EP1641829A1 - Emitter-bindende peptide die eine ver nderung der spektralen emissionseigenschaften des emitters bewirken - Google Patents
Emitter-bindende peptide die eine ver nderung der spektralen emissionseigenschaften des emitters bewirkenInfo
- Publication number
- EP1641829A1 EP1641829A1 EP04729434A EP04729434A EP1641829A1 EP 1641829 A1 EP1641829 A1 EP 1641829A1 EP 04729434 A EP04729434 A EP 04729434A EP 04729434 A EP04729434 A EP 04729434A EP 1641829 A1 EP1641829 A1 EP 1641829A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- emitter
- seq
- binding peptide
- nos
- fluorescence
- 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.)
- Withdrawn
Links
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- HAFZTWYRKGACLU-UHFFFAOYSA-K tetrasulfocyanine Chemical compound [Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C(C)(C)C(\C=C\C=C(\C=C\C=C\3C(C4=CC(=CC=C4N/3CCS([O-])(=O)=O)S([O-])(=O)=O)(C)C)/C)=[N+](CCS([O-])(=O)=O)C2=C1 HAFZTWYRKGACLU-UHFFFAOYSA-K 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
Definitions
- the present invention relates to emitter-binding peptides which, when their antigen-binding pocket interacts with the emitter, cause a change in the spectral emission properties of the emitter.
- the emitter-binding peptides of the invention are in particular components of antibodies and antibody fragments.
- peptides, proteins, antibodies or oligonucleotides are based, which have a high affinity for a substance to be determined. Proteins and peptides are preferably used for this purpose, and antibodies and antibody fragments are particularly preferably used.
- Certain in-vitro diagnostic methods are based on the combination of different antibodies against the substance to be determined, one antibody being used to separate the substance to be determined from the test sample and the other antibody carrying the diagnostically proven signal molecule.
- the labeled antibody is optically detected [Grayeski, ML 3 Anal Chem. 1987, 59, 1243].
- light-induced phosphorescence and fluorescence can also be used as the optical property of molecules for diagnostic measurement methods.
- fluorescence in particular as the optical property of molecules offers the advantage of high detection sensitivity and high linearity of the measurement signal over a large dynamic range.
- Other detection methods are based on a change in the polarization level or the detection of phosphorescence.
- the anti-substance antibodies used serve different purposes. On the one hand they are used for the separation of the substance to be determined from the sample, on the other hand they also perform the task of localizing or positioning different signal transmitters used on the substance to be examined.
- acoustic see e.g. Cooper MA, et al. Direct and sensitive detection of a human virus by rupture event scanning. Nat Biotechnol. 2001 Sep; 19 (9): 833-7.
- magnetic measuring methods are known.
- Optical measurement methods have become most widespread [Nakamura, R.M., Dito, W.R., Tucker, E.S. (Eds.). Immunoassays: Clinical Laboratory Techniques for the 1980s. A. R. Liss, New York. Edwards, R. (ed.). Immunoassays: Essential Data, 1996, Wiley Europe.].
- the anti-substance antibody is labeled with a fluorophore. This marking takes place through specific and non-specific chemical coupling.
- the labeled antibody is added in excess to the test sample. This is necessary to bind all substance molecules to be examined.
- This method is further generally based on the fact that the one anti-substance antibody serves to separate the substance to be examined and the second anti-substance antibody, which recognizes another binding site of the test substance, is labeled with a signaling molecule. In this way, a falsification of the measurement result by the unbound, but signaling antibody can be avoided.
- this procedure is associated with increased methodological and technical effort and higher costs.
- the high technical effort which prevents this method from being established for rapid diagnostics, proves to be particularly disadvantageous.
- Antibodies and peptides directed against molecules of small molecular weight are already known. This also includes antibodies and peptides against dye molecules.
- Simeonov et al. Give no indication of a red shift while maintaining the fluorescence quantum yield for cyanine dyes in the wavelength range from 600-1200 nm.
- Rozinov M.N. et al. (Chem. Biol. 1998, 5, 713-728) describe the selection of 12-mer peptides from phage libraries which bind the dyes Texas Red, Rhodamine Red, Oregon Green 514 and fluorescein. A red shift in absorption and fluorescence was observed for Texas Red, but only by 2.8 nm and 1.4 nm, respectively.
- antibodies against various dyes are already commercially available, e.g. B. against fluorescein, tetramethylrhodamine, Texas Red, Alexa Fluor 488, BODIPY FL, Lucifer yellow and Cascade Blue, Oregon Green (Molecular Probes, Inc., USA).
- these are polyclonal IgG antibodies for bioanalytical purposes, some of which have uncontrollable cross-reactivities and have not resulted from a strict selection process.
- Emitter-binding peptides which have a red color Shift while maintaining the fluorescence quantum yield for cyanine dyes in the wavelength range of 600-1200 nm.
- an emitter-binding peptide which is characterized in that, when its antigen binding pocket interacts with the emitter, this causes a change in the spectral emission properties of the emitter, comprising a method for producing an emitter-binding peptide according to the invention the immunization of a suitable organism with an emitter, comprising a dye which is selected from the group of polymethine dyes, such as dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium and oxonol dyes and rhodamine dyes, phenoxazine or Phenothiazine dyes and corresponding uses of an emitter-binding peptide, a nucleic acid, a host cell or an antibody or conjugate according to the invention as a diagnostic for in vitro diagnostics.
- Appropriate configurations are listed in the dependent claims.
- a first aspect of the present invention thus relates to an emitter-binding peptide, characterized in that this causes a change in the spectral emission properties of the emitter when its antigen binding pocket interacts with the emitter.
- an emitter-binding peptide according to the invention is preferred, the emitter comprising a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 nm, preferably at least one absorption maximum and fluorescence maximum within the spectral range from 750 to 900 nm.
- an emitter-binding peptide according to the invention, the change in the emission properties of the part of the emitter being selected from a change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift in the absorption maximum and / or the fluorescence maximum.
- the invention is not restricted to these special phenomena; the term “change in emission properties” in the context of the present invention is intended to encompass all physical phenomena or effects in which the energy-rich radiation striking the emitter is changed in its properties and these Change depends on the binding / non-binding of the substance-emitter conjugate or substance-recognizing agent-emitter conjugate with its emitter binding partner and the substance.
- the substance is, for example, a peptide, protein, oligonucleotide and in particular an antibody or an antibody fragment.
- the antibody fragments are fragments which comprise at least the antigen-binding regions which contain the so-called "complementarity-determining regions"("CDRs").
- the antigen-binding regions preferably comprise the complete variable chains VL and VH.
- the antibody or the antibody fragment is selected from polyclonal or monoclonal antibodies, humanized antibodies, Fab fragments, in particular monomeric Fab fragments, scFV fragments, synthetic and recombinant antibodies, scTCR Chains and mixtures thereof.
- antibody fragments can be present either as complete immunoglobulins or antibodies in one of the naturally occurring formats (IgA, IgD, IgE, IgG, IgM) or as antibody fragments, the antibody fragments at least the amino acid positions 4 to 103 for VL and 5 to 109 for VH , preferably comprise the amino acid positions 3 to 107 for VL and 4 to 111 for VH and particularly preferably the complete variable chains VL and VH (amino acid positions 1 to 109 for VL and 1 to 113 for VH) (numbering according to WO 97/08320).
- the antibody or the antibody fragment comprises at least one of the sequences SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 contain CDR areas (VL: CDR1 positions 24-34, CDR2 positions 50-56, CDR3 positions 89-96; VH: CDR1 positions 26-35, CDR2 positions 50-65, CDR3 positions 95-102), especially VL CDR3 or VH CDR3.
- VL CDR1 positions 24-34, CDR2 positions 50-56, CDR3 positions 89-96
- VH CDR1 positions 26-35, CDR2 positions 50-65, CDR3 positions 95-102
- An antibody which contains one of the sequences SEQ-ID NOs: 2, 6, 10, 14, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 is particularly preferred variable chains VL or a variable chains VH contained in the sequences SEQ-ID NOs: 1, 5, 9 and 13 (or a fragment of such an antibody).
- an antibody comprising one of the VH / NL pairs contained in the following sequence pairs: SEQ ID Os: 1 + 2; SEQ ID ⁇ Os: 5 + 6; SEQ ID ⁇ Os: 9 + 10; SEQ ID ⁇ Os: 13 + 14; SEQ ID ⁇ Os: 5 + 17; SEQ ID ⁇ Os: 5 + 19; SEQ ID ⁇ Os: 5 + 37; SEQ ID ⁇ Os: 9 + 21; SEQ ID ⁇ Os: 9 + 23; SEQ ID ⁇ Os: 9 + 25; SEQ ID ⁇ Os: 9 + 27; SEQ ID ⁇ Os: 9 + 29; SEQ ID ⁇ Os: 9 + 31; SEQ ID ⁇ Os: 9 + 33; SEQ ID ⁇ Os: 9 + 39; SEQ-ID ⁇ Os: 13 + 35 (or a fragment of such an antibody).
- CDR regions of both the heavy and the light chain are responsible for the affinity, selectivity and specificity.
- the CDR3 area from NH and the CDR3 area from VL play a role, followed by CDR2 from VH and CDR1 from VL, while CDR1 from VH and CDR2 from VL mostly play a subordinate role.
- the CDR ranges are therefore particularly suitable for optimizing the affinity and selectivity and specificity of antibodies (see also, for example, Schier et al., J. Mol. Biol. (1996) 263, 551).
- one or more of the CDR areas could be exchanged, for example specifically for CDR areas of other antibodies already showing the properties according to the invention, or by libraries of corresponding CDR sequences (see the optimization in Example 1), which either produce completely random variations or contain a more or less strong preference (tendency) towards certain amino acids or combinations thereof.
- the person skilled in the art is also able to exchange entire variable chains in the same way for corresponding chains of other defined antibodies or for various libraries of such chains. Methods are also known to the person skilled in the art to specifically exchange one or more amino acid residues in the CDRs by mutagenesis. The identification of amino acid residues to be changed in this way takes place, for. B.
- the specialist mom When making changes to the CDRs, the specialist mom also has knowledge of the so-called “canonical structures" (Al-Lazikani et al., J. Mol. Biol. (2000) 295, 979); Knappik et al. J. Mol. Biol. (2000) 296, 57), which have an influence on the three-dimensional arrangement of the CDR areas and which can be taken into account in the design of corresponding optimization strategies.
- nucleic acid molecules which encode one of the antibodies according to the invention or an antibody fragment.
- these are nucleic acid molecules which are one of the sequences SEQ-ID NOs: 2, 6, 10, 14, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 contained variable chains VL or a variable chain VH contained in the sequences SEQ-ID NOs: 1, 5, 9 and 13.
- the sequences according to SEQ-ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 are particularly preferred and 40.
- the emitter-binding peptide of the present invention optimally has a binding affinity of less than 50 nM and preferably less than 10 nM.
- an emitter-binding peptide of the present invention comprising a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 nm, preferably at least one absorption maximum and fluorescence maximum within the spectral range from 750 to 900 nm.
- the bathochromic shift of the dye is chosen so that the shift of the absorption and / or fluorescence maximum to higher wavelengths after interaction with the means for recognizing the emitter by a value greater than 15 nm, preferably greater than 25 nm, and most preferably about 30 nm.
- a shift that is a property of the dye need not necessarily be considered as such.
- the shift would be measured as a change in an emission value adapted to the dye, that is to say at a certain singular wavelength.
- Suitable optical means for measurement are provided for this purpose, which are known to the person skilled in the art. This also applies to the measurement of the change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift in the absorption maximum and / or the fluorescence maximum.
- the emitter used comprises a dye which is selected from the group of polymethine dyes such as dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium and oxonol dyes and rhodamine dyes , Phenoxazine or phenothiazine dyes.
- the emitter of the substance-emitter conjugate according to the invention contains a cyanine dye of the general formula (I)
- R 1 and R 2 independently of one another are a C 1 -C sulfoalky! chain 3 a saturated or unsaturated, branched or straight-chain Ci-Cso-alkyl chain, optionally with from 0 to 15 acidic atoms and / or from 0 up to 3 carbonyl groups is interrupted and / or can be substituted with 0 to 5 hydroxyl groups, means R 3 and R 4 independently of one another for the group -COOE 1 , -CONE ⁇ 2 , -NHCOE 1 , -NHCONHE 1 , - N- ⁇ E 2 , -OE 1 , -OSO 3 E 1 , -SOsE 1 , -SO ⁇ HE 1 or -E 1 , where E 1 and E 2 independently of one another are a hydrogen atom, a C 1 -C sulfoalkyl chain, a saturated or represents unsaturated, branched or straight-chain Ci-Cso-alky
- the emitters can form conjugates with substances.
- those of the general formula are used as substance-emitter conjugates
- Suitable structural components of the conjugates according to the invention include Dyes which have at least one absorption maximum and fluorescence maximum within the spectral range from 600 to 1200 nm. Dyes with at least one absorption maximum and fluorescence maximum within the spectral range from 700 to 1000 nm are preferred.
- Dyes that meet these criteria are, for example, those from the following classes: polymethine dyes, such as dicarbocyanine, tricarbocyanine, merocyanine and oxonol dyes, rhodamine dyes, phenoxazine or phenothiazine dyes, tetrapyrrole dyes, in particular benzopophyrins, chorines, bacteriochlorins, pheophorbhorides Pu ⁇ urine and Phthalocyanine.
- polymethine dyes such as dicarbocyanine, tricarbocyanine, merocyanine and oxonol dyes, rhodamine dyes, phenoxazine or phenothiazine dyes, tetrapyrrole dyes, in particular benzopophyrins, chorines, bacteriochlorins, pheophorbhorides Pu ⁇ urine and Phthalocyan
- Preferred dyes are the cyanine dyes with absorption maxima between 750 and 900 nm, with particular advantage indotricarbocyanines.
- Structural constituents of the conjugates according to the invention are also the substances whose concentration is to be determined using the method according to the invention. These are selected, for example, from antigens such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight, in particular sugars, dyes or other compounds with a molecular weight of less than 500 daltons.
- Preferred dyes are the cyanine dyes with absorption maxima between 750 and 900 nm, with particular advantage indotricarbocyanines.
- the dyes contain structural elements via which the covalent coupling to the substance structures takes place. These are e.g. B. Linkers with carboxy groups, amino groups, hydroxy groups.
- an optical measurement this can be done in different ways and depends mainly on the type of characteristic change in the spectral properties of the emitter (e.g. fluorophore). Generally preferred is a detection of the shift in the absorption wavelength and emission wavelength or the measurement of the absorption and / or fluorescence intensity at a wavelength which for the most part detects the portion of the fluorophore bound to the antibody. Depending on the change in the spectral properties of the antibody-bound fluorophore, other properties, such as. B. the photon lifetime, the polarization and the fading behavior can be used for optical measurement.
- Another aspect of the present invention relates to the use of an emitter-binding peptide according to the invention for in vitro diagnostics.
- the emitter-binding peptide according to the invention can also be present in a diagnostic kit, optionally together with other auxiliaries. All kits according to the invention can furthermore contain special instructions and documents (e.g. calibration curves, instructions for quantification, etc.).
- FIG. 1 the CysDisplay screening vector pMORPH23 (vector map and sequence),
- Figure 2 The expression vector pMORPHX9 MS (vector map and sequence), and
- FIG. 3 Abso ⁇ tionspektrum (left) and fluorescence spectrum of the dye from Example 2 in the absence and in the presence of antibody MOR02965 in PBS.
- Example 1 Selection, production and characterization of emitter-binding antibodies: Selection of HuCAL GOLD Fab antibody fragments against the cyanine dye Fuji 6-4 (ZK203468) [T- ⁇ iatri ⁇ -m-3,3-dimethyl-2 - ⁇ 4- ⁇ - ⁇ ethyl-7- [3 ⁇ - dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indoliu ⁇ n-2-ylJ- ⁇ epta-2,4,6-lrien-l-ylidenJ- l- (2-sul onatoethyl) -2,3-dihydro-lH-indole-5-sulfonate, inner salt]
- HuCAL GOLD is a fully synthetic, modular human antibody library in Fab antibody format. HuCAL GOLD is based on the HuCAL consensus antibody genes which have been described for the HuCAL-scFvl library (WO 97/08320; Knappik, (2000), J. Mol. Biol. 296, 57-86; Krebs et al. J Immunol Methods. 2001 Aug 1,254 (1-2): 67-84). In HuCAL GOLD, all six CDR areas are nucleic by using so-called trinucleotide mutagenesis (Virnelas et al. (1994)) Acids Res.
- HuCAL GOLD also uses a modified screening process, the so-called CysDisplay (WO 01/05950).
- CysDisplay WO 01/05950.
- the vector pMORPH23 used for the screening process can be found in Figure 1.
- the infected cells were pelleted and resuspended in 2xYT / 34 ⁇ g / ml chloramphenicol / 10 ⁇ g / ml tetracycline / 50 ⁇ g / ml kanamycin / 0.25 mM IPTG and cultivated at 22 ° C. overnight.
- the phages were precipitated twice from the supernatant with PEG and harvested by centrifugation (Ausubel (1998) Current protocols in molecular biology. John Wiley Sons, Inc., New York, USA).
- the phages were resuspended in PBS / 20% glycerin and stored at -80 ° C.
- the phagemid amplification between the individual selection rounds took place as follows: log-phase E. coli TG1 cells were infected with the selected phages and plated on LB agar plates with 1% glucose / 34 ⁇ g / ml chloramphenicol. After overnight incubation, the bacterial colonies were scraped off, re-cultured and infected with VCSM13 helper phages.
- the purified and concentrated phagemids from the HuCAL GOLD Antikö ⁇ er library were used in a standard selection process.
- ZA203468 or transferrin-coupled ZK203468 were used alternately as antigens.
- the antigens were taken up in PBS and applied in concentrations of 50 ⁇ g / ml to Maxiso ⁇ TM Mikrotite ⁇ latten F96 (Nunc).
- the Maxiso ⁇ plates were incubated at 4 ° C. overnight ("coating"). After blocking the Maxiso ⁇ plates with 5% milk powder in PBS, approx. 2E + 13 HuCAL GOLD phages were placed in the antigen-loaded, blocked wells and incubated there overnight or for two hours at room temperature.
- bound phages were eluted with 20 mM DTT or 100 ⁇ M unconjugated ZK203468. A total of three successive selection rounds were carried out, the phage amplification taking place between the selection rounds, as described above.
- the Fab-coding inserts of the isolated HuCAL clones were subcloned into the expression vector pMORPHX9_MS (see FIG. 2) in order to facilitate the subsequent expression of the Fab fragments.
- the purified plasmid DNA of the selected HuCAL Fab clones was digested with the restriction enzymes Xbal and EcoRI.
- the Fab-coding inserts were purified and ligated into the correspondingly digested vector pMORPHX9 VIS. This cloning step leads to the Fab expressing vector pMORPHX9_Fab_MS.
- Fab fragments expressed by this vector carry two C-terminal tags (Myc-Tag and Strep-Tag II) for purification and detection. Screening and characterization of ZK203468-binding Fab fragments
- the plasmid DNA of the four parenteral clones MOR02628, MOR02965, MOR02969 and MOR02977 was digested with the restriction enzymes EcoRI and Xbal and the resulting complete Fab insert from the expression vector pMORPHX9_MS was subcloned into the correspondingly cut display vector pMORPH23. This step is necessary in order to provide the genlll which is used to present the Fab fragment on the phage surface.
- the four parenteral clones (now in pMORPH23) were digested with Bpil and Sphl. The LCDR3 region and the constant Clambda region were removed from the vector backbone.
- the corresponding vector DNA fragment was isolated and purified.
- Several ⁇ g of vector DNA and compatible insert DNA were ligated in a molar ratio of 1: 2 with T4 DNA ligase and, after a purification step, transformed into electrocompetent TOP10F 'cells. Library sizes of 5E + 8 to approximately 1E + 9 clones were achieved per parenteral antibody.
- the libraries based on the clones MOR02628, MOR02969 and MOR02977 were combined ("Pool”).
- the MOR02965 library was treated separately ("lead”).
- the corresponding phagemids were produced by infection with VCSM13 Helfe ⁇ hagen using these TOPIOF 'maturation libraries.
- the purified and concentrated phagemids from "lead” and “pool” libraries were used in a maturation selection process under stringent conditions (long washing periods, displacement by purified, parental Fab proteins).
- ZA203468 or transferrin-coupled ZK203468 were used alternately as antigens. These antigens were taken up in PBS and applied in small concentrations of 100-250 ng / ml on Maxi-so ⁇ TM microtitre plates F96 (Nunc). The Maxiso ⁇ plates were incubated at 4 ° C. overnight ("coating"). After blocking the Maxiso ⁇ plates with 5% milk powder in PBS, approx.
- 2E + 11 phages were added in small concentrations of 60 or 12 ng / ml Antigen-phage solutions were incubated overnight or 2 hours at room temperature In order to increase the stringency, an additional 0.5 ⁇ g / ml of purified Fab fragments of the parent clones or 40 ng / ml of ZK203468 were added during this incubation Solutions containing antigen-bound phages were then applied to blocked neutravidin strips and incubated for 30 min in order to bind to the solid phase via the bio- to allow tinrest of the antigen. After several washing steps, bound phages were eluted with 20 mM DTT. A total of two successive selection rounds were carried out, the phage amplification between the selection rounds taking place as described above.
- the Fab-coding inserts of the isolated HuCAL clones were sub-cloned into the expression vector pMORPHX9_MS in order to facilitate the subsequent expression.
- the purified plasmid DNA of the selected HuCAL Fab clones was digested with the restriction enzymes Xbal and EcoRI.
- the Fab coding insert was purified and ligated into the correspondingly digested vector pMORPHX9_MS. This cloning step fills to the Fab expressing vector pMORPHX9_Fab_MS.
- Fab fragments expressed by this vector carry two C-terminal tags (Myc-Tag and Strep-Tag II) for purification and detection.
- the affinity of the parenteral MOR02977 could be improved by a factor of 140 compared to MOR03267. All other clones identified showed improvements of factor 2-70 compared to the respective parenteral Fab.
- Example 3 Photophysical characterization of the dye-antibody complexes and determination of the spectral shifts / fluorescence quantum yields
- Example 4 Construction of expression vectors for the expression of HuCAL immunoglobulins cloning of the heavy chain: The "multiple cloning site" of the vector pCDNA3.1 + (Invitrogen) is removed (Nhel / Apal), and a placeholder, which corresponds to the restriction sites from the HuCAL design is used for the ligation of the leader sequence (Nhel / EcoRI), the VH domain from the Fab fragment (Muni /), and the constant immunoglobulin regions (Blpl / Apal) , The leader sequence (EMBL 83133) is equipped with a Kozak sequence (Kozak, 1987).
- the constant regions of human IgG (PIR J00228), IgG4 (EMBL K01316), and serum IgAl (EMBL J00220) are divided into overlapping oligonucleotides approximately 70 bases in length. "Silent mutations” are introduced to remove restriction sites that are not compatible with the HuCAL design. The oligonucleotides are linked by "overlap extension-PCR".
- the heavy chain of the Fab fragment is cut out over Mfel / Blpl and ligated into the vector, which is opened with EcoRI / BlpI.
- EcoRI (g / aattc) and Mfel (c / aattg) both have compatible ones cohesive ends (aatt), and the sequence of the original Mfel cleavage site in the Fab fragments changes from: c / aattg to g / aattg after ligation into the IgG expression vector, thereby on the one hand both the Mfel and the EcoRI - Interface are destroyed, and on the other hand an amino acid exchange from Q (codon: caa) to E (codon: gaa) takes place.
- the "multiple cloning site" of pCDNA3.1 / Zeo + (Invitrogen) is replaced by two different placeholders.
- the k placeholder contains restriction interfaces for the incorporation of a k leader sequence (Nhel / EcoRV), the HuCAL Fab Vk domain (EcoRV / BsiWI), and the constant region of the k chain (BsiWI / Apal).
- the corresponding interfaces in the 1 placeholder are Nhel / EcoRV (1 leader), EcoRV / Hpal (VI domain), and Hpal / Apal (constant region 1 chain).
- the k-leader (EMBL Z00022) and the 1-leader (EMBL J00241) are both provided with Kozak sequences.
- the constant regions of human k- (EMBL L00241) and 1-chains (EMBL Ml 8645) are both assembled by "overlap extension-PCR" as described above.
- IgG-expressing CHO cells CHO-Kl cells are co-transfected with an equimolar mixture of expression vectors for the heavy and light IgG chains. Double-resistant transfectants are selected with 600 mg / ml G418 and 300 mg / ml Zeocin (Invitrogen) followed by limiting dilution. The supernatant from individual clones is checked for IgG expression by "capture ELISA". Positive clones are grown in RPMI-1640 medium, which is provided with 10% "ultra-low IgG-FCS" (Life Technologies). After adjusting the pH of the supernatant to 8.0 and sterile filtration, the solution is subjected to a standard ProteinA column chromatography (Porös 20 A, PE Biosystems).
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DE2003131054 DE10331054A1 (de) | 2003-07-09 | 2003-07-09 | Emitter-bindende Peptide die eine Veränderung der spektralen Emissionseigenschaften des Emitters bewirken |
US48723403P | 2003-07-16 | 2003-07-16 | |
PCT/EP2004/004403 WO2005005482A1 (de) | 2003-07-09 | 2004-04-26 | Emitter-bindende peptide die eine veränderung der spektralen emissionseigenschaften des emitters bewirken |
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EP04740875A Expired - Lifetime EP1641830B1 (de) | 2003-07-09 | 2004-07-09 | Emitter-bindende peptide, die eine veränderung der spektralen emissionseigenschaften des emitters bewirken |
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CA (1) | CA2531962A1 (de) |
DE (1) | DE502004011269D1 (de) |
EA (1) | EA010289B1 (de) |
HK (1) | HK1094209A1 (de) |
IL (1) | IL172548A0 (de) |
WO (2) | WO2005005482A1 (de) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2111553B1 (de) | 2007-01-24 | 2018-09-19 | Carnegie Mellon University | Optische biosensoren |
WO2009079212A2 (en) * | 2007-12-03 | 2009-06-25 | Carnegie Mellon University | Linked peptide fluorogenic biosensors |
KR101335560B1 (ko) * | 2009-11-19 | 2013-12-31 | 우시오덴키 가부시키가이샤 | 형광 면역 측정 방법 |
CN102993305B (zh) * | 2012-11-16 | 2015-05-13 | 上海赛伦生物技术有限公司 | 人源抗人表皮生长因子受体抗体及其编码基因与应用 |
US9862682B2 (en) | 2016-01-08 | 2018-01-09 | BroadPharm | Functionalized pegylated cyanine compounds, pharmaceutical compositions, and methods of use thereof |
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US4837003A (en) * | 1984-09-13 | 1989-06-06 | Mallinckrodt, Inc. | Radiolabeled antibody fragments |
US5268486A (en) * | 1986-04-18 | 1993-12-07 | Carnegie-Mellon Unversity | Method for labeling and detecting materials employing arylsulfonate cyanine dyes |
US5569587A (en) * | 1986-04-18 | 1996-10-29 | Carnegie Mellon University | Method for labeling and detecting materials employing luminescent arysulfonate cyanine dyes |
US6492160B1 (en) * | 1991-05-15 | 2002-12-10 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
ES2241710T3 (es) * | 1991-11-25 | 2005-11-01 | Enzon, Inc. | Procedimiento para producir proteinas multivalentes de union a antigeno. |
US6437099B1 (en) * | 1998-01-07 | 2002-08-20 | Hamamatsu Photonics K.K. | Fluorescene—activating antisera and IgG fraction therefrom |
AU2001295042A1 (en) * | 2000-09-13 | 2002-03-26 | The Scripps Research Institute | Anti-stilbene antibodies |
DE60328808D1 (de) * | 2002-09-19 | 2009-09-24 | Hamamatsu Photonics Kk | Fluoreszenzanalyseverfahren mit verwendung eines fluoreszenzantikörpers |
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2004
- 2004-04-26 WO PCT/EP2004/004403 patent/WO2005005482A1/de active Application Filing
- 2004-04-26 EP EP04729434A patent/EP1641829A1/de not_active Withdrawn
- 2004-07-09 WO PCT/EP2004/007596 patent/WO2005005483A1/de active Application Filing
- 2004-07-09 US US10/887,231 patent/US20050064512A1/en not_active Abandoned
- 2004-07-09 CA CA002531962A patent/CA2531962A1/en not_active Abandoned
- 2004-07-09 KR KR1020067000340A patent/KR20060067949A/ko not_active Application Discontinuation
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- 2004-07-09 DE DE502004011269T patent/DE502004011269D1/de not_active Expired - Fee Related
- 2004-07-09 EP EP04740875A patent/EP1641830B1/de not_active Expired - Lifetime
- 2004-07-09 BR BRPI0412374-3A patent/BRPI0412374A/pt not_active IP Right Cessation
- 2004-07-09 AT AT04740875T patent/ATE470678T1/de not_active IP Right Cessation
- 2004-07-09 AU AU2004255358A patent/AU2004255358A1/en not_active Abandoned
- 2004-07-09 JP JP2006518165A patent/JP2007526750A/ja active Pending
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Also Published As
Publication number | Publication date |
---|---|
WO2005005483A1 (de) | 2005-01-20 |
EA200600158A1 (ru) | 2006-08-25 |
AU2004255358A1 (en) | 2005-01-20 |
IL172548A0 (en) | 2006-04-10 |
ATE470678T1 (de) | 2010-06-15 |
EP1641830A1 (de) | 2006-04-05 |
BRPI0412374A (pt) | 2006-09-05 |
JP2007526750A (ja) | 2007-09-20 |
DE502004011269D1 (de) | 2010-07-22 |
KR20060067949A (ko) | 2006-06-20 |
CA2531962A1 (en) | 2005-01-20 |
EP1641830B1 (de) | 2010-06-09 |
WO2005005482A1 (de) | 2005-01-20 |
EA010289B1 (ru) | 2008-08-29 |
US20050064512A1 (en) | 2005-03-24 |
HK1094209A1 (en) | 2007-03-23 |
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