ANTI-CSF-IR ANTIBODY
20220235139 · 2022-07-28
Inventors
Cpc classification
C07K16/2866
CHEMISTRY; METALLURGY
G01N33/57492
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/40
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a monoclonal antibody, or fragment thereof, which binds to CSF-1R (Colony stimulating factor 1 receptor), in particular to human CSF-1R. The present invention further relates to the in vitro use of the monoclonal antibody, or fragment thereof, of the present invention for the detection of CSF-1R in a sample. Further encompassed by the present invention is a complex comprising the monoclonal antibody, or fragment thereof, of the present invention and CSF-1R such as the human CSF-1R polypeptide.
Claims
1. A monoclonal antibody, or fragment thereof, which binds to CSF-1R (Colony stimulating factor 1 receptor), wherein said monoclonal antibody, or fragment thereof, comprises a light chain variable domain that is at least 85% identical to the light chain variable domain having a sequence as shown in SEQ ID NO: 2, and wherein said monoclonal antibody, or fragment thereof, comprises a heavy chain variable domain that is at least 85% identical to the heavy chain variable domain having a sequence as shown in SEQ ID NO: 3.
2. The monoclonal antibody, or fragment thereof, of claim 1, wherein CSF1-R is human CSF-1R.
3. The monoclonal antibody, or fragment thereof, of claim 1, wherein the epitope of the monoclonal antibody, or fragment thereof, comprises a sequence as shown in SEQ ID NO: 1 (YKNIHLEKKY).
4. The monoclonal antibody, or fragment thereof, of claim 3, wherein at least one of the two Tyrosine residues of said epitope is phosphorylated.
5. The monoclonal antibody, or fragment thereof, of claim 4, wherein both Tyrosine residues of said epitope are phosphorylated.
6. The monoclonal antibody, or fragment thereof, of claim 1, wherein said monoclonal antibody, or fragment thereof, comprises a light chain variable domain that is at least 90% identical to the light chain variable domain having a sequence as shown in SEQ ID NO: 2, and/or wherein said monoclonal antibody, or fragment thereof, comprises a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain having a sequence as shown in SEQ ID NO: 3.
7. The monoclonal antibody, or fragment thereof, of claim 1, wherein said monoclonal antibody, or fragment thereof, comprises a light chain variable domain that is at least 95% identical to the light chain variable domain having a sequence as shown in SEQ ID NO: 2, and/or wherein said monoclonal antibody, or fragment thereof, comprises a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain having a sequence as shown in SEQ ID NO: 3.
8. A monoclonal antibody, or fragment thereof, comprising (a) a light chain variable domain comprising (a1) a light chain CDR1 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) a light chain CDR2 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5 (EASKVAS), and/or (a3) a light chain CDR3 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 6 (AGGYDVSDDA), (b) a heavy chain variable domain comprising (b1) a heavy chain CDR1 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) a heavy chain CDR2 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and/or (b3) a heavy chain CDR3 that differs by not more than a total of three amino acid additions, substitutions, and/or deletions from a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 9 (GGQNNGYDL), or (c) both the light chain variable domain as defined under (a) and the heavy chain variable domain as defined under (b).
9. The monoclonal antibody, or fragment thereof, of claim 8, wherein the monoclonal antibody, or fragment thereof, comprises (a) a light chain variable domain comprising (a1) a light chain CDR1 that differs by not more than one amino acid addition, substitution or deletion from a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) a light chain CDR2 that differs by not more than one amino acid addition, substitution or deletion from a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5 (EASKVAS), and/or (a3) a light chain CDR3 that differs by not more than one amino acid addition, substitution or deletion from a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 6 (AGGYDVSDDA), (b) a heavy chain variable domain comprising (b1) a heavy chain CDR1 that differs by not more than one amino acid addition, substitution or deletion from a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) a heavy chain CDR2 that differs by not more than one amino acid addition, substitution or deletion from a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and/or (b3) a heavy chain CDR3 that differs by not more than one amino acid addition, substitution or deletion from a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 9 (GGQNNGYDL), or (c) both the light chain variable domain as defined under (a) and the heavy chain variable domain as defined under (b).
10. The monoclonal antibody, or fragment thereof, of claim 8, wherein the monoclonal antibody, or fragment thereof, comprises (a) a light chain variable domain comprising (a1) a light chain CDR1 having a sequence as shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) a light chain CDR2 having a sequence as shown in SEQ ID NO: 5 (EASKVAS), and (a3) a light chain CDR3 having a sequence as shown in SEQ ID NO: 6 (AGGYDVSDDA), and (b) a heavy chain variable domain comprising (b1) a heavy chain CDR1 having a sequence as shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) a heavy chain CDR2 having a sequence as shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and (b3) a heavy chain CDR3 having a sequence as shown in SEQ ID NO: 9 (GGQNNGYDL).
11. In vitro use of the monoclonal antibody, or fragment thereof, of claim 1 for the detection of CSF-1R in a sample.
12. The in vitro use of claim 11, wherein (a) the sample has been contacted with a candidate compound for the treatment of cancer, or wherein the sample has been obtained from a subject who has been contacted with said candidate compound, (b) the sample is a cancer cell or cancer tissue, (c) the subject is a mammalian subject such as a human subject, (d) the detection of CSF-1R is the quantitative detection of CSF-1R, and/or (e) the detection of CSF-1R is the immunohistochemical detection of CSF-1R.
13. A method for detecting CSF-1R in a sample, comprising (a) contacting a sample comprising CSF-1R with the monoclonal antibody, or fragment thereof, of claim 1, thereby forming a complex comprising CSF-1R and said monoclonal antibody, or fragment thereof, and (b) detecting the complex formed in step (a), thereby detecting CSF-1R in said sample.
14. A complex comprising the monoclonal antibody, or fragment thereof, of claim 1 and CSF-1R.
15. The in vitro use of claim 11, wherein said CSF-1R comprises a phosphorylated Tyrosine residue at a position which corresponds to position 699 of human CSR-1R and/or a phosphorylated Tyrosine residue at a position which corresponds to position 708 of human CSR-1R.
16. The in vitro use of claim 12, wherein said CSF-1R comprises a phosphorylated Tyrosine residue at a position which corresponds to position 699 of human CSR-1R and/or a phosphorylated Tyrosine residue at a position which corresponds to position 708 of human CSR-1R.
17. The in vitro use of the method of claim 13, wherein said CSF-1R comprises a phosphorylated Tyrosine residue at a position which corresponds to position 699 of human CSR-1R and/or a phosphorylated Tyrosine residue at a position which corresponds to position 708 of human CSR-1R.
18. The in vitro use of the complex of claim 14, wherein said CSF-1R comprises a phosphorylated Tyrosine residue at a position which corresponds to position 699 of human CSR-1R and/or a phosphorylated Tyrosine residue at a position which corresponds to position 708 of human CSR-1R.
Description
[0145] The Figures show:
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[0148]
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[0150]
TABLE-US-00003 SEQ ID NO: 13: P-E-G-G-V-D-pY-K-N-I-H-L-E-K-K, SEQ ID NO: 14: E-G-G-V-D-pY-K-N-I-H-L-E-K-K-pY SEQ ID NO 15: G-G-V-D-pY-K-N-I-H-L-E-K-K-pY-V SEQ ID NO 16: G-V-D-pY-K-N-I-H-L-E-K-K-pY-V-R SEQ ID NO 17: V-D-pY-K-N-I-H-L-E-K-K-pY-V-R-R SEQ ID NO 18: D-pY-K-N-I-H-L-E-K-K-pY-V-R-R-D SEQ ID NO 19: pY-K-N-I-H-L-E-K-K-pY-V-R-R-D-S SEQ ID NO 20: K-N-I-H-L-E-K-K-pY-V-R-R-D-S-G SEQ ID NO 21: N-I-H-L-E-K-K-pY-V-R-R-D-S-G-F [0151] The 1H11 epitope sequence was determined by a single amino acid step analysis through the CSF-1R sequences.
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[0153]
EXAMPLES
[0154] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.
Example 1: Preparation of the Immunogen
[0155] TtSlyD-CSF1R and TtSlyD-sh3 ORFs were synthetized by GeneArt and delivered within an ampicillin resistance cloning vector. TtSlyD-CSF1R encodes the amino acids sequence:
TABLE-US-00004 >TtSlyD-CSF1R (SEQ ID NO: 11) MRSKVGQDKV VTIRYTLQVE GEVLDQGELS YLHGHRNLIP GLEEALEGRE EGEAFQAHVP AEKAYGAGSM LGPSLSPGQD PEGGVDYKNI HLEKKYVRRD SGFSSQGVDT YVEMRPVSTS SNDSFSEQDL DKEDGRPGSS GKDLDFQVEV VKVREATPEE LLHGHAHGGG SRPLLPPLPG GGSRKHHHHH HHH
[0156] TtSlyD-sh3 was applied as insertion free control protein and encodes the amino acid sequence:
TABLE-US-00005 >TtSlyDsh3 (SEQ ID NO: 12) MRSKVGQDKV VTIRYTLQVE GEVLDQGELS YLHGHRNLIP GLEEALEGRE EGEAFQAHVP AEKAYGAGSG SSGKDLDFQV EVVKVREATP EELLHGHAHG GGSRPLLPPL PGGGSRKHHH HHHHH
[0157] Restriction Enzymes and “Rapid DNA Ligation Kit” were obtained from Roche. E. coli XL1-Blue Supercompetent Cell and BL21 Codon Plus were obtained from Stratagene. To purify the DNA “High Pure Plasmid Isolation Kit” and “High Pure PCR Product Purification Kit” (Roche) were used. pQE80L was used as cloning and expression vector.
[0158] The bacteria were grown in Lysogeny Broth (LB: 5 g/l Yeast Extract, 10 g/l Trypton, 5 g/l NaCl, pH7.0) with selection antibiotic (100 μg/ml Ampicillin). For improved growing during protein expression LB medium was exchanged to Super Broth medium (SB: 20 g/l Yeast Extract, 32 g/l Trypton, 5 g/l NaCl, pH7.0).
[0159] TtSlyD-CSF1R was released from the delivering vector with EcoRI and HindIII and ligated into the expression vector pQE80-L, the later digested with EcoRI and HindIII. After transformation of E. coli XL1-Blue bacteria, plasmidic DNA was obtained and transformed in E. coli BL21 Codon Plus. In brief, for recombinant expression and purification, cells were grown in SB medium at 37° C. When the exponential phase was reached, TtSlyD variants expression was induced using 0.5 mM isopropil-β-D-thiogalactoside (IPGT) for at least 3 h. Inclusion bodies from the cell pellet were resuspended in chilled sodium phosphate buffer (pH8.0) containing 7.0M GdmCl, and stirred for 2 h until complete cell lysis. The precleared lysate was applied into a Ni-NTA column and 10-15 column volumes of wash buffer (phosphate buffer pH8.0, 7.0M GdmCl, 10 mM imidazole) were applied. To avoid reactivation of copurifying proteases, a protease inhibitors cocktail (Complete EDTA-free, Roche) was included in the refolding buffer (phosphate buffer pH8.0, 20 mM imidazole). A total of 20-25 column volumes of refolding buffer were applied slowly over night. The inhibitors cocktail was removed, with 5-10 column volumes of further washing with refolding buffer, before eluting the protein in a gradient of 250 mM imidazole. Protein containing fractions were pooled and purified again through a size-exclusion chromatography column (HiLoad™ 26/60 Superdex™ 75 size exclusion chromatography column, Amersham Pharmacia) in Storage Buffer (50 mM KH.sub.2P04 pH6.95, 100 mM KCl, 0.5 mM EDTA). Only monomer containing fractions were recovered and assessed for purity in SDS denaturizing gels. Novex® NuPAGE® SDS-PAGE Gel Systems (Invitrogen) were used for Western Blot analysis. Commassie-like protein staining was performed using SimplyBlue™ Safe-Stain (Invitrogen). Protein concentration measurements were performed with a DU®7400 Spectrophotometer (Beckman Coulter™). The molar extinction coefficients (ε.sub.280) for fusion proteins were calculated by bioinformatics
Example 2: Circular Dichroism Spectra Analysis
[0160] Protein concentration measurements were performed with a DU®7400 Spectrophotometer (Beckman Coulter™). The molar extinction coefficients (ε.sub.280) for fusion proteins were calculated by bioinformatics.
[0161] Near-UV CD spectra were recorded using a Jasco-720 spectropolarimeter with a thermostatic cell holder set to 20° C. and converted to mean residue ellipticity. The buffer was 50 mM potassium phosphate (pH6.95), 100 mM KCl and 0.5 mM EDTA. The spectra was recorded between 330-250 nm with a path length of 0.2 cm, and the protein concentration was 500 μM. The bandwidth was 1 nm, the scanning speed was 20 nm/min at a resolution of 0.5 nm, and the response was 1 s. To improve the signal to noise ratio, spectra were measured nine times and averaged. FAr-UV CD spectra were recorded using a Jasco-720 spectropolarimeter with a thermostatic cell holder set to 20° C. and converted to mean residue ellipticity. The buffer was 10 mM potassium phosphate (pH6.95) and 10 mM KCl. The spectra was recorded between 250-190 nm with a path length of 0.2 cm, and the protein concentration was 5 μM. The bandwidth was 1 nm, the scanning speed was 20 nm/min at a resolution of 0.5 nm, and the response was 1 s. To improve the signal to noise ratio, spectra were measured nine times and averaged.
[0162] For the thermal unfolding transitions of fusion proteins, the proteins were measured at 500 μM in 50 mM potassium phosphate (pH6.95), 100 mM KCl and 0.5 mM EDTA. Thermally induced unfolding-refolding transitions were recorded at 278 nm, and the path length of the cuvette was 0.2 cm. Heating and cooling rages were 1° C./min, and the response time was 4 s. To assess the reversibility of the unfolding, near-UV CD spectra of the fusion proteins were recorded before and after the thermally induced unfolding-refolding cycle.
[0163] Protein concentration measurements were performed with a DU®7400 Spectrophotometer (Beckman Coulter™). The molar extinction coefficients (ε.sub.280) for fusion proteins were calculated by bioinformatics according to Gasteiger et al 2005.
[0164] Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian) with a thermostatic cell holder set to 20° C. The buffer was 50 mM potassium phosphate (pH6.95), 100 mM KCl and 0.5 mM EDTA. The samples were excited at 280/290/295 nm and the spectra were recorded between 300-425 nm with a path length of 1 cm. The protein concentration was 10-30 μM. The bandwidth was 5 nm, the scanning speed was 120 nm/min at a resolution of 1 nm, and the response was 0.5 s.
[0165] For the thermal unfolding transitions of fusion proteins, the same concentration and buffer were used. The full spectra were recorded for thermally induced unfolding transitions, with the same specifications as for single measurements. Heating intervals were set to 5° C., and the stabilization period between temperature changes was set to 10 min. To assess the reversibility of the unfolding, fluorescence spectra of the fusion proteins were recorded before and after the thermally induced unfolding-refolding cycle.
Example 3: Phosphorylation and Purification of TtSlyD-CSF1R
[0166] Buffer components were obtained from Merck, Roche and Sigma. Src (1-530) active kinase was purchased from Upstate (Millipore). According to manufacture instructions Src kinase was diluted (20 mM MOPS-NaOH pH7.0, 1 mM EDTA, 0.01% Brij-30, 5% glycerol, 0.1% 3-mercaptoethanol and 1 mg/ml BSA), aliquoted and stored at −80° C. Novex® NuPAGE® SDS-PAGE Gel Systems (Invitrogen) were used for Western Blot analysis. To detect phosphorylated protein, several pan-pTyr primary antibodies were used: P-Tyr-4G10 (Millipore), P-Tyr-100 and P-Tyr-102 (Cell Singaling), and an HRP-conjugated goat-anti-mouse-IgG (Invitrogen) as secondary. The results were analyzed by ChemiDoc™ MP (Bio-Rad), incubating the membrane with Lumi-Light PLUS Western Blotting Substrate (IRoche).
[0167] 4.5 mg TtSlyD-CSF1R was diluted to a 60 μM concentration with reaction buffer (10 mM MOPS-NaOH pH7.0, 50 mM NaCl, 0.3 mM EDTA, 0.001% Brij-30, 0.5% glycerol, 10 mM MgAc, 0.1 mM ATP, 0.25 mM orthovanadate and 0.1 mg/ml BSA). 4 units of Src were added (1.97 U/μg) and the reaction was incubated for 2 h at 30° C. Afterwards the reaction was stopped by chelating Mg.sup.2+ cations using EDTA. To remove the buffer and contaminating proteins, the solution was loaded in a HiLoad™ 16/60 Superdex™ 200 size exclusion chromatography column (Amersham, Pharmacia) using sterile-filtrated PBS pH6.95 as a sheath solution. TtSlyD-CSF1R containing fractions were recovered and the protein was concentrated using Ultracell™ 10 k Amicon (Millipore). 81% of the initial protein was recovered.
Example 4: Rabbit Monoclonal Antibody Production
[0168] Rabbits were immunized subcutaneous each 30 days with 100 μg antigen. Serum was taken starting at day 45 and the antibody titers against the antigens were tested. After 2 month the immunogen titer was over 200.000. Peripheral blood was monthly taken 5 to 6 days after the boost. The blood was treated with citrate to avoid coagulation, and was freshly processed the same day. The Peripheral Blood Mononuclear Cells (PBMCs) were required for either obtaining antibody-producing B-cells or macrophages for their secreted grown factors. PBMCs were obtained from peripheral rabbit blood and antigen specific monoclonal antibodies were generated as it is described in Seeber et al. (2014), PLoS One. 2014 Feb. 4; 9(2). 5.10e7 PMBCs/ml were resuspended in FACS Puffer (PBS+0.1% BSA) with 250 nM of the biotinylated antigen. After 15-20′ incubation at 4° C., the cells were washed with 40 ml PBS, and resuspended to 10e8 PMBCs/ml in Labeling Buffer (PBS+2 mM EDTA). 10% volume of Streptavidin Beads was added (MACS Miltenyi Biotec) and they were incubated 15-20′ at 4° C. The cells were washed with 40 ml PBS, and resuspended to 2.10e8 PMBCs/ml in MACS Buffer (PBS+2 mM EDTA+0.5% BSA). The suspension was loaded in a preequilibrated MS Columns (MACS Mitenyi Biotec), washed with three volumes of MACS Buffer, and the bound cells were recovered in 1 ml MACS Buffer. To discriminate between cell types, the recovered cells were stained with a fluorescent antibody against rabbit IgG (AbD Serotec), and the IgG positive cells were single cell sorted using a FACSAria I cell sorter (BD Biosciences). The cells were incubated in B-cell medium as described in Seeber et al. for one week. After one week, the supernatant of the clones were tested for IgG production and antigen specificity using HitELISA techniques. The positive clones were selected and stored at −80° C. with RNA lysis buffer. HitELISAs with cell culture supernatans and purified mAbs. ELISA plates (Roche) were coated with 100-250 ng/ml of antigens in carbonate buffer, pH9.6. Biotinylated antigens were bound directly to Streptavidin coated plates (Roche) using the same antigen concentration. After washing, plates were blocked with Incubation Buffer (IB: 1% BSA 0.05% Tween-PBS). The plates were incubated with cell supernatants or purified mAbs diluted with IB. After washing, the plates were developed by incubation for 1 h with HRP-conjugated F(ab′)2 fragment goat anti-rabbit IgG (Dianova) and adding 100 μl ABTS solution (Roche). Optical densities were measured at the appropriate wavelength using an ELISA microplate reader. IgG concentration sandwich-ELISA. ELISA plates were coated with 3 μg/ml of goat anti-rabbit IgG in carbonate buffer, pH9.6. After washing, plates were blocked with IB. The plates were incubated with cell supernatants diluted in D3. Subsequent ELISAs were performed similarly as described above.
[0169] The B-cell mRNA was purified with RNeasy® Plus Mini Kit (Qiagen®) from the positive single memory B-cells cultures frozen at −80° C. Reverse transcription was performed with Transcription Universal cDNA Master (Roche). The cDNA plates were stored at −20° C. until further use. The IgH, Igλ and Igκ variable genes were amplified independently by PCR with the appropriate primers, starting from 2 μl cDNA as a template with Expand High Fidelity PCR System (Roche). The purified single amplified bands and the plasmids containing the IgG constant regions were digested with T4 DNA Polymerase (Roche) to generate 5′ overhangs, followed by RecA treatment (NE Biolabs) to be religated by Sequence and Ligation-Independent Cloning (SLIC). The recombined plasmids were transformed and tested for correct insertion cloning using standard digestion and sequencing protocols.
[0170] Full length IgG mAbs were produced by transient cotransfection of the paired heavy and light chain TIPE plasmids into FreeStyle 293-F cells (Invitrogen) grown in serum-free FreeStyle™ 293 Expression Medium (Gibco®Invitrogen) using 293-Free™ Transfection Reagent (Novagen®). Cells were cultivated one week at 37° C./5-8% CO.sub.2 with continuous shaking at 180 rpm. The supernatants were collected by centrifugation and stored at −20° C.
Example 5: Interaction Analysis
[0171] A Biacore B3000 instrument (GE Healthcare) was used to kinetically assess the rabbit antibodies for kinetics and binding specificity for TtSlyD-CSF1R. A CMS series sensor was mounted into the system and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to the manufacturer's instructions. The sample buffer was the system buffer supplemented with 1 mg/ml CMD (Carboxymethyldextran, Sigma #86524). The system operated at 25° C. 10000 RU GAR<F(ab)2> (relative units of goat anti rabbit F(ab)2/Jackson Laboratories, cat. No. 100018) were immobilized according to the manufacturer's instructions using EDC/NHS chemistry on all flow cells. The sensor was saturated with 1M ethanolamine. The binding activity of the antibodies against the analytes were kinetically tested. Analytes in solution were two 2 kDa peptides CSF1R (716-729) with Y723 phosphorylated and non-phosphorylated, a 14 kDa TtSlyDsh3 control protein with a CSF1R unrelated insertion domain and the 21 kDa TtSlyD-CSF1R protein. Antibodies were captured by a 2 min injection at 10 μl/min of cell culture HEK supernatant diluted 1:2 in sample buffer. The flow rate was set to 100 μl/min. The analyte was injected at different concentration steps of 0 nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM, 100 nM and 300 nM for 2 min. The dissociation was monitored for 5 min. Kinetic signatures were monitored and evaluated using the Biaevaluation Software and a binary Langmuir Fitting model with R.sub.MAX local. Acidic regeneration of the sensor surface was achieved using three consecutive injections of 10 mM Glycine pH 1.7 at 30 μl/min for 60 sec.
Example 6: Linear Epitope Mapping
[0172] Peptide based epitope mappings were carried out as described and commercially offered by Intavis, Cologne Germany, http://www.intavis.com) using the CelluSpots™ technology. Epitope mappings were carried out by means of a library of overlapping, immobilized peptide fragments (length: 15 amino acids) corresponding to the sequences of human CSF1R KID domain. Each peptide synthesized was shifted by one amino acid, i.e. it had 14 amino acids overlap with the previous and the following peptide, respectively. For preparation of the peptide arrays the Intavis CelluSpots™ technology was employed. In this approach, peptides are synthesized with an automated synthesizer (Intavis MultiPep RS) on modified cellulose disks which are dissolved after synthesis. The solutions of individual peptides covalently linked to macromolecular cellulose are then spotted onto coated microscope slides. The CelluSpots™ synthesis was carried out stepwise utilizing 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on amino-modified cellulose disks in a 384-well synthesis plate. In each coupling cycle, the corresponding amino acids were activated with a solution of DIC/HOBt in DMF. Between coupling steps un-reacted amino groups were capped with a mixture of acetic anhydride, diisopropylethyl amine and 1-hydroxybenzotriazole. Upon completion of the synthesis, the cellulose disks were transferred to a 96-well plate and treated with a mixture of trifluoroacetic acid (TFA), dichloromethane, triisoproylsilane (TIS) and water for side chain deprotection. After removal of the cleavage solution, the cellulose bound peptides are dissolved with a mixture of TFA, TFMSA, TIS and water, precipitated with diisopropyl ether and re-suspended in DMSO. The peptide solutions were subsequently spotted onto Intavis CelluSpots™ slides using an Intavis slide spotting robot.
[0173] For linear epitope analysis, the slides prepared as described above were treated using the BenchMark XT Automated Slide Preparation System (Ventana). The slides were developed with OptiView DAB IHC Detection Kit plus OptiView Amplification Kit (Ventana), according to the standardized protocols. Briefly described, the slides were “wet-loaded” in the system, and blocked for 32 minutes with 1% BSA in Phosphate Buffer (PBS). The antibody was diluted to 1 μg/ml in Antibody Diluent (Ventana) and applied manually on the slide for 1 h at room temperature; both Amplifier and Amplification Multimer reagents were incubated for 8 minutes each without counterstain staining. To analyze the colorimetric staining, a ChemiDoc Analyzer (BioRAD) was used.
[0174] When a high signal was desired, the slides were treated manually with chemiluminiscent reagents. Briefly, the slides were washed with ethanol and then with Tris-buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8) before blocking for 16 h at 4° C. with 5 mL 10× Western Blocking Reagent (Roche Applied Science), 2.5 g sucrose in TBS, 0.1% Tween 20. The slide was washed with TBS and 0.1% Tween 20 and incubated afterward with 1 μg/mL of the corresponding 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 slide was incubated with anti-rabbit/anti-mouse secondary HRP-antibody (1:20000 in TBS-T) followed by incubation with chemiluminescence substrate 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.
Immunohistochemistry
[0175] Immunohistochemistry was performed using the BenchMark XT Automated Slide Preparation System (Ventana). The slides were developed with iVIEW DAB Detection Kit (Ventana), according to the standardized protocols. Briefly described, the slides were deparafined with Cell Conditioning 1 (CC1, Ventana), and blocked for 32 minutes with 1% BSA in Phosphate Buffer (PBS). The antibody was diluted to 1 μg/ml in Antibody Diluent (Ventana) and applied manually on the slide for 1 h at room temperature.