WO2013007667A1 - An antibody specifically binding to neuropilin-1 - Google Patents

Abstract

The present invention relates to an isolated antibody that specifically binds to 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.

Description

An antibody specifically binding to neuropilin-1 Background of the Invention

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.

Neuropilins are transmembrane glycoproteins having multiple biological functions.

Human neuropilin-1 (NRP1) 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).

Despite the fact that neuropilins are known since more than ten years it has been and even today is extremely difficult to locate the protein neuropilin 1 in a tissue sample. However, localization of NRP-1 in a tissues sample is crucial to correlate structure, morphology, intracellular distribution and/or concentration of NRP-1, tissue distribution and/or concentration of NRP1 to a biological function.

While several anti-neuropilin-1 antibodies are available as research reagents from various companies, no satisfactory staining of a tissue sample, especially of a tissue sample that has been formalin-fixed and paraffin-embedded would appear to be possible using those reagents. It was the task of the present invention to identify an antibody that can at least partially overcome the problems known in the art. Surprisingly it has been found that an isolated antibody binding to an epitope comprised in amino acids 672 to amino acid 688 of human neuropilin-1 (SEQ ID NO:2) can be used with great advantage in an immunohistological staining procedure. It is especially surprising and worth mentioning that the antibody according to the present invention yields excellent staining results even with a formalin-fixed paraffin-embedded tissue (FFPET) sample.

Summary of the Invention

It has surprisingly been found that antibodies binding to a rather short partial sequence of neuropilin-1 represented by SEQ ID NO: 2 have quite advantageous properties and can overcome at least some of the problems known in the art.

In one embodiment 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).

In one embodiment of the present invention, 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.

In one embodiment 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.

Detailed Description of the Invention

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. 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).

In addition to the full-length transmembrane form, four naturally occurring human soluble NRP1 (sNRPl) isoforms were cloned. These sNRPl proteins contain only the al, a2 and bl, b2 domains and lack the MAM, transmembrane and intracellular domains. They are generated by alternative splicing or by reading through into the intron, resulting in premature truncation.

The present invention relates to an isolated antibody binding within amino acids 672-688 of neuropilin-1 (SEQ ID NO: 2). With other words, the isolated antibody according to the present invention binds to at least one epitope comprised in SEQ

ID NO: 2.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention disclosed herein belongs. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "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. 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) typically are also called linear epitopes. 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. In some embodiments, 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. In one preferred embodiment 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. In case 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.

Whether 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. In one preferred way 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 10⁷ l/mol for this molecule. Also preferred it has an affinity of 10⁸ 1/mol or better or of 10⁹ 1/mol or better.

As the skilled artisan will appreciate 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. Preferably, 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.

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 (NRP-1 672-688) was kinetically tested. Antibodies were captured by a 1 min injection at 10 μΐ/min of crude hybridoma supematants diluted 1 :3 in sample buffer.

The flow rate was set to 80 μΐ/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. 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 30μ1/ηιίη for 30 sec. Data was evaluated using the Biaevaluation software V.4.1 according to the manufacturer's instructions. Since the association rate constant ka (1/Ms) was out of the instruments specifications the affinity was calculated according to a steady state model from the equilibrium binding phase. The steady state dissociation constants KDss are given in the table. The dissociation rate constants kd (1/s) were calculated and tl/2 diss was calculated tl/2diss = ln(2)/60*kd [min].

As described above, 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. In a preferred embodiment the antibody binding to an epitope comprised in SEQ ID NO: 2 is a monoclonal antibody.

Two of the best monoclonal antibodies generated surprisingly bind to almost the identical epitope within SEQ ID NO: 2, i.e. they bind to the epitope represented by SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

In one embodiment 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). 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: 12, i.e. to those peptides comprising an epitope consisting of the amino acids 673 to 680 of neuropilin-1 (SEQ ID NO: 4).

Whether 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 preferred hybridoma cell lines according to the invention, the hybridoma cell line <Nrpl>mAb clone 3.004.020 (=MAK<h-NRPl>M-3.4.20), the hybridoma cell line <Nrpl>mAb clone 4.002.023 (=MAK<h-NRPl>M-4.2.23) and the hybridoma cell line <Nrpl>mAb clone 4.002.013 (=MAK<h-NRPl>M-4.2.13)were deposited, under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany: Cell line Deposition No. Date of Deposit

MAK<h-NRP 1 >M-3.4.20 DSM ACC 3119 2011-02-15 MAK<h-NRPl>M-4.2.23 DSM ACC 3120 2011-02-15 MAK<h-NRP 1 >M-4.2.13 DSM ACC 3118 2011-02-15

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

3.004.020 deposited with the DSM under accession number DSM ACC 3119.

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

4.002.023 deposited with the DSM under accession number DSM ACC 3120.

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

4.002.013 deposited with the DSM under accession number DSM ACC 3118.

The antibodies according to the present invention have proven extremely useful in the immunohistochemical detection of neuropilin-1. In one embodiment 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).

For successful immunostaining of an antigen in a cell or tissue sample at least three criteria have to be met: a) retention of the antigen at its original site, b) accessibility of the antigen and c) correct conformation/preservation of the antigen/epitope of interest. What is very surprising and most relevant to clinical routine testing is the fact that the antibodies according to the present invention also work excellent with tissues samples that had been fixed with formalin and embedded in paraffin.

In one embodiment 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).

Several fixatives are available and used in the routine of a clinical pathology laboratory, like glutardialdehyde, formaldehyde and acetone, or other organic solvents. The vast majority of fixation procedures, however, are based on the use of cross-linking agents, like formaldehyde. 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.

As mentioned before, 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. These positive effects, however, go to the expense of permeability of the sample, the accessibility and/or conformation of an antigen/epitope of interest, damage of nucleic acids and inactivation of enzyme activity.

For long term storage 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")). One of the advantages of performing immunohistochemistry on FFPE cell or tissue samples is that neuropilin-1 in such specimen substantially maintains its position relative to other components e.g. its location within the cell or tissue sample.

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). 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.g., adenocarcinomas and mixed Mullerian tumors (carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina (such as adenocarcinoma and squamous carcinoma of each of same), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, melanoma, and skin appendage tumors), esophageal carcinoma, carcinomas of the nasopharynx and oropharynx (including squamous carcinoma and adenocarcinomas of same), salivary gland carcinomas, brain and central nervous system tumors (including, for example, tumors of glial, neuronal, and meningeal origin), tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage.

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 BIACORE™ chips.

Cross-linking due to formaldehyde fixation in general is likely to mask or to destroy epitopes, leading to a false negative immunostaining. It has been found that an epitope on SEQ ID NO:2 as recognized by an antibody according to the present invention can easily be retrieved, e.g. by the standard procedures practiced automatically on the BenchMark-analyzer (Roche Tissue Diagnostics, Tucson, USA). In one embodiment 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.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Sequence Listing

SEQ ID NO: 1 Sequence of human neuropilin-1

SEQ ID NO: 2 Partial sequence of human neuropilin-] (pos ttions 672 to 688)

SEQ ID NO: 3 Partial sequence of human neuropilin-] (pos ttions 674 to 680)

SEQ ID NO: 4 Partial sequence of human neuropilin-] (pos ttions 673 to 680)

SEQ ID NO: 5 Partial sequence of human neuropilin-] (pos ttions 666 to 680)

SEQ ID NO: 6 Partial sequence of human neuropilin-] (pos ttions 667 to 681)

SEQ ID NO: 7 Partial sequence of human neuropilin-] (pos ttions 668 to 682)

SEQ ID NO: 8 Partial sequence of human neuropilin-] (pos ttions 669 to 683)

SEQ ID NO: 9 Partial sequence of human neuropilin-] (pos ttions 670 to 684)

SEQ ID NO: 10 Partial sequence of human neuropilin-] (pos ttions 671 to 685)

SEQ ID NO: 11 Partial sequence of human neuropilin-] (pos ttions 672 to 686)

SEQ ID NO: 12 Partial sequence of human neuropilin-] (pos ttions 673 to 687)

SEQ ID NO: 13 Partial sequence of human neuropilin-] (pos ttions 674 to 688)

SED ID NO: 14 Partial sequence of human neuropilin-] (pos ttions 295 to 306)

SED ID NO: 15 Partial sequence of human neuropilin-] (pos ttions 731 to 750)

SED ID NO: 16 Partial sequence of human neuropilin-] (pos ttions 748 to 762) Description of the Figures

Figure 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. 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.

Figure 2 Western Blotting using the four newly developed <NRP-1> monoclonal antibodies

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,

3.004.020, 4.002.023 and 4.002.013, respectively. Each antibody was used at a concentration of 1.25 ng/ml. All antibodies detect NPvP-1 and show no significant cross reaction with neuropilin-2 or other proteins. Molecular weights are indicated on the left side of each Blot.

Figure 3 Results of comparative Western Blotting

Molecular weight standards, 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. For each primary antibody a membrane with the four samples given above was separately used. The monoclonal antibody <Nrpl>mAb clone 4.002.023 was used 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.

Figure 4 Epitope analysis by 15-mer synthetic peptides

For epitope analysis 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. In addition the 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.

In this experiment also the reproducibility of the method has been investigated and both sequence motives (human/mouse) spotted in duplicate,

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.

Immunohistochemistry on cell line samples using the monoclonal antibody <NRP-l>mAb clone 4.002.023

A) 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.

B) 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.

Immunohistochemistry on FFPET from breast cancer using the monoclonal antibody <NRP-l>mAb clone 4.002.023

Formalin-fixed paraffin-embedded tissue (FFPET) samples obtained from a patient with breast cancer (BC) were stained with the monoclonal antibody produced by <Nrpl>mAb clone 4.002.023. There is a strong membrane staining on tumor cells. Example 1

Overview over neuropilin-1

An overview over the various subunits and iso forms known from neuropilin-1 is given in Figure 1. As indicated in Figure 1 , the sequence that proved surprisingly advantageous for immunization and generation of appropriate antibodies (SEQ ID

NO: 2) ranges from amino acids 672 to 688 of the human neuropilin-1 sequence (SEQ ID NO: 1).

Example 2

Generation of antibodies to neuropilin-1. For the generation of a monoclonal antibody initially four peptide sequences, three of them within the MAM domain were selected: a) Peptide SQYSTNWSAERS (SEQ ID NO: 14) from the NRP-1 Bl-domain (273- 427), representing amino acids 295-306 of neuropilin. b) Peptides KWS VLT S KT GPI QDHT G (SEQ ID NO: 2); SHVGTLRVKLRYQKPEEYDQ (SEQ ID NO: 15); and

YDQLVWMAIGHQGDH (SEQ ID NO: 16) representing amino acid positions 672-688; 731-750 and 748-762, respectively, of the NRP-1 MAM domain.

According to standard procedures 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.

Example 3

Testing of hybridoma culture supernatants by Biacore-analysis. An A 100 instrument (GE Healthcare, Biacore) was used to kinetically characterize the hybridoma culture supernatants with binding specificity for NRP-1 peptides. A CM5 series S sensor was mounted into the system and was hydrodynamically addressed in HBSN buffer (10 mM HEPES pH 7.4, 150 niM 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.

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, respectively, on the sensitive spots 1, 2, 4 and 5 on each flow cell. The spots were deactivated using 1M ethanolamine.

The binding activity of the antibody against the peptide of SEQ ID NO: 2 (NRP-1 672-688) was kinetically tested. Antibodies were captured by a 1 min injection at 10 μΐ/min of crude hybridoma supematants diluted 1 :3 in sample buffer. 300 nM of the respective peptide was injected for 5 min at 30 μΐ/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 30μ1/ηιίη.

Data was evaluated using the Biaevaluation A100 software V. l .l according to the manufacturer's instructions. 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. 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 activity of the antibody against the peptide of SEQ ID NO:2 (NRP-1 672-688) was kinetically tested. Antibodies were captured by a 1 min injection at 10 μΐ/min of crude hybridoma supernatants diluted 1 :3 in sample buffer.

The flow rate was set to 80 μΐ/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 30μ1/ηιίη for 30 sec.

Data was evaluated using the Biaevaluation software V.4.1 according to the manufacturer's instructions. Since the association rate constant ka (1/Ms) was out of the instruments specifications the affinity was calculated according to a steady state model from the equilibrium binding phase. The steady state dissociation constants KDss are given in the table. The dissociation rate constants kd (1/s) were calculated and tl/2 diss was calculated tl/2diss = ln(2)/60*kd [min]. Results and Discussion:

High titers in immunized mice were only observed with the immunogens comprising SEQ ID NO: 2 and SEQ ID NO: 15, respectively. With SEQ ID NO: 15 one hybridoma/monoclonal antibody showing nanomolar binding affinity was obtained. This MAb could be used in Western Blotting, see 1.004.012 in Figure 2. However, the monoclonal antibodies produced with this hybridoma did not yield a specific signal for NRP-1 if used in IHC with tissue sections. Surprisingly, only with the immunogen spanning amino acids 672-688 (SEQ ID NO: 2) antibodies with good IHC-reactivity have been obtained.

Biacore:

Table 1:

mAb peptide KDss (nM) Rmax (RU) Chi2 kd (1/s) t1/2 diss (min)

3.1.20 NRP1 [672-688] 2 23.3 2.12 3.80E-04 30

4.2.23 NRP1 [672-688] 0.8 23.1 6.04 7.25E-04 16

4.2.13 NRP1 [672-688] 2 36.4 0.89 < 1E-05 > 1155

Kinetic parameters of <Nrpl>mAb clones 3.1.20, 4.2.23 and 4.2.13, respectively. All three monoclonal antibodies show a high steady state affinity KDss at 25°C and a complex stability tl/2 diss sufficient for an application on the Ventana

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.

As obvious from Table 1 the slowest dissociation is observed with the monoclonal antibody produced by hybridoma (clone) 4.2.13 (=4.002.013).

Example 4

Western Blotting.

For generation of positive and negative controls, lysates from HEK293, transiently transfected with plasmids coding for Nrpl or Nrp2, were prepared. Untransfected HEK293 cells were used as negative controls. For western blotting 5μg 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.

Comparative Western Blotting analysis was also performed using e.g. the newly generated antibody <Nrpl>mAb clone 4.002.023 and several commercially available antibodies against neuropilin-1. The list of commercial antibodies as well as the type of labeled secondary antibody used is given in Table 2. Table 2: Primary and secondary antibodies used for Western Blotting

For comparative evaluation of the specificity and reactivity of various anti- neuropilin-1 antibodies essentially the procedure as given above was used, however, dilutions of primary antibodies were made as recommended by its manufacturer and choice of secondary (detection) antibody adapted as required. Protein lysates from Nrpl, Nrp2 or untransfected HEK293 cells were separated by SDS-PAGE, transferred to a membrane and stained with the above listed primary and secondary antibodies. In this comparative experiment the antibody <Nrpl>mAb clone 4.002.023 was used at a concentration of 2 ng/ml. Results of the individual blots are compiled and given in Figure 3. The comparative Western Blot analysis would indicate that the newly developed antibody <Nrpl>mAb clone 4.002.023 has an excellent specificity for NRP-1 and at the same time exhibits a strong binding to NRP-1. Example 5

Epitope analysis by Peptide Scan

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

(Muitisyntech, Syro I) according to the SPOT method of Frank, R. (Tetrahedron 48 ( 1992 ) 9217-9232) on amino-PEG derivatized cellulose membranes (AIMS, Germany) with a β-Ala linker. Assembly of the peptides was carried out stepwise utilizing 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (Frank, R. and Overwin H., In: Methods in Molecular Biology, "Epitope Mapping Protocols", Vol. 66, Morris G.E. (ed.), Humana Press, Totovva ( 1996) 149- 169). For each amino acid coupling a 0.3 M solution of protected amino acid pentafluorophenyl esters in N- methylpyrrolidinone (NMP) was employed. Between coupling steps un-reacted amino groups were capped with a mixture of acetic anhydride, diisopropyiethyl amine and 1-hydroxybenzotriazoie. Completion of the peptide sequences was monitored with Bromophenol blue (BPB) by a color change from yellow to blue.

Finally, the peptide sequences were deprotected with trifluoroacetic acid (TFA), triisoproyisiiane (TIS) and water (95:2.5:2.5).

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 μg/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. For detection, 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.

In addition the 15-mers of the mouse homologue of neuropilin-1 have been analyzed on the same membrane. The corresponding mouse sequences are shifted by one amino acid towards the C-terminus of the Nrpl sequence per spot. The membrane coated with the above mentioned 15-mer partial sequences of human and mouse neuropilin-1 was blocked as described above.

The anti-neuropilin-1 monoclonal antibodies <Nrpl>mAb clone 4.002.023 and <Nrpl>mAb clone 3.004.020, respectively, both were used at a concentration of 0.5 ug/ml. Detection of the bound primary antibody (the respective monoclonal antibody) was preformed as described above.

The results of the corresponding PepScan analyses are shown in Figure 4 and given in the tables below, respectively. a) anti-neuropilin-1 monoclonal antibody (<Nrpl>mAb) from clone 3.004.020

The 15-mers out of the human neuropilin-1 sequence bound by monoclonal <Nrpl>mAb clone 3.004.020 antibody are given in the Table 3 below.

Table 3: 15-mers out of the human neuropilin-1 sequence bound by

<Nrpl>mAb 3.004.020 - common epitope in bold

Conclusion: The monoclonal antibody secreted by hybridoma clone 3.004.020 strongly bind to 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) ofNRP-1. b) anti-neuropilin-1 monoclonal antibodies (<Nrpl>mAb) clone 4.002.023

The 15-mers out of the human neuropilin-1 sequence bound by the monoclonal antibody produced by hybridoma clone 4.002.023 are given in Table 4 below.

Table 4: 15-mers out of the human neuropilin-1 sequence bound by

<Nrpl>mAb 4.002.023 - common epitope in bold

Conclusion: The monoclonal antibody secreted by hybridoma clone 4.002.023 strongly binds to 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) ofNRP-1.

Example 6

Immunohistochemistry. For the IHC-scrccning of the newly developed <Nrpl>mAb and for establishment of staining procedures, FFPE cell line controls have been used. Therefore Nrpl, Nrp2 transfected or untransfected HEK293 cells, respectively, were fixed with 4% PBS buffered formaldehyde and were subsequently embedded in paraffin.

All staining procedures were performed on the Ventana Benchmark XT automated IHC stainer using Ventana buffers and reagents.

As obvious from Figure 5 (A) the monoclonal antibody produced by <Nrpl>mAb clone 4.002.023 shows specific membrane staining only on Nrpl -transfected cells. Both Nrp2-transfected cells and control cell are negative. All three monoclonal antibodies to NRP-1 that had been obtained using the peptide of SEQ ID NO: 2 as an immunogen showed good IHC-staining with NRP-1 transfected cells if used according to standard protocols on the Ventana Benchtop analyzer (see Figure 5 (B)).

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. As demonstrated in Figure 6, 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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Claims

Patent Claims

1. An isolated antibody binding to an epitope comprised within amino acids 672-688 of neuropilin-1 (SEQ ID NO: 2).

2. The antibody of claim 1, wherein said antibody is a monoclonal antibody.

3. The antibody of claim 1 or 2, wherein said antibody binds with an epitope comprising the amino acids 674 to 680 of neuropilin-1 (SEQ ID NO: 3).

4. The antibody of claim 1 or 2, wherein said antibody binds with an epitope comprising the amino acids 673 to 680 of neuropilin-1 (SEQ ID NO: 4).

5. The antibody according to claim 3, wherein said antibody is 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.

6. The hybridoma cell line deposited under DSM ACC 3119.

7. The antibody according to claim 4, wherein said antibody is 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.

8. The hybridoma cell line deposited under DSM ACC 3120.

9. The antibody according to claim 1, wherein said antibody is 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.

10. The hybridoma cell line deposited under DSM ACC 3118.

11. A method for performing immunohistochemistry the method comprising the steps of a) incubating a tissue sample with an antibody according to any of claims 1 to 5, 7 and 9, 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).

12. The method according to claim 11, wherein said tissue sample is a tissue sample that had been formaldehyde-fixed and paraffin-embedded (FFPE).

13. Use of an antibody according to any of claims 1 to 5, 7 and 9 in the immunohistochemical detection of neuropilin-1.

14. Use of an antibody according to any of claims 1 to 5, 7 and 9 in the detection of neuropilin-1 in a formaldehyde-fixed paraffin-embedded tissue (FFPET) sample.