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WO2013007667A1 - Anticorps se liant spécifiquement à la neuropiline-1 - Google Patents

Anticorps se liant spécifiquement à la neuropiline-1 Download PDF

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Publication number
WO2013007667A1
WO2013007667A1 PCT/EP2012/063344 EP2012063344W WO2013007667A1 WO 2013007667 A1 WO2013007667 A1 WO 2013007667A1 EP 2012063344 W EP2012063344 W EP 2012063344W WO 2013007667 A1 WO2013007667 A1 WO 2013007667A1
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Prior art keywords
antibody
neuropilin
seq
nrpl
binding
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Ceased
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PCT/EP2012/063344
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English (en)
Inventor
Sebastian Dziadek
Aljoscha Michael FLOHR
Michael Gerg
Tiantom JARUTAT
Michael Schraeml
Michael Tacke
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an isolated antibody that specifically binds within the stretch of amino acids ranging from amino acid 672 to amino acid 688 as known for human neuropilin-1.
  • the novel antibodies are of great utility since they allow for the sensitive and specific detection of neuropilin-1. Detection of neuropilin-1 is e.g. possible in a tissue sample, even when such tissue sample is a formalin-fixed paraffin embedded tissue (FFPET) sample.
  • FFFPET formalin-fixed paraffin embedded tissue
  • Neuropilins are transmembrane glycoproteins having multiple biological functions.
  • Human neuropilin-1 has a molecular weight of 130-140 kDa and the sequence given in SEQ ID NO: 1.
  • Neuropilin-1 was initially identified as a receptor for class 3 semaphorins which are negative mediators of neuronal guidance (He, Z. et al, Cell 90 (1997) 739-751 and Kolodkin, A.L., Cell 90 (1997) 753-762).
  • NRP1 also functions as a co-receptor for vascular endothelial growth factor (VEGF) and enhances VEGF165 activities, such as endothelial cell migration (Soker, S. et al, Cell 92 (1998) 735-745).
  • VEGF vascular endothelial growth factor
  • the present invention relates to an isolated antibody binding to an epitope comprised within amino acids 672-688 of neuropilin-1 (SEQ ID NO: 2).
  • monoclonal antibodies binding to an epitope comprised in SEQ ID NO: 2, or to a partial sequence within this stretch of amino acids, e.g. ranging from amino acids 674 to 680 (SEQ ID NO: 3) or from amino acids 673 to 680 (SEQ ID NO: 4), respectively, are disclosed.
  • the present invention also relates to a method for performing immunohistochemistry the method comprising the steps of incubating a tissue sample with an antibody according to the present invention, whereby binding of said antibody to neuropilin-1 in said tissue takes place and staining said tissue sample for the anti-neuropilin-1 antibody bound to the tissue sample.
  • the present invention relates to the use of an antibody according to the present invention in the immunohistochemical detection of neuropilin-1, especially in a formaldehyde-fixed paraffin-embedded tissue (FFPET) sample.
  • FPET formaldehyde-fixed paraffin-embedded tissue
  • the present invention relates to an isolated antibody binding to an epitope comprised within amino acids 672-688 of neuropilin-1 (SEQ ID NO: 2).
  • Neuropilin-1 (NRPl) has a molecular weight of 130-140 kDa.
  • Neuropilin-1 has a large extracellular domain (ECD), a single transmembrane domain and a short cytoplasmic tail that does not contain a kinase domain, but may mediate signaling.
  • ECD extracellular domain
  • the extracellular portion of NRPl contains several subdomains: al, a2 (two CUB motifs), a semaphorin 3 ligand-binding domain; bl, b2 (two coagulation factor V/VIII domains), a VEGF binding domain; and the c (MAM) domain implicated in NRPl dimerization (cf. Figure 1).
  • sNRPl human soluble NRP1
  • the present invention relates to an isolated antibody binding within amino acids 672-688 of neuropilin-1 (SEQ ID NO: 2).
  • the isolated antibody according to the present invention binds to at least one epitope comprised in SEQ ID NO: 2.
  • an antibody means one antibody or more than one antibody.
  • An “epitope” is a site on a target molecule (e.g., an antigen, such as a protein or nucleic acid molecule) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, scaffold protein containing antibody binding regions, or aptamer) binds.
  • a target molecule e.g., an antigen, such as a protein or nucleic acid molecule
  • an antigen-binding molecule e.g., an antibody, antibody fragment, scaffold protein containing antibody binding regions, or aptamer
  • Epitopes can be formed both from contiguous or juxtaposed noncontiguous residues (e.g., amino acids) of the target molecule.
  • Epitopes formed from contiguous residues e.g., amino acids
  • An epitope typically includes at least 5 and up to about 12 residues, mostly between 7 and 10 residues (e.g. amino acids).
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • an antibody is purified to greater than 70% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 80%, 90, 95%, 96%, 97%, 98% or 99% by weight.
  • the isolated antibody according to the present invention is purified to a purity of greater than 90% as determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.
  • a polyclonal antibody binding to a sequence comprised in SEQ ID NO: 2 can e.g. obtained by immunoadsorption using an affinity column containing this sequence as immunosorbent material.
  • the isolated antibody is a monoclonal antibody
  • such antibody in some embodiments is purified (1) to greater than 90% as determined by, for example, the Lowry method, and in some embodiments, to greater than 95%, 96%, 97%, 98% or 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain.
  • an antibody binds to an epitope comprised within the amino acid sequence of SEQ ID NO: 2, i.e. to an epitope within amino acids 672 and 688 of neuropilin-1 is easily assessed by an appropriate immuno assay.
  • the peptide of SEQ ID NO: 2 will N-terminally be labeled with Biotin.
  • One preferred labeling agent is using Fmoc-Glu(biotinyl-PEG)-OH, which incorporates a flexible, hydrophilic spacer between biotin residue and the peptide chain. This biotinylated peptide is, bound to a streptavidin coated solid phase. If an antibody to be analyzed contains a binding site to an epitope within SEQ ID NO: 2, such antibody binds to the peptide and can be detected by any appropriate means.
  • the antibody according to the present invention binding to an epitope within SEQ ID NO: 2 preferably has at least a binding affinity of 10 7 l/mol for this molecule. Also preferred it has an affinity of 10 8 1/mol or better or of 10 9 1/mol or better.
  • the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the antibody that is (specifically) binding to neuropilin-1.
  • the level of binding to a biomolecule other than neuropilin-1 results in a binding affinity which is only 10% or less, more preferably only 5% or less of the affinity to neuropilin-1, respectively.
  • the antibody specifically binding to neuropilin-1 will e.g. not bind neuropilin-2, i.e. have a binding affinity thereto that is at least 10-fold or also preferred at least 20- fold worse as the binding affinity for neuropilin-1.
  • the binding affinity is determined using a Biacore 3000 instrument (GE Healthcare, Biacore).
  • a Biacore 3000 instrument (GE Healthcare) was used to kinetically characterize the hybridoma culture supematants with binding specificity for NRP-1 peptides.
  • a CM5 series S sensor was mounted into the system and was normalized in HBSN buffer (10 mM HEPES pH 7.4, 150 mM NaCl) according to the manufacturer's instructions.
  • the system buffer was changed to HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05 % TWEEN 20).
  • the sample buffer was the system buffer supplemented with 1 mg/ml CMD (Carboxymethyldextrane, Sigma #86524). The system was driven at 25 °C.
  • RAMIgGFC 10000 RU RAMIgGFC (relative units of rabbit-anti-mouse F(c)gamma- fragment of the respective mouse immunoglobulin G subclass / Jackson Laboratories) were immobilized according to the manufacturer's instructions using EDC/NHS chemistry on the flow cells FC1 (anti-mouse F(c)gamma of subclass 1), FC2, FC3 and FC4. The sensor was deactivated using 1M ethanolamine.
  • the binding properties of the antibody against the peptide of SEQ ID NO:2 was kinetically tested.
  • Antibodies were captured by a 1 min injection at 10 ⁇ /min of crude hybridoma supematants diluted 1 :3 in sample buffer.
  • the flow rate was set to 80 ⁇ /min.
  • the peptide SEQ ID NO:2 (NRP-1 672-688) was injected at different concentration steps of 0 nM, 1.2 nM, 3.7 nM, 11 nM , 33 nM and 100 nM, respectively, for 2 min.
  • antibodies according to the present invention i.e. binding to an epitope comprised in SEQ ID NO: 2 can e.g. be isolated from the serum of an immunized animal by immunoadsorption using the peptide of SEQ ID NO: 2).
  • An antibody according to the present invention only detects the membrane bound isoform of neuropilin-1 and not a soluble isoform of neuropilin-1.
  • Monoclonal antibodies can be produced with constant quality and in almost unlimited quantity.
  • the antibody binding to an epitope comprised in SEQ ID NO: 2 is a monoclonal antibody.
  • the antibody of the present invention binds to an epitope comprising the amino acids 674 to 680 of neuropilin-1 (SEQ ID NO: 3). In one embodiment the antibody of the present invention binds to the synthetic 15-mer peptides of SEQ ID NO: 5 through SEQ ID NO: 13, i.e. to those peptides comprising an epitope consisting of the amino acids 674 to 680 of neuropilin-1 (SEQ ID NO: 3). In one embodiment the antibody of the present invention binds to an epitope comprising the amino acids 673 to 680 of neuropilin-1 (SEQ ID NO: 4).
  • the antibody of the present invention binds to the synthetic 15-mer peptides of SEQ ID NO: 5 through SEQ ID NO: 12, i.e. to those peptides comprising an epitope consisting of the amino acids 673 to 680 of neuropilin-1 (SEQ ID NO: 4).
  • an antibody binds to an epitope of the amino acid sequence given in SEQ ID NO: 3 or SEQ ID NO: 4, respectively, is preferably assessed by PepScan- analysis as described in the Examples section. Binding to the same epitope is for example acknowledged, if the PepScan-peptides comprising the sequence of SEQ ID NO: 3 test positive with the antibody in such analysis.
  • the present invention relates to the monoclonal antibody produced by the hybridoma cell line ⁇ Nrpl>mAb clone 3.004.020 deposited with the DSM under accession number DSM ACC 3119.
  • the present invention relates to the hybridoma cell line ⁇ Nrpl>mAb clone
  • the present invention relates to the monoclonal antibody produced by the hybridoma cell line ⁇ Nrpl>mAb clone 4.002.023 deposited with the DSM under accession number DSM ACC 3120.
  • the present invention relates to the hybridoma cell line ⁇ Nrpl>mAb clone
  • the present invention relates to the monoclonal antibody produced by the hybridoma cell line ⁇ Nrpl>mAb clone 4.002.013 deposited with the DSM under accession number DSM ACC 3118.
  • the present invention relates to the hybridoma cell line ⁇ Nrpl>mAb clone
  • the present invention relates to a method for performing immunohistochemistry the method comprising the steps of a) incubating a tissue sample with an antibody according to the present invention, whereby binding of said antibody to neuropilin-1 in said tissue takes place and b) staining said tissue sample for the anti-neuropilin-1 antibody bound in step (a).
  • the present invention relates to an immunohistochemical method for detection of neuropilin-1 with an antibody according to the present invention wherein the tissue sample on which immunostaining is performed is a tissue sample that had been formaldehyde-fixed and paraffin-embedded (FFPE).
  • FFPE formaldehyde-fixed and paraffin-embedded
  • fixatives are available and used in the routine of a clinical pathology laboratory, like glutardialdehyde, formaldehyde and acetone, or other organic solvents.
  • the fixative solution usually is an aqueous formaldehyde solution that contains sodium phosphates, contrived to provide buffering (minimal pH change following addition of a small amount of strong acid or base) to pH 7.2-7.6 and an approximately isotonic solution (one whose osmotic pressure is the same as that of mammalian extracellular fluids, often based on physiological saline).
  • the terms formaldehyde and formalin are used interchangeably.
  • fixation in formaldehyde is most widely used in clinical pathology. The major reason most likely is that by fixation with formaldehyde the antigen of interest is trapped at the sites it occupied in the living organism. By building of methylene bridges introduced upon formaldehyde fixation the morphology of a cell or tissue sample is well preserved.
  • a fixed cell or tissue sample usually has to be de-hydrated and embedded in an appropriate embedding medium. Paraffin embedding is usually preferable to either plastic embedding or cutting un-embedded specimens with a vibrating microtome or a cryostat.
  • This disclosure provides, among other things, methods for detecting neuropilin-1 in biological samples (e.g., isolated cells or tissues) that have been mounted on a solid surface (e.g., a microscope slide) and treated (e.g., formalin-fixed and paraffin- embedded ("FFPE”)).
  • FFPE formalin-fixed and paraffin- embedded
  • Biological samples useful in a disclosed method are isolated, analyzed in vitro and include any cell preparation or tissue preparation that can be fixed and mounted on a solid surface.
  • Exemplary samples include, without limitation, blood smears, cytocentrifuge preparations, cytology smears, core biopsies, fine-needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).
  • tissue sections e.g., cryostat tissue sections and/or paraffin-embedded tissue sections.
  • Exemplary biological samples may be isolated from normal cells or tissues, or from neoplastic cells or tissues.
  • Neoplasia is a biological condition in which one or more cells have undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and where cells may be capable to metastasize.
  • Exemplary neoplastic cells or tissues may be isolated from solid tumors, including breast carcinomas (e.g. lobular and duct carcinomas), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial carcinomas (including, e
  • a solid support useful in a disclosed method need only bear the biological sample and, optionally, but advantageously, permit the convenient detection of components (e.g., proteins and/or nucleic acid sequences) in the sample.
  • exemplary supports include microscope slides (e.g., glass microscope slides or plastic microscope slides), coverslips (e.g., glass coverslips or plastic coverslips), tissue culture dishes, multi-well plates, membranes (e.g., nitrocellulose or polyvinylidene fluoride (PVDF)) or BIACORETM chips.
  • the present invention relates to the use of an antibody as disclosed in the present invention in the detection of neuropilin-1 in a formaldehyde-fixed paraffin-embedded tissue (FFPET) sample.
  • FPET formaldehyde-fixed paraffin-embedded tissue
  • FIG. 1 Schematic representation of neuropilin-1 domains and isoforms:
  • Neuropilin-1 has a large extracellular domain (ECD), a single transmembrane domain and a short cytoplasmic tail that does not contain a kinase domain, but may mediate signaling.
  • ECD extracellular domain
  • the extracellular portion of NRP1 contains several subdomains: al, a2 (two CUB motifs), a semaphorin 3 ligand-binding domain; bl, b2 (two coagulation factor V/VIII domains), a VEGF binding domain; and the c (MAM) domain implicated in NRP1 dimerization.
  • Proteins lysates from Nrpl-, Nrp2 -transfected or untransfected HEK293 cells were separated by SDS-PAGE, transferred to a membrane and stained with ⁇ Nrpl>mAbs clones 1.004.012,
  • proteins lysates from Nrpl-, Nrp2- transfected or untransfected HEK293 cells were separated by SDS-PAGE, transferred to a membrane and stained with ⁇ Nrpl>mAb clone 4.002.023.
  • ⁇ Nrpl>mAb clone 4.002.023 was used for each primary antibody at a concentration of 2 ng/ml. The dilutions for the other primary antibody reagents are given underneath the corresponding Blot. Molecular weights are indicated on the left side of the Figure.
  • contiguous peptide sequences with 15 amino acids in length spanning the amino acids comprised in SEQ ID NO: 2 have been synthesized and are represented in the top line of the membranes.
  • 15-mers of the mouse homologue of neuropilin-1 have been spotted to the membrane below the 15-mers from human NRP-1.
  • the corresponding mouse sequences are shifted by one spot (i.e. one amino acid position) to the right.
  • the monoclonal antibody secreted by hybridoma clone 3.004.020 strongly binds to the dots representing the 15-mer peptides of SEQ ID NO: 5 through SEQ ID NO: 13. This corresponds to a shared epitope consisting of the amino acids 674 through 680 (SEQ ID NO: 3) of NRP-1.
  • the monoclonal antibody secreted by hybridoma clone 4.002.023 strongly binds to the dots representing the 15-mer peptides of SEQ ID NO: 5 through SEQ ID NO: 12. This corresponds to a shared epitope consisting of the amino acids 673 through 680 (SEQ ID NO: 4) of NRP-1.
  • Nrpl, Nrp-transfected or untransfected HEK293 cells were fixed with 4% PBS buffered formaldehyde and were subsequently embedded in paraffin.
  • the monoclonal antibody produced by ⁇ Nrpl>mAb clone 4.002.023 shows specific membrane staining only on Nrpl-transfected cells HEK 293 - Nrpl . Both Nrp2-transfected cells (HEK 293 - Nrp2) and control cells (HEK 293) are negative.
  • Nrp-transfected HEK293 cells were fixed with 4% PBS buffered formaldehyde and were subsequently embedded in paraffin. Staining is shown for the monoclonal antibodies 3.004.020; 4.002.023 and 4.002.013, respectively.
  • FPET paraffin-embedded tissue
  • amino acids 672 to 688 of the human neuropilin-1 sequence ranges from amino acids 672 to 688 of the human neuropilin-1 sequence (SEQ ID NO: 1).
  • YDQLVWMAIGHQGDH (SEQ ID NO: 16) representing amino acid positions 672-688; 731-750 and 748-762, respectively, of the NRP-1 MAM domain.
  • these peptides were coupled to KLH as a carrier and experimental animals were immunized following routine protocols. After a final booster immunization single cell preparations of the spleen were made and spleen cells were fused to myeloma cells as e.g. described in Koehler and Milstein (1975). Resulting hybridomas were screened with ELISA for reactivity against a biotinylated screening peptide with a sequence corresponding to the used immunogen. The binding affinity of the selected hybridomas was tested by Biacore analysis. Hybridomas with high affinity for their respective immunogen sequence were cloned by single cell deposition using a F ACS Aria III. The resulting monoclonal subclones were again tested by ELISA and Biacore analysis.
  • the binding activity of the antibody against the peptide of SEQ ID NO: 2 was kinetically tested.
  • Antibodies were captured by a 1 min injection at 10 ⁇ /min of crude hybridoma supematants diluted 1 :3 in sample buffer. 300 nM of the respective peptide was injected for 5 min at 30 ⁇ /min and dissociation was monitored for 3 min. Acidic regeneration of the sensor surface using 3 consecutive injections of 10 mM Glycine pH 1.5 at 30 ⁇ 1/ ⁇ .
  • Binding Late (RU), Stability Late (RU), Rmax, kd (1/s) and MR (Molar Ratio) were calculated. Clones were scored according to their antigen complex stability and MR.
  • a Biacore 3000 instrument (GE Healthcare) was used to kinetically characterize the hybridoma culture supematants with binding specificity for NRP-1 peptides.
  • a CM5 series S sensor was mounted into the system and was normalized in HBSN buffer (10 mM HEPES pH 7.4, 150 mM NaCl) according to the manufacturer's instructions.
  • the system buffer was changed to HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05 % TWEEN 20).
  • the sample buffer was the system buffer supplemented with 1 mg/ml CMD (Carboxymethyldextrane, Sigma #86524). The system was driven at 25 °C.
  • the flow rate was set to 80 ⁇ /min.
  • the peptide of SEQ ID NO:2 (NRP-1 672-688) was injected at different concentration steps of 0 nM, 1.2 nM, 3.7 nM, 11 nM , 33 nM and 100 nM for 2 min.
  • the dissociation was monitored for 320 sec using a kinject command.
  • Acidic regeneration of the sensor surface was achieved using 3 consecutive injections of 10 mM Glycine pH 1.5 at 30 ⁇ 1/ ⁇ for 30 sec.
  • Benchmark XT platform Especially the rather slow dissociation (tl/2 diss from 16 minutes to more than 1155 minutes) is of critical importance for IHC-staining using the automated Ventana Benchmark XT platform.
  • Western blotting 5 ⁇ g protein lysates were loaded per lane on 4 -12 % NuPage SDS gels (Invitrogen). Western blotting was performed according to standard protocols with standard NuPage buffers and reagents (Invitrogen). The monoclonal antibodies ⁇ Nrpl>mAb clones 1.004.012, 3.004.020, 4.002.023 and 4.002.013, respectively, were used at a concentration of 1.25 ng/ml. Primary antibody incubation was performed for 30 min at RT. The membrane was developed on the Lumilmager (Roche Applied Science) using LumiLight reagent (Roche Applied Science).
  • Results are given in Figure 2.
  • the newly developed monoclonal antibodies i.e. ⁇ Nrpl>mAb clones 1.004.012, 3.004.020, 4.002.023 and 4.002.013, respectively, specifically detect NRP-1 and show no significant cross reactions with neuropilin-2 or other proteins.
  • Epitope mappings were carried out by means of a library of overlapping peptides (length: 15 amino acids), immobilized on a solid surface .
  • the library consists of peptides covering the sequence (666-703) of human as well as murine NRPl . Peptides were simultaneously assembled with an automated synthesizer
  • the prepared membrane consisted of overlapping pentadecapeptides spanning the sequence of human and murine NRP1 (residues 666-703), with an offset of one amino acid residue.
  • the membrane was washed with ethanol and then with Tris- buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KC1, pH 8) before blocking for 16 h at 4°C with 5 mL lOx Western Blocking Reagent (Roche Applied Science), 2.5 g sucrose in TBS, 0.1% Tween 20.
  • the membrane was washed with TBS and 0.1% Tween 20 and incubated afterwards with 100 ng/niL - 1 ⁇ g/mL of the corresponding anti-NRP-1 antibodies in TBS and 0.1% Tween 20 at ambient temperature for 2 h and subsequently washed with TBS + 0.1 % Tween 20.
  • the membrane was incubated with anti-rabbit / anti-mouse secondary HRP-antibody (1 :20,000 in TBS-T) followed by incubation with chemiluminescence luminol and visualized with a Lumilmager (Roche Applied Science).
  • ELISA-positive SPOTs were quantified and through assignment of the corresponding peptide sequences the antibody binding epitopes were identified.
  • Nrpl Nrp2 transfected or untransfected HEK293 cells, respectively, were fixed with 4% PBS buffered formaldehyde and were subsequently embedded in paraffin.
  • the monoclonal antibody produced by ⁇ Nrpl>mAb clone 4.002.023 was further evaluated for suitability of immunohistochemistry staining from formalin-fixed paraffin-embedded tissue (FFPET) samples.
  • FFPET samples obtained from a patient with breast cancer (BC) were stained. The staining was performed on the Ventana Benchmark XT platform using standard reagents and procedures.
  • the newly developed mAb produced by ⁇ Nrpl>mAb clone 4.002.023 shows strong membrane staining on tumor cells. Additionally, weak membrane staining was detected on endothelial cells.

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Abstract

Cette invention concerne un anticorps isolé qui se lie spécifiquement à un tronçon d'acides aminés connu de la neuropiline-1 humaine allant de l'acide aminé 672 à l'acide aminé 688. Ces nouveaux anticorps sont d'une grande utilité dans la mesure où ils permettent la détection sensible et spécifique de la neuropiline-1. La détection de la neuropiline-1 est, par ex., possible dans un échantillon de tissu, même quand ledit échantillon de tissu est un échantillon de tissu fixé dans le formol et inclus en paraffine (FFPET).
PCT/EP2012/063344 2011-07-11 2012-07-09 Anticorps se liant spécifiquement à la neuropiline-1 Ceased WO2013007667A1 (fr)

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Cited By (4)

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US9540439B2 (en) 2012-10-08 2017-01-10 St. Jude Children's Research Hospital Therapies based on control of regulatory T cell stability and function via a neuropilin-1:semaphorin axis
WO2017209351A1 (fr) * 2016-06-03 2017-12-07 삼성전자 주식회사 Anticorps bispécifique anti-c-met/anti-nrp1
EP4403578A1 (fr) * 2023-01-19 2024-07-24 Theranovir Anticorps contre un epitope d'un neuropiline-1-peptide modifiée par glycosaminoglycane et procédés d'utilisation de celui-ci
WO2024153818A1 (fr) * 2023-01-19 2024-07-25 Theranovir Anticorps à base de peptides neuropiline-1, nouvel épitope de mise en évidence associé à la neuropiline-1 modifiée par glycosaminoglycane et ses procédés d'utilisation

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US9540439B2 (en) 2012-10-08 2017-01-10 St. Jude Children's Research Hospital Therapies based on control of regulatory T cell stability and function via a neuropilin-1:semaphorin axis
WO2017209351A1 (fr) * 2016-06-03 2017-12-07 삼성전자 주식회사 Anticorps bispécifique anti-c-met/anti-nrp1
KR20170137470A (ko) * 2016-06-03 2017-12-13 삼성전자주식회사 항-c-Met/항-Nrp1 이중 특이 항체
KR101985299B1 (ko) 2016-06-03 2019-09-03 삼성전자주식회사 항-c-Met/항-Nrp1 이중 특이 항체
EP4403578A1 (fr) * 2023-01-19 2024-07-24 Theranovir Anticorps contre un epitope d'un neuropiline-1-peptide modifiée par glycosaminoglycane et procédés d'utilisation de celui-ci
WO2024153818A1 (fr) * 2023-01-19 2024-07-25 Theranovir Anticorps à base de peptides neuropiline-1, nouvel épitope de mise en évidence associé à la neuropiline-1 modifiée par glycosaminoglycane et ses procédés d'utilisation

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