AU2002215194A1 - Method for in vivo identification of intracellular epitopes - Google Patents
Method for in vivo identification of intracellular epitopesInfo
- Publication number
- AU2002215194A1 AU2002215194A1 AU2002215194A AU1519402A AU2002215194A1 AU 2002215194 A1 AU2002215194 A1 AU 2002215194A1 AU 2002215194 A AU2002215194 A AU 2002215194A AU 1519402 A AU1519402 A AU 1519402A AU 2002215194 A1 AU2002215194 A1 AU 2002215194A1
- Authority
- AU
- Australia
- Prior art keywords
- peptide
- antigen
- cells
- intracellular
- antibody
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 46
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 44
- 238000001727 in vivo Methods 0.000 title claims abstract description 20
- 239000000427 antigen Substances 0.000 claims abstract description 43
- 108091007433 antigens Proteins 0.000 claims abstract description 43
- 102000036639 antigens Human genes 0.000 claims abstract description 43
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 20
- 230000003993 interaction Effects 0.000 claims abstract description 9
- 239000013598 vector Substances 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 108010026424 tau Proteins Proteins 0.000 claims description 30
- 102000013498 tau Proteins Human genes 0.000 claims description 30
- 102000004169 proteins and genes Human genes 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 22
- 210000005253 yeast cell Anatomy 0.000 claims description 8
- 102000029749 Microtubule Human genes 0.000 claims description 7
- 108091022875 Microtubule Proteins 0.000 claims description 7
- 210000004688 microtubule Anatomy 0.000 claims description 7
- 108010029485 Protein Isoforms Proteins 0.000 claims description 5
- 102000001708 Protein Isoforms Human genes 0.000 claims description 5
- 230000007170 pathology Effects 0.000 claims description 5
- 108010033276 Peptide Fragments Proteins 0.000 claims description 4
- 102000007079 Peptide Fragments Human genes 0.000 claims description 4
- 108010067902 Peptide Library Proteins 0.000 claims description 4
- 108091023037 Aptamer Proteins 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 description 18
- 238000012217 deletion Methods 0.000 description 11
- 230000037430 deletion Effects 0.000 description 11
- 230000027455 binding Effects 0.000 description 9
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 8
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 8
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 8
- 239000012634 fragment Substances 0.000 description 7
- 101100235354 Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440) lexA1 gene Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- 101150047523 lexA gene Proteins 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000010396 two-hybrid screening Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 230000004568 DNA-binding Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101150009006 HIS3 gene Proteins 0.000 description 3
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108020001580 protein domains Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000000467 phytic acid Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 1
- 102000001805 Bromodomains Human genes 0.000 description 1
- 108050009021 Bromodomains Proteins 0.000 description 1
- 102000017589 Chromo domains Human genes 0.000 description 1
- 108050005811 Chromo domains Proteins 0.000 description 1
- 101000854943 Enterobacteria phage T4 Valyl-tRNA ligase modifier Proteins 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000021236 Hereditary diffuse leukoencephalopathy with axonal spheroids and pigmented glia Diseases 0.000 description 1
- 108700005087 Homeobox Genes Proteins 0.000 description 1
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 1
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 description 1
- 101000703464 Homo sapiens SH3 and multiple ankyrin repeat domains protein 2 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102100029571 Immunoglobulin J chain Human genes 0.000 description 1
- 101710132152 Immunoglobulin J chain Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 1
- 102100040243 Microtubule-associated protein tau Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000000470 PDZ domains Human genes 0.000 description 1
- 108050008994 PDZ domains Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010036631 Presenile dementia Diseases 0.000 description 1
- 108010034634 Repressor Proteins Proteins 0.000 description 1
- 102000009661 Repressor Proteins Human genes 0.000 description 1
- 102100030680 SH3 and multiple ankyrin repeat domains protein 2 Human genes 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 208000036969 diffuse hereditary with spheroids 1 leukoencephalopathy Diseases 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 230000009760 functional impairment Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- -1 helix-loop- helix Proteins 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 108010041420 microbial alkaline proteinase inhibitor Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 108700042226 ras Genes Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Food Science & Technology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method is described for the in vivo identification of epitopes of an intracellular antigen comprising the steps of :a) co-transforming of cells by a first vector including the nucleotide sequence encoding for the region of an antibody able to recognise and bind the intracellular antigen and by a second vector comprising the nucleotide sequence encoding for a peptide; b) growing co-transformed cells in such an environment that only cells wherein the antibody region and peptide recognise and interact each other are able to replicate and/or be recognised because: the antibody region able to recognise and bind the intracellular antigen is associated with a first molecule; the peptide is associated with a second molecule; the interaction of the first with the second molecule generates a selectable phenotype and/or recognisable signal; and the interaction of the first with the second molecule occurs only when the antibody region and peptide recognise and interact each-other; c) selecting the b) cells and identify the peptide as epitope.
Description
METHOD FOR IN VIVO IDENTIFICATION OF INTRACELLULAR
EPITOPES
The present invention relates to a method for the in vivo identification of intracellular epitopes. More particularly the invention relates to a method for in vivo mapping epitopes of an antigen, which is normally present in a cell, using intracellular antibodies.
An essential element for efficiently utilize antibodies, as well as intracellular antibodies, is their epitope(s) identification, i.e. the antigen portion recognised by the antibody itself. In fact for research, diagnostic and therapy it is desirable to know the epitope of an antigen and select antibodies able to bind to the same.
Many different approaches have been used to map the epitopes of an antigen, all using in vitro technologies and very laborious procedures. The limitation of in vitro technologies derives from the fact that the antigen- antibody binding is not investigated in the natural functional context of the antigen, i.e. the intracellular environment.
A method (peptide scanning) makes use of: a) synthesis of peptides which overlap by few amino acids and, as a whole, comprise the complete amino acid sequence of the investigated antigen, and b) the challenging of each peptide with antibodies. The method is very time-consuming, laborious, expensive and, overall, not always provides satisfactory results. In fact not always an epitope recognised by an antibody consists of a linear amino acid sequence. Presently the most useful technology for the identification of epitopes recognised by different antibodies is the use of peptides having random sequences, either selected from peptide-phage libraries or synthetically synthesized. The strategy further allows to identify peptides active as epitopes having no homology with the antigen primary sequence, thus resulting in a discontinuous or conformational epitope. The methodology, therefore, allows to map a virtual collection of epitopes recognised by a given antibody, even with no structural correlation with the starting antigen.
Alternatively mutated, truncated or deleted forms of the target antigen can be engineered, expressed in suitable cellular systems, purified, and individually assayed. Obviously this procedure is very laborious too. The authors of the present invention now provide, a method which identifies in vivo, in the natural environment, epitopes of intracellular antigens by means of antibodies which are expressed and active in the cellular environment (intracellular antibodies).
The use of intracellular antibodies, i.e. antibodies or portions thereof, expressed and active within a cell, represents a successful technology to interact with the function of a protein, within its physiological intracellular environment (Cattaneo & Neuberger, 1987; Carlson, 1988; Biocca & Cattaneo, 1995; Cattaneo & Biocca, 1997; Cattaneo & Biocca, 1999; Marasco, 1997). Thus the technology allows to achieve the functional knock-out of the target protein. It is therefore possible to confer a given phenotype to a cell or organism by means of suitable intracellular antibodies.
The technology of intracellular antibodies is based on two advantageous aspects: 1 ) the virtually unlimited availability of the immune system (natural or artificial) repertoire, providing a source of molecules suitable for high affinity and specificity reactions against any protein, and 2) the possibility to direct a protein (and then an antibody) into different intracellular compartments by appropriated, autonomous and dominant intracellular localising signals.
The technology is validly used for research and biotechnology applications, particularly for gene therapy of human and animal pathologies, to provide experimental models of pathologies, and in plant biotechnology to produce pathogen resistant transgenic plants.
The recent big development of both human and other species genome sequencing technologies determined an increasing demand to provide technologies suitable to clarify the function of identified genes, thus resulting in a new research field, so called functional genomics. It includes methods and technologies suitable to identify gene functions in a way as possible as parallel, at high throughput and suitable to be
automated. Presently this step represents a remarkable bottleneck for the industry and particularly for the procedures leading from genes to new classes of drugs.
Recently a development of the intracellular antibody technology was introduced, allowing its use as an elective methodology for programs of functional genomics: the so-called ITT (intrabody trap technology). The methodology allows to isolate or select, among all the antibodies directed against a target protein, the sub-population which is able to operate efficiently in vivo, in the intracellular environment. The technology is described in the International Patent Application PCT n. WO00/54057, herein incorporated by reference. In summary it consists of a method to select antibodies from phage-libraries or from immune splenocytes, given their ability to bind an antigen in the intracellular environment. The method allows to overcome a limitation of the intracellular antibody technology, i.e. the fact that not all of antibodies able to bind in vitro an antigen are then able to bind the same antigen when expressed as cytoplasmic antibodies. Antibodies selected by the ITT procedure, based on the two-hybrid system, are able to fold correctly in the intracellular environment and to bind the target antigen therein. Therefore they are "validated" to be used as intracellular antibodies (Visintin et al., 1999). In all of ITT's applications, the antigen is challenged with an antibody library.
The authors of the present invention advantageously adapted and modified this technology for the identification of epitopes, i.e. the antigen portions able to recognise and bind an antibody. Such epitopes, in virtue of the selection technique, are in vivo recognised by the antibody itself. According to the technology of the invention, opposite to ITT, the antibody is the constant element to be challenged with a library, or a series of antigens. This new technique of in vivo epitope mapping of a specific intracellular antibody (IVEM) represents a valid alternative to the known in vitro techniques.
The simplicity of the technique makes the same applicable for large scale screenings. The advantage of the method results also from the fact that the epitope identification occurs in vivo,, without the need of peptide
synthesis, but with the same if not higher accuracy than known methods. The method is able to accelerate the identification of epitopes to be useful for clinical, diagnostic and pharmacological applications, with large scale screeneings within a physiological contest. Furthermore because it is becoming evident that active sites of a protein are more conserved than the whole structure, by means of the method of the invention, it is possible to identify ancestral proteins having similar functions.
Therefore, the method allows the in vivo identification of the epitopes of an intracellular protein. It is known that many intracellular proteins include structurally homologous domains or modules, involved in protein-protein (e.g. SH2, SH3, PH, WW, PTB, PDZ domains; Pawson, 1965) or protein-DNA interactions (e.g. zinc finger, homeo-box, helix-loop- helix, chromo domains, bromo-domains). Being able to know the antibody specificity for a domain with respect to similar domains belonging to other proteins represents an essential prerequisite of the applications designed for intracellular antibodies.
The information is essential also in all of more conventional uses of antibodies and the simplicity of the instant method provides an easy solution to this need. Accordingly an antibody of interest, targeting one of these protein modules or domains, is challenged with a library of protein domains of the same family.
Analogously the method of the invention allows to identify the specificity of a given antibody for a family of correlated proteins, as well as isoforms of a protein (i.e., isoforms resulting from alternative splicing), and the specificity of a given antibody for families of evolutionary correlated proteins. This allows phylogenetic studies to be carried out very easily.
A further very interesting application made feasible by the present invention is represented by the more easier isolation of specific antibodies for mutated forms of a given protein. In this aspect, the antigen library consists of a collection of point mutants of the antigenic protein of interest. Isolation of antibodies for mutated forms of tau intracellular proteins, present in neurodegenerative pathologies, as taupathies, (Hong, M., et al.,
1998; Spillantini MG, et al., 1998; Goedert et al., 1999), or of p21-ras, present in many human cancers (Barbacid, 1987; Grand and Owen, 1991) can be carried out more easily than previously. " . ■■ • •-
As exemplary experimental system the authors used tau protein, a protein involved in Alzheimer's disease. However the invention is directed to a general method for epitope identification.
It is therefore an object of the present invention a method for in vivo identification of epitopes of an intracellular antigen comprising the steps of: a) co-transforming cells with a first vector including the nucleotide sequence encoding the region of an antibody able to recognise and bind the intracellular antigen and with a vector family, each one comprising a nucleotide sequence encoding a peptide and forming a peptide library as a whole; b) growing co-transformed cells in such an environment that only cells wherein the antibody region and the peptide able to recognize and interact each other are able to replicate and/or be identified, given that the antibody region able to recognize and bind the intracellular antigen is associated with a first molecule; the peptide is associated with a second molecule; the interaction of the first and of the second molecule generates a selectable phenotype and/or recognisable signal; and the interaction of the first and of the second molecule occurs only when the antibody region and peptide recognize and interact each other; c) selecting cells of b) and identify the peptide as epitope. According to a preferred embodiment the peptide library is a library of peptide fragments of an antigen; or a library of antigens; or a series of point mutants of an antigen; or a number of phylogenetic variants of an anatigen; or a series of antigens, each comprising a variant of at least one common protein domain (as, for example, SH2, SH3, PDZ, PH, PTB, WW, SHANK, ankirine repeated domains, microtubule-binding repeated domains, ZINC-FINGER domains, omeodomains, helix-loop-helix domains) or a library of aptamer peptide fragments, also fused, with a stable framework (as for example TRXA Thioredoxin, ROP).
According to a preferred embodiment the cells of the method are yeast cells.
According to a preferred embodiment the intracellular antigen is the tau protein, or phylogenetic variants thereof, or pathology associated mutants thereof, or splicing isoforms thereof, or other proteins able to bind microtubules (as, for example, MAP proteins, namely MAPI , MAP2).
The invention is now described according to preferred embodiments, with reference to the following figures:
- figure 1 is a scheme of tau protein deletion mutants; - figure 2 shows western blot analysis of lexA-tau deletion mutant fusion proteins revealed by using a polyclonal anti-lexA antibody (panel a); a generic anti-tau monoclonal antibody (Tau-1 ) (panel a); another generic anti-tau monoclonal antibody (7.51 ) (panel c).
- figure 3 shows the map of tau deletion mutants as described in Fasulo et al. 200 (panel a) and the anti-tau specificity of scFv2, scFv14 and scFv52 fragments, with respect to various deletion mutants in vivo (panel b). The epitope-scFv binding is detected by ITT technology measuring the trans-activation of HIS3 and lacZ genes and therefore the ability of the cells to grow in medium without histidine and synthesise beta-galactosidase.
Example 1 Tau mutants and fusion constructs with lexA
Deletion mutants of the tau protein were fused in frame to lexA binding domain. Such DNA binding domain is derived by an E. coli repressor protein (Brent & Ptashne, 1985) (Golemis & Brent, 1992) (Silver et al., 1986). LexA binding sites are located upstream to reporter genes, at the transcription activating region or promoter. The binding of lexA-tau deletion mutant proteins can reach and bind lexA binding sites but are unable to activate the transcription of reporter genes because of lacking of transcriptional activation domain. Tau fragments are illustrated in figure 1 and are:
-Tau 151-274 including the proline rich domain and the R1 repeated sequence able to bind to microtubules;
-Tau 151-305 including the proline rich domain and R1 and R2 repeated sequences able to bind to microtubules;
-Tau 151-336 including the proline. rich domain and R1 , R2 and R3 repeated sequences able to bind to microtubules; -Tau 151-368 including the proline rich domain and R1 , R2, R3 and R4 repeated sequences able to bind to microtubules;
-Tau 151-391 including the proline rich domain and R1 , R2, R3 and R4 repeated sequences extended up to Glu 391 residue;
-Tau 151-402 including the proline rich domain and all the repeated sequences extended up to Asp 402 residue;
-Tau 151-412 including the proline rich domain and all the repeated sequences extended up to Ser 412 residue;
-Tau 151-422 including the proline rich domain and all the repeated sequences extended up to Ser 422 residue; -dGAE including the minimal region constituting PHF filament core observed in Alzheimer's patients.
Example 2 Transformation with Tau mutants and expression thereof
Plasmid DNA enconding lexA domain fused to tau protein deletion mutants is utilized to transform the L40 yeast strain (Hollenberg et al, 1995) using a standard method including lithium acetate to make permeable yeast cells. The method was provided by Clontech on the base of previously published protocols (Gietz et al., 1992; Hill et al., 1991 ;
Schiestl & Gietz, 1989). Yeast cells are transformed using 0.1 μg of plasmid DNA and 100 μg of salmon sperm as carrier, in a solution including lithium acetate, polyethylene glycol and dimethyl sulphoxide.
Transformed cells are plated on minimal medium (synthetic dropout) containing all the amino acids but tryptophan to identify the obtained phenotype. In fact the vector used to express lexA domain fused to tau deletion mutant proteins is able to complement the nutritional deficit resulting from the lack of tryptophan, thus allowing yeast growth without said amino acid.
Hybrid proteins produced by yeast cells are extracted from transformed yeast culture using an extraction buffer containing 10% β- mercaptethanol, 2% SDS, 0,1 % bromophenol blue and 10% glycerol
(Sambrook et al., 1990). The expression level of fusion proteins was evaluated by western blot analysis, using anti lexA-protein polyclonal antibodies (Fig. 2a), anti
Tau mAbs: Tau-1 (Roche) (Fig. 2b) and 7.51 (Novak et al., 1991) (Fig. 2c).
The experiment shows that tau deletion mutants are differently recognised by the two anti-tau antibodies, thus indicating that well expressed proteins have a suitable molecular weight and are then correctly translated and correctly express known epitopes, recognised by various used antibodies.
Example 3 Co-transformation of tau mutants with single chain (ScFv) anti- tau antibody fragments, selected by means of the ITT method The experiment uses a panel of single chain Fv fragments (scFv) selected by ITT technology (Visintin et al., 1999 and PCT WO00/54057). A scFv fragment library exposed on the surface of filamentous bacteriophages was screened with the purified recombinant tau protein, bound to a solid phase. After two adsorption cycles, elution, growth of eluted phages in E. coli, the "polyclonal" population of anti-tau protein enriched antibody fragments is subcloned in the two-hybrid expression vector and tested for the tau protein binding ability in two-hybrid system. From yeast cells selected in the absence of histidine, anti-tau scFv antibody fragments were isolated, able to bind tau intracellularly. Yeast colonies, transformed with tau mutants and with each of the
ITT selected scFvs, were plated on medium selective for the identification of phenotype from nutritional reporter HIS3, which is under the control of lexA binding sites, whose expression allows cells to grow in the absence of histidine. The reconstitution of the transcriptional factor, by antigen-antibody specific interaction allows the transcription of both HIS3 and lacZ-reporter genes. The identification of such interaction is performed by qualitative analysis of colonies grown in the absence of histidine, and by colour
change (from white to blue) of the same colony in a colorimetric assay for β-galactosidase expression.
The specificity of three different scFv (2, 14, 52) fragments against the panel of tau antibody fragments is showed in Fig. 3 and summarised in Table 1.
Table 1 Antigen 151-274 151-305 151-336 151-368 151-391 151-402 151-412 151-422 dGAE ScFv2 - - - - - - +
+
ScFv14 _ - - _ + + +
+ +
ScFv52 - - - - - . + +
Fig. 3 shows the identification of tau protein epitopes by anti-tau single chain variable fragments, scFv2, scFv14 and scFv52. The growth on His" dishes and the β-gal phenotype shows Tau peptides interacting with one or more of the three scFV fragments. In particular S412-S422 and N368-E391 fragments do contain an in vivo recognisable epitope.
Example 4 Selection with the IACT method
According to the method as disclosed in the PCT application WO
00/54057, a series of 18 new anti tau ScFvs (A to Y) is selected and characterized by means of the same panel of Tau deletion mutants of the
Example 1 , but dGAE.
Each antibody has been challenged in vivo with each of Tau deletion mutants, according to the method of WO 00/54057. Results are shown in Table 2 and demonstrate that the method of the invention (IVEM) allowed the identification of three main regions (1 151 -K 274; N 368-E 391 ;
D 402-S 412) of Tau, recognized by antibodies selected with the IACT method.
Table 2
Then the selection method (IVEM) allows a rapid identification of epitopes recognized in vivo by ScFvs previously selected with the IACT method, thus representing a valid alternative to traditional expensive and time consuming methods. Moreover only by means of the instant technique an easy and precise identification of the in vivo recognizing antigen region is achieved.
BIBLIOGRAPHY
- Barbacid (1987) ras genes. Annu. Rev. Biochem. 56, 779-828.
- Biocca, S. & Cattaneo, A. (1995). Intracellular immunization: antibody targeting to subcellular compartments. Trends Cell Biol 5, 248-252.
- Brent, R. & Pthashne, M. (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43 (3 Pt 2), 729-36.
- Carlson, J. R. (1988). A new means of inducibly inactivating a cellular protein. Mol Cell Biol 8(6), 2638-46.
- Cattaneo, A. & Biocca, S. (1997). Intracellular Anribodies: Development and Applications (Springer, Ed.). - Cattaneo, A. & Biocca, S. (1999). The selection of intracellular antibodies. Trends Biotechnol 17(3), 115-21.
- Cattaneo, A & Neuberger, M.S. (1987). Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain. Embo J 6(9), 2753-8. - Fasulo, A. et al. (2000). J. Neurochem. 75, 624-633.
- Gietz, D., et al., (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20(6), 1425.
- Goedert et al., (1999). Tau gene mutation in familial progressive subcortical gliosis. Nature Medicine, 5, 454-457. - Golemis, E.A. & Brent, R. (1992). Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol 12(7), 3006-14
- Grand and Owen (1991 ). The biochemistry of ras p21. Biochem. J. 279, 609-631.
- Hill, J. et al., (1991). DMSO-enhanced whole cell yeast transformation [published erratum appears in Nuclei Acids Res 1991 Dec 11 ;
19(23):6688] Nucleic Acids Res 19(20), 5791.
- Hollenberg, S.M., et al., (1995). Identification of a new family of tissue- specific basic helix-loop-helix proteins with a two-hybrid system. Molecular and Cellular Biology 15(7), 3813-3822. - Hong, M., et al. (1998). Mutation-specific functional impairments in Distinct Tau isoforms of Heriditary FTDP-17. Science, 282, 1914-1917.
- Marasco, W.A. (1997). Intrabodies: turning the humoral immune system outside in for intracellular immunization. Gene Ther 4(1), 11-5.
- Novak, M. et al. (1991). Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51. Proc Natl
Acad Sci USA 88(13), 5837-41.
- Sambrook, J., Fritsch, E. F. & Maniatis, T (1990). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor NY edit (Press, C.S.H.I., Ed.)
- Schiestl, R.H. & Gietz, R.D. (1989). High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier Curr
Gen 16(5-6), 339-46.
- Silver, P.A., Brent, R. & Ptashne, M. (1986). DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae. Mol Cell Biol 6(12), 4763-6. - Spillantini, M.G. et al. (1998). Mutation in the gene in familial multiple system taupathy with presenile dementia. Proc. Natl. Acad. Sci USA, 95, 7737-7741.
- Visintin et al. (1999). Selection of antibodies for intracellular function using a two-hybrid system. Proc. Natl. Acad. Sci. USA 96, 11723- 11728.
Claims
1. Method for in vivo identification of epitopes of an intracellular antigen comprising the steps of : a) co-transforming of cells by a first vector including the nucleotide sequence encoding for the region of an antibody able to recognise and bind the intracellular antigen and by a vector family, anyone comprising a nucleotide sequence encoding for a peptide and forming a peptide library as a whole ; b) growing co-transformed cells in such an environment that only cells wherein the antibody region and peptide recognise and interact each other are able to replicate and/or be recognised because: the antibody region able to recognise and bind the intracellular antigen is associated with a first molecule; the peptide is associated with a second molecule; the interaction of the first with the second molecule generates a selectable phenotype and/or recognisable signal; and the interaction of the first with the second molecule occurs only when the antibody region and peptide recognise and interact each other; c) selecting the b) cells and identifying the peptide as epitope.i
2. Method for in vivo identification of epitopes of an intracellular antigen according to claim 1 wherein the peptide library is a library of antigen peptide fragments; or a library of antigens; or a number of antigen punctiform mutants; or a number of antigen phylogenetic variants; or a number of antigens, anyone comprising a variant of at least one common protein or a library of aptamer peptide fragments, also fused, with a stable framework.
3. Method for in vivo identification of epitopes of an intracellular antigen according to claim 1 or 2 wherein the cells are yeast cells.
4. Method for in vivo identification of epitopes of an intracellular antigen according to anyone of preceding claims wherein the intracellular antigen is the tau protein or phylogenetic variants or pathology associated mutants or splicing isoforms thereof or other proteins able to bind microtubules.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM200A000561 | 2000-10-24 | ||
| IT2000RM000561A IT1317922B1 (en) | 2000-10-24 | 2000-10-24 | METHOD TO IDENTIFY INTRACELLULAR EPITOPES IN VIVO. |
| PCT/IT2001/000535 WO2002035237A2 (en) | 2000-10-24 | 2001-10-22 | Method for in vivo identification of intracellular epitopes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2002215194A1 true AU2002215194A1 (en) | 2002-05-06 |
Family
ID=11454967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2002215194A Abandoned AU2002215194A1 (en) | 2000-10-24 | 2001-10-22 | Method for in vivo identification of intracellular epitopes |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US20030235850A1 (en) |
| EP (1) | EP1328814B1 (en) |
| JP (1) | JP2004512529A (en) |
| AT (1) | ATE342511T1 (en) |
| AU (1) | AU2002215194A1 (en) |
| CA (1) | CA2426744A1 (en) |
| DE (1) | DE60123819T2 (en) |
| DK (1) | DK1328814T3 (en) |
| ES (1) | ES2272553T3 (en) |
| IT (1) | IT1317922B1 (en) |
| PT (1) | PT1328814E (en) |
| WO (1) | WO2002035237A2 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7799535B1 (en) | 1997-12-09 | 2010-09-21 | Arch Development Corporation | Methods for identifying factors that control the folding of amyloid proteins of diverse origin |
| ATE382704T1 (en) | 2001-02-15 | 2008-01-15 | Univ Chicago | METHOD FOR DETECTING IN YEAST FOR MEANS OF INFLUENCING PROTEIN FOLDING |
| US9506051B2 (en) * | 2011-05-20 | 2016-11-29 | Oligomerix, Inc. | Tau protease compositions and methods of use |
| JP2014533242A (en) * | 2011-10-28 | 2014-12-11 | ユニバーシティー オブ メリーランド | Methods and compositions for intracellular neutralization by IgG |
| SG10201800800YA (en) | 2013-05-06 | 2018-03-28 | Scholar Rock Inc | Compositions and methods for growth factor modulation |
| CA2967595A1 (en) | 2014-11-12 | 2016-05-19 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
| US9879087B2 (en) | 2014-11-12 | 2018-01-30 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
| ITUB20155272A1 (en) | 2015-11-02 | 2017-05-02 | Scuola Normale Superiore | Intracellular antibody |
| CA3002097A1 (en) | 2015-11-12 | 2017-05-18 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
| EP3541847A4 (en) | 2016-11-17 | 2020-07-08 | Seattle Genetics, Inc. | GLYCAN INTERACTING COMPOUNDS AND METHOD FOR USE |
| JP2020510671A (en) | 2017-03-03 | 2020-04-09 | シアトル ジェネティックス， インコーポレイテッド | Glycan interacting compounds and methods of use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2344886B (en) * | 1999-03-10 | 2000-11-01 | Medical Res Council | Selection of intracellular immunoglobulins |
| DK1242457T3 (en) * | 1999-12-28 | 2004-12-20 | Esbatech Ag | Intracellular antibodies [intracellular antibodies] of defined structure that are stable in a reducing environment and use thereof |
-
2000
- 2000-10-24 IT IT2000RM000561A patent/IT1317922B1/en active
-
2001
- 2001-10-22 ES ES01983776T patent/ES2272553T3/en not_active Expired - Lifetime
- 2001-10-22 AU AU2002215194A patent/AU2002215194A1/en not_active Abandoned
- 2001-10-22 PT PT01983776T patent/PT1328814E/en unknown
- 2001-10-22 AT AT01983776T patent/ATE342511T1/en not_active IP Right Cessation
- 2001-10-22 JP JP2002538169A patent/JP2004512529A/en active Pending
- 2001-10-22 CA CA002426744A patent/CA2426744A1/en not_active Abandoned
- 2001-10-22 EP EP01983776A patent/EP1328814B1/en not_active Expired - Lifetime
- 2001-10-22 DE DE60123819T patent/DE60123819T2/en not_active Expired - Lifetime
- 2001-10-22 DK DK01983776T patent/DK1328814T3/en active
- 2001-10-22 WO PCT/IT2001/000535 patent/WO2002035237A2/en not_active Ceased
-
2003
- 2003-04-18 US US10/418,388 patent/US20030235850A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| PT1328814E (en) | 2007-01-31 |
| IT1317922B1 (en) | 2003-07-15 |
| ITRM20000561A1 (en) | 2002-04-24 |
| ATE342511T1 (en) | 2006-11-15 |
| EP1328814B1 (en) | 2006-10-11 |
| DE60123819T2 (en) | 2007-08-02 |
| WO2002035237A2 (en) | 2002-05-02 |
| ES2272553T3 (en) | 2007-05-01 |
| JP2004512529A (en) | 2004-04-22 |
| US20030235850A1 (en) | 2003-12-25 |
| EP1328814A2 (en) | 2003-07-23 |
| WO2002035237A3 (en) | 2002-10-10 |
| CA2426744A1 (en) | 2002-05-02 |
| DE60123819D1 (en) | 2006-11-23 |
| ITRM20000561A0 (en) | 2000-10-24 |
| DK1328814T3 (en) | 2007-01-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2714641C (en) | Methods for identification of an antibody or a target | |
| Gangwani et al. | Interaction of ZPR1 with translation elongation factor-1α in proliferating cells | |
| Poon et al. | Yeast Taf170 is encoded by MOT1 and exists in a TATA box-binding protein (TBP)-TBP-associated factor complex distinct from transcription factor IID. | |
| EP1328814B1 (en) | Method for in vivo identification of intracellular epitopes | |
| Di Niro et al. | Characterizing monoclonal antibody epitopes by filtered gene fragment phage display | |
| JP3738308B2 (en) | Cloning method of antigen-specific lymphocyte antigen receptor gene | |
| JP2000505652A (en) | New CREBa isoform | |
| JP2004108943A (en) | Analysis method and materials for organelle-localized proteins | |
| CA2474880C (en) | Production of hybridoma producing antihuman cenp-a peptide monoclonal antibody and method of using the same | |
| Meanor et al. | Yeast display guided selection of pH-dependent binders | |
| CN102766202A (en) | PHF14 C-terminal protein, its polyclonal antibody and application thereof | |
| Rhyner et al. | Direct Selection of cDNAs from Filamentous Phage Surface Display Libraries: An Update | |
| Gargano et al. | From phage libraries to intracellular immunization | |
| Traincard et al. | Evidence for the presence of an NF-κB signal transduction system in Dictyostelium discoideum | |
| Bradbury | Functional genomics, an integrated approach. Final report for the Trieste component | |
| Gangwani | eScholarship@ UMassChan | |
| Gorsch | Characterization of two Saccharomyces cerevisiae genes implicated inmRNA export: RAT7, encoding a novel nuclear pore complex protein and NPL3, encoding a putativehnRNP protein | |
| Lin-Fa et al. | Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5 | |
| Svetlov | Functional characterization and interactions between yeast Saccharomyces cerevisiae transcription factors involved in regulation of nitrogen metabolism | |
| Gangwani et al. | Let us know how access to this document benefits you. | |
| JPH10286094A (en) | Novel dlg family molecule, polynucleotide encoding the molecule, antibody to the molecule, and method for detecting dlg gene |