, DOI: 10.4049/jimmunol.181.10.6779 *
MONTOYA C J ET AL: "Increased IFN-gamma production by NK and CD3+/CD56+ cells in sexually HIV-1-exposed but uninfected individuals", CLINICAL IMMUNOLOGY, ACADEMIC PRESS, US, vol. 120, no. 2, 1 August 2006 (2006-08-01), pages 138 - 146, XP024941265, ISSN: 1521-6616, [retrieved on 20060801], DOI: 10.1016/J.CLIM.2006.02.008 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11578331B2 (en) 2015-09-09 2023-02-14 Gilead Sciences, Inc. Combination comprising immunostimulatory oligonucleotides
US11583581B2 (en) 2015-09-21 2023-02-21 Gilead Sciences, Inc. Methods of treating a retroviral infection
EP3392345A1 (en) 2017-04-22 2018-10-24 Mologen AG Biomarker for small cell lung cancer therapy
WO2018193137A1 (en) 2017-04-22 2018-10-25 Mologen Ag Biomarker for small cell lung cancer therapy
CN110914428A (en) * 2017-04-22 2020-03-24 莫洛根股份公司 Biomarkers for small cell lung cancer therapy
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)

Also Published As

Publication number Publication date
GB201309657D0 (en) 2013-07-17
GB2514591A (en) 2014-12-03
CN105247365A (en) 2016-01-13
JP2016526164A (en) 2016-09-01
ES2795929T3 (en) 2020-11-25
CN105247365B (en) 2019-06-18
AU2014273400A1 (en) 2015-10-08
BR112015029852A2 (en) 2017-07-25
KR101937442B1 (en) 2019-01-11
DK3004875T3 (en) 2020-06-08
CA2907980A1 (en) 2014-12-04
ZA201506813B (en) 2017-01-25
RU2015150739A (en) 2017-07-06
MX2015016305A (en) 2016-03-21
MX365512B (en) 2019-06-05
PL3004875T3 (en) 2020-11-16
RU2672358C2 (en) 2018-11-14
HK1216779A1 (en) 2016-12-02
EP3004875B1 (en) 2020-03-04
CN110201156A (en) 2019-09-06
US20160115479A1 (en) 2016-04-28
EP3004875A1 (en) 2016-04-13
US10006032B2 (en) 2018-06-26
JP6441320B2 (en) 2018-12-19
KR20150129860A (en) 2015-11-20
IL242551A (en) 2017-07-31
AU2014273400B2 (en) 2017-02-23

Similar Documents

Publication Publication Date Title
US10006032B2 (en) Predictive biomarker for cancer therapy
Droemann et al. Human lung cancer cells express functionally active Toll-like receptor 9
JP6247253B2 (en) Leukemia stem cell marker
Pashenkov et al. Phase II trial of a toll-like receptor 9–activating oligonucleotide in patients with metastatic melanoma
Krug et al. Toll‐like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL‐12
Weide et al. Results of the first phase I/II clinical vaccination trial with direct injection of mRNA
Fűri et al. Epithelial toll-like receptor 9 signaling in colorectal inflammation and cancer: clinico-pathogenic aspects
Schuh et al. Human plasmacytoid dendritic cells display and shed B cell maturation antigen upon TLR engagement
JP2020517665A (en) Biomarkers for the treatment of small cell lung cancer
EP2012772A1 (en) Reducing cancer cell invasion using an inhibitor of toll like receptor signaling
US20210155933A1 (en) Tlr-9 agonists for modulation of tumor microenvironment
Karbach et al. Therapeutic administration of a synthetic CpG oligodeoxynucleotide triggers formation of anti-CpG antibodies
EP3491387A1 (en) Methods of treatement of cancer disease by targetting tumor associated macrophage
TW201823471A (en) Methods of treating patients with a retinoic acid receptor-[alpha] agonist and an anti-CD38 antibody
Xu et al. Toll-like receptor 9 ligands increase type I interferon induced B-cell activating factor expression in chronic rhinosinusitis with nasal polyposis
Ren et al. Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances anti-tumor responses of peripheral blood mononuclear cells from human lung cancer patients
JP2024521514A (en) Sequential innate and adaptive immune modulation for cancer treatment
Kurtinović Exploring the tumor microenvironment to improve immunotherapy for bladder cancer
Abdulmajed Identification of Tumour Associated Antigens as Potential Targets for the Immunotherapy of B-cell Chronic Lymphocytic Leukaemia (B-CLL)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14725114

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2907980

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2014273400

Country of ref document: AU

Date of ref document: 20140515

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20157029934

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2014725114

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 242551

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 14891869

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: MX/A/2015/016305

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2016515715

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112015029852

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2015150739

Country of ref document: RU

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112015029852

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20151127