WO2008092442A1 - Stratification et commande thérapeutique d'un patient au moyen de biopuces protéiniques - Google Patents

Stratification et commande thérapeutique d'un patient au moyen de biopuces protéiniques Download PDF

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Publication number
WO2008092442A1
WO2008092442A1 PCT/DE2008/000186 DE2008000186W WO2008092442A1 WO 2008092442 A1 WO2008092442 A1 WO 2008092442A1 DE 2008000186 W DE2008000186 W DE 2008000186W WO 2008092442 A1 WO2008092442 A1 WO 2008092442A1
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WIPO (PCT)
Prior art keywords
protein
tag
stratifying
binders
therapy control
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PCT/DE2008/000186
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German (de)
English (en)
Inventor
Jens Beator
Angelika LÜKING
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Protagen Aktiengesellschaft
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Publication of WO2008092442A1 publication Critical patent/WO2008092442A1/fr

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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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to a method for stratification and therapy control of a patient by means of protein biochips.
  • Protein biochips are gaining increasing industrial importance in analytics and diagnostics as well as in pharmaceutical development.
  • High-throughput cloning of defined open reading frames is one possibility (Heyman, JA, Cornthwaite, J., Foncerrada, L., Gilmore, JR, Gontang, E., Hartman, KJ, Hernandez, CL., Hood, R., Hill HM, Lee, WY, Marcil, R., Marsh, EJ, Mudd, KM, Patino, MJ, Purcell, TJ, Rowland, JJ, Sindici, ML and Hoeffler, JP (1999) Genome-scale cloning and expression of individual Genome Res, 9, 383-392, Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor , MI, Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H.
  • expression vectors have Sequences for so-called affinity epitopes or proteins, which on the one hand allow the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, on the other hand, the specific purification via affinity chromatography (IMAC) allows.
  • affinity epitopes or proteins which on the one hand allow the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, on the other hand, the specific purification via affinity chromatography (IMAC) allows.
  • the gene products of a cDNA expression library of human fetal brain tissue in the bacterial expression system Escherichia coli in high density format were placed on a membrane and successfully screened with different antibodies. It could be shown that the proportion of full-length proteins is at least 66%.
  • the recombinant proteins of this library could be expressed and purified at high throughput (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J.
  • Antibody arrays An embryonic but growing technology, DDT, 7, 143-149, Kusnezow et al., (2003) Antibody microarrays: An evaluation of production parameters, Proteomics, 3, 254-264).
  • the invention relates to the object of providing a method for stratifying or for therapy control of a patient.
  • the object is achieved by providing a method for stratification or therapy control of a patient, wherein an array of protein binders on a solid support is mixed with body fluid of a patient and the binding success is detected.
  • the invention therefore relates to a method for stratifying or for therapy control of a patient as defined in the patent claims (hereinafter: method according to the invention).
  • Stratification also: stratification or therapy control
  • the method according to the invention allows decisions for treatment and therapy of the patient, be it hospitalization of the patient, use and / or dosage of one or more drugs, a therapeutic measure or the Monitoring a disease course or etiology or classification of a Disease, for example, in a subtype or differentiation of diseases.
  • the term "stratification” also includes risk stratification with the prognosis of an "outcome” of a detrimental health event.
  • protein binder in the sense of this invention means that a protein to be bound in the presence of a protein binder contacts it or binds to it or at least interacts with it
  • the protein to be bound is addressed to the protein binder or the protein to be bound recognizes the protein binder or the protein binder has the potential to interact with a protein (eg, antigen (epitope) / antibody (paratope) interaction).
  • Protein binders can be proteins, peptides, modified proteins / peptides, recombinant proteins / peptides, antibodies or antigens, or other proteins which can be represented on a protein biochip according to the invention.
  • the protein binders have a recognition signal to which a protein to be bound is addressed.
  • the recognition signal is preferably an epitope and / or paratope and / or hapten.
  • the protein binder is therefore a (native) antigen (epitope) or (native) antibody (paratope), in particular also a monoclonal or polyclonal antibody.
  • the protein binder is an antigen, in particular a biomarker, wherein the respective antigen or the biomarker correlates with a disease (indication). Therefore, those protein binders (or biomarkers) which are specific for an indication are preferred according to the invention. According to the invention, such proteins to be bound to a protein binder are constituents of a body fluid, in particular blood, whole blood, blood plasma, blood serum, patient serum-ie not conclusive: proteins, peptides, modified proteins / peptides, antibodies or antigens or other proteins, proteids.
  • the respective protein binder may be represented in different amounts in one or more regions on the solid support. This allows a variation of the sensitivity.
  • the regions may each comprise a total of protein binders, i. a sufficient number of different protein binders. At least 96 to 25,000 (numerically) or more of different protein binders are preferred. Preferably, however, more than 2,500, more preferably 10,000 or more protein binders.
  • arrangement synonymously means “array” and insofar as this "array” is used to identify proteins to be bound to protein binders, this is to be understood as an “assay”.
  • the arrangement is such that the protein binders represented on the array are in the form of a grid on a solid support. Further, such arrangements are preferred which allow a high density array of protein binders. Such high density arrangements are disclosed, for example, in WO 99/57311 and WO 99/57312.
  • the protein binders of the assembly are fixed to a solid support, spotted or immobilized even printed.
  • One or more protein binders may be present multiple times in the totality of all protein binders and be present in different amounts relative to one spot. Further, the protein binders may be standardized on the solid support (eg, by serial dilution series of human globulins as internal calibrators for data normalization).
  • the protein binders are present as clones. Such clones can be obtained, for example, by means of a cDNA expression library according to the invention (Büssow et al., 1998 (supra)). In a preferred embodiment, such expression libraries containing clones are obtained by means of expression vectors from an expressing cDNA library.
  • These expression vectors preferably contain inducible promoters.
  • the induction of expression can be done, for example, by means of an inducer, such as IPTG.
  • an inducer such as IPTG.
  • Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol 2003 Jan; 60 (5): 523-33).
  • Expression libraries are known to the person skilled in the art, and these can be prepared according to standard works, such as Sambrook et al., Molecular Cloning, Laboratory Handbook, 2nd edition (1989), CSH press, CoId Spring Harbor, New York Further preferred are expression libraries which are tissue-specific.
  • expression libraries which can be obtained by exon trapping are also included in the invention, and instead of an expression library it is possible to speak synonymously of an expression library.
  • Uniclone® library protein biochips or corresponding expression libraries which have no redundancy
  • Uniclone® library protein biochips or corresponding expression libraries which have no redundancy
  • WO 99/57311 and WO 99/57312 protein biochips or corresponding expression libraries which have no redundancy
  • These preferred Uniclone libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.
  • the clones may not be such as transformed bacteria, recombinant phage or transformed cells of mammals, insects, fungi, yeasts or plants.
  • the clones are fixed on a solid support, spotted or immobilized.
  • the protein binders in the particular form may be in the form of a fusion protein containing, for example, at least one affinity epitope or "tag".
  • the tag may be one such as c-myc, His-tag, Arg-tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or Strep tag, HAT tag, NusA, S-tag, SBP tag , Thioredoxin, DsbA, a fusion protein, preferably a cellulosic binding domain, green fluorescent protein, maltose binding protein, calmodulin binding protein, glutathione S-transferase or lacZ.
  • solid support includes embodiments such as a filter, a membrane, a magnetic bead, a silicon wafer, glass, metal, a chip, a mass spectrometric target, or a matrix.
  • a filter is preferred according to the invention.
  • PVDF polyvinyl styrene
  • nitrocellulose polystyrene
  • nylon polystyrene
  • this corresponds to a grid having the size of a microtiter plate (96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometric target or a matrix.
  • the evaluation of the binding success is carried out, for example using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience)).
  • the visualization of protein-protein interactions according to the invention can be carried out, for example, by means of Fluorescent labeling, biotinization or radio-isotope labeling in a conventional manner.
  • Bound antibodies are detected using secondary antibodies labeled with commercial reporter molecules (eg, Cy, Alexa, Dyomics, FITC or similar fluorescent dyes), or with reporter enzymes such as alkaline phosphatase, horseradish peroxidase, etc., and the corresponding colorimetric, fluorescent or chemiluminescent substrates.
  • a reading is carried out for example by means of a microarray laser scanner.
  • the invention relates to a method for the differential screening of protein binders which can be used for the inventive method.
  • a method for the differential screening of protein binders which can be used for the inventive method.
  • Corresponding protein binders are selected and are newly placed on a solid support.
  • the invention also relates to a method for
  • This method advantageously allows the identification of suitable disease-specific or disease-associated protein binders.
  • the arrangement contains protein binders which, for one, are known antigens and / or biomarkers for an indication or are disease-associated or are identified by means of the abovementioned differential screening for such an indication.
  • Table 1 Known disease-associated protein binders of defined indication:
  • NOL8 protein [Homo sapiens] Alopecia areata specific endosulfine alpha isoform 3 [Homo sapiens] Alopecia areata specific
  • Signal recognition particle 14kDa homologous Alu RNA binding protein [Homo sapiens] Alopecia areata specific
  • PRG2 protein [Homo sapiens] frame_3 ak641f21 frame_2 ak644k22 similar to chromosomes
  • FK506 binding protein 3 25kDa [Homo sapiens]
  • DCM IgG specific hs low density lipoprotein-related protein associated protein 1 (LRPAPl).
  • DCM IgG specific nuclear mitotic apparatus protein 1 [Homo sapiens] DCM IgG3 Specific
  • Homo sapiens NDRG family member 4 DCM IgG3 Specific heterogeneous nuclear ribonucleoprotein Al isoform b [Homo sapiens] RA specific unnamed protein product [Homo sapiens] RA specific regulator of G-protein signaling GAIP [Homo sapiens] RA specific frame_3 ak627kO8 TRAF interacting protein TANK isoform a [Homo sapiens] RA specific
  • ROA1_HUMAN Heterogeneous nuclear ribonucleoprotein Al helix destabilizing protein
  • Chromodomain helicase DNA binding protein 8 (helicase with SNF2 domain 1) RA specific
  • Hnrpal protein [Rattus norvegicus] RA specific Alopecia areata Specific erythrocyte membrane protein band 4.9 fibroblast growth factor receptor 3 (FGFR3) transcript variant 2 alopecia areata specific
  • Homo sapiens cDNA clone GLCDAC05 Alopecia areata specific
  • PRG2 protein [Homo sapiens] MS specific
  • MAPlB protein [Homo sapiens] MS specific
  • nuclear mitotic apparatus protein 1 [Homo sapiens] MS specific
  • Protein binders may be disposed on a solid support.
  • the obtained data of the reading are evaluated bioinformatorisch.
  • the bioinformatory evaluation can preferably take place by means of "Support Vector Machines.” This is a purely mathematical method of pattern recognition, which is converted into a corresponding computer program (Vapnik and Chervonenkis, Theory of Pattern Recognition, 1979).
  • the invention relates to a method of stratifying one or more patients according to any one of the preceding embodiments, wherein stratifying allows classification of patients or patient groups. This makes it particularly advantageous to create / modify and execute study designs for a particular indication.
  • a further subject of the invention therefore relates to the use of an array of protein binders on a solid support, according to one of the preceding embodiments, for stratifying or classifying one or more patients for a particular indication, such as not concluding for hospitalization of the patient, use and / or dosage one or more medicines, a therapeutic measure or the monitoring of a disease course or etiology or classification of a disease or the differentiation of diseases and for risk stratification.
  • a study design first takes place, in which groups of patients are formed depending on the question (for example, disease progression x, drug therapy x).
  • a study objective is the stratification (classification) of the patients in different disease forms. This stratification allows different therapies.
  • the samples (body fluid, e.g., serum, plasma, cerebrospinal fluid, etc.) of these specified groups of patients are incubated on a solid support with numerous protein binders according to one of the previously described embodiments.
  • body fluid e.g., serum, plasma, cerebrospinal fluid, etc.
  • the bioinformatic evaluation takes place by means of support vector machines (SVM) to a classification result.
  • SVM support vector machines
  • FIG. 2 This is illustrated in FIG. 2 by way of example for a study of the disease multiple sclerosis.
  • the classification into the groups A, B and C may be a decision criterion for the choice of a suitable therapy.
  • bioinformatic evaluation can be used to calculate significance values (for example as pValue) for the individual protein binders, which indicate how significant they are for the classification (see Table 3).
  • Table 3 Section of a list of proteins sorted according to their significance for the classification.
  • Figure 1 shows an embodiment of a l ⁇ -fold array of protein binders on a solid support.
  • FIG. 2 shows the bioinformatory evaluation of patient data by means of protein biochips and their classification based on 229 samples.
  • Figure 3 classification result of healthy subjects and multiple sclerosis patients using highly significant protein binders.

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Abstract

L'invention concerne un procédé de stratification et de commande thérapeutique d'un patient au moyen de biopuces protéiniques.
PCT/DE2008/000186 2007-02-01 2008-02-01 Stratification et commande thérapeutique d'un patient au moyen de biopuces protéiniques WO2008092442A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2437060A1 (fr) * 2010-10-01 2012-04-04 Protagen AG Séquences de marqueurs pour la sclérose multiple et leur utilisation

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DE102010042359A1 (de) * 2010-10-12 2012-04-12 Protagen Ag Markersequenzen für Multiple Sklerose und deren Verwendung

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2437060A1 (fr) * 2010-10-01 2012-04-04 Protagen AG Séquences de marqueurs pour la sclérose multiple et leur utilisation
WO2012042062A3 (fr) * 2010-10-01 2012-06-21 Protagen Ag Séquences de marqueurs pour la sclérose multiple et utilisation desdites séquences de marqueurs

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