HEPATITIS C VIRUS SPECIFIC ANTIBODY
20170313764 · 2017-11-02
Inventors
- Tim Beaumont (Amsterdam Zuidoost, NL)
- Sabrina Julia Louisa MERAT (Amsterdam Zuidoost, NL)
- Cornelia Johanna SCHINKEL (Amsterdam Zuidoost, NL)
- Richard MOLENKAMP (Amsterdam Zuidoost, NL)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to isolated, synthetic or recombinant antibodies and functional parts thereof specific for hepatitis C virus (HCV). The invention further relates to the use of such antibodies for diagnosis, treatment and prevention of HCV infection.
Claims
1. An antibody comprising: at least one of: a heavy chain CDR1 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-5 and SEQ ID NOs: 81-84; a heavy chain CDR2 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs:6-10 and SEQ ID NOs: 85-88; a heavy chain CDR3 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-15 and SEQ ID NOs: 89-92; a light chain CDR1 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-20 and SEQ ID NOs: 93-96; a light chain CDR2 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs:21-25 and SEQ ID NOs: 97-100; a light chain CDR3 sequence comprising a sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs:26-30 and SEQ ID NOs: 101-104; wherein said antibody is isolated, synthetic or recombinant.
2. The antibody of claim 1, having the sequence of: SEQ ID NOs 1, 6, 11, 16, 21 and 26; SEQ ID NOs 2, 7, 12, 17, 22 and 27; SEQ ID NOs 3, 8, 13, 18, 23 and 28; SEQ ID NOs 4, 9, 14, 19, 24 and 29; SEQ ID NOs 5, 10, 15, 20, 25 and 30; SEQ ID NOs 81, 85, 89, 93, 97 and 101; SEQ ID NOs 82, 86, 90, 94, 98 and 102; SEQ ID NOs 83, 87, 91, 95, 99 and 103; or SEQ ID NOs 84, 88, 92, 96, 100 and 104.
3. The antibody of claim 2, further comprising at least one of: a heavy chain variable region sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:31-35 and SEQ ID NOs 105-108; or a light chain variable region sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:36-40 and SEQ ID NOs: 109-112.
4. The antibody of claim 1, wherein said antibody specifically binds to an epitope of hepatitis C virus (HCV) protein E2, said epitope comprising amino acids corresponding to amino acids F442, Y527, W529, G530, D535 and W616 of the H77 E2 amino acid sequence, wherein said epitope inhibits binding of HCV protein E1E2 to CD81.
5. The antibody of claim 1, wherein said antibody competes with antibody AT12-011 for binding to HCV protein E2.
6. The antibody of claim 1, wherein said antibody competes with antibody AT12-007 for binding to HCV protein E2.
7. The antibody of claim 1, wherein said antibody competes with antibody AT12-009 for binding to HCV protein E2.
8. The antibody of claim 1, wherein said antibody competes with antibody AT12-010 for binding to HCV protein E2.
9. The antibody of claim 1, wherein said antibody competes with antibody AT13-021 for binding to HCV protein E2.
10. The antibody of claim 1, wherein said antibody competes with antibody AT15-009 for binding to HCV protein E2.
11. The antibody of claim 1, wherein said antibody competes with antibody AT15-011 for binding to a HCV E1E2 heterodimer.
12. The antibody of claim 1, wherein said antibody competes with antibody AT15-012 for binding to HCV protein E2.
13. The antibody of claim 1, wherein said antibody competes with antibody AT15-015 for binding to a HCV E1E2 heterodimer.
14. A nucleic acid molecule, comprising a sequence encoding at least one CDR sequence of the antibody of claim 1.
15. The nucleic acid molecule of claim 14, wherein said nucleic acid molecule encodes at least one of the heavy chain variable region sequence; or the light chain variable region sequence of the antibody of claim 1.
16. The nucleic acid molecule of claim 15, comprising at least one of: a heavy chain CDR1 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 41-45 and SEQ ID NOs 113-116; a heavy chain CDR2 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 46-50 and SEQ ID NOs 117-120; a heavy chain CDR3 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 51-55 and SEQ ID NOs 121-124; a light chain CDR1 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 56-60 and SEQ ID NOs 125-128; a light chain CDR2 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 61-65 and SEQ ID NOs 129-132; a light chain CDR3 sequence comprising a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 66-80 and SEQ ID NOs 133-136.
17. The nucleic acid molecule of claim 14, comprising a heavy chain variable region encoding sequence that has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs 71-75 and SEQ ID NOs 137-140.
18. The nucleic acid molecule of claim 14, comprising a light chain variable region encoding sequence that has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs 76-80 and SEQ ID NOs 141-144.
19. A vector comprising the nucleic acid molecule of claim 14.
20. A pharmaceutical composition comprising: an antibody of claim 1, a nucleic acid molecule of claim 14, or a vector of claim 19, and at least one of a pharmaceutically acceptable carrier, diluent and/or excipient.
21. An antibody of claim 1 for use in diagnosis of HCV infection.
22. A method for determining whether HCV is present in a sample comprising: contacting said sample with the antibody of claim 1, allowing said antibody to bind HCV or part thereof if present, and determining whether HCV or part thereof is bound to said antibody, whereby bound HCV indicates HCV is present.
23. The antibody of claim 1, the nucleic acid molecule of claim 14, or the vector of claim 19 for use as a medicament or prophylactic agent, for treating and/or preventing HCV infection, an HCV related disorder or a disorder caused by HCV infection.
24. An isolated or recombinant cell comprising the nucleic acid molecule of claim 14 or the vector of claim 19.
25. A method for determining whether an individual is suffering from a HCV infection, comprising: contacting a sample from said individual with an antibody of claim 1, and allowing said antibody to bind HCV, if present, and determining whether or not HCV is bound to said antibody, thereby determining whether or nor said individual is suffering from HCV infection.
26. Use of the antibody of claim 1 for determining whether a sample comprises HCV, HCV protein E2 or a HCV E1E2 heterodimer.
27. A method for producing the antibody of claim 1, the method comprising: providing a cell with a nucleic acid molecule or a vector of claim 14; allowing said cell to translate the nucleic acid sequence comprised by said nucleic acid molecule or vector, thereby producing said antibody of claim 1; harvesting, purifying and/or isolating said antibody of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0139]
[0140] The H77 sequence corresponds to Genbank accession number AAB67037 with three amino acid changes: R564C, V566A, and G650E.
[0141]
[0142] Cell lysates of 293T cells transfected with different E1E2 constructs were incubated (1:5 diluted) on GNA pre-coated plates before addition of antibodies (1 μg/mL). Next to a lysate of non-transfected 293T cells, the RSV-F protein specific mAb D25 was used as a negative control. On the Y-axis the mean optical density (OD450 nm) is depicted and the standard errors of the means (SEM) is included. The E1E2 sequences are indicated between brackets and the assay was performed in duplicate and repeated twice.
[0143]
[0144] The antibodies were added to E2-his6-ST pre-coated wells at 1 μg/mL. PBS treated and the RSV-F protein specific mAb D25 were used as negative controls. On the Y-axis the mean optical density (OD450 nm) is depicted and the standard errors of the means (SEM) is included. The assay was performed in duplicate and performed twice.
[0145]
[0146] Cell lysate containing H77 derived E1E2 was denatured with DTT and SDS and added (diluted 1:5) to GNA pre-coated plates before the antibodies were added at 1 μg/mL. Non-transfected cell lysate was used as a control for non-E1E2 specific binding and native E1E2 lysate as positive control for the binding of antibodies. The RSV-F protein specific mAb D25 was used as negative control. On the Y-axis the mean optical density (OD450 nm) is depicted and the standard errors of the means (SEM) is included. The assay was performed in duplicate and performed twice.
[0147]
[0148] E1E2 transfected 293T cells were pre-incubated with different concentrations of antibody before CD81-LEL was added. D25 was used as negative control. The y-axis indicates the percentage of CD81 binding inhibition and the errors bars represent one standard error of the mean (SEM). The assay was performed in duplicate and repeated in one separate experiment.
[0149]
[0150] Cell lysates of 293T/17 cells transfected with different E1E2 constructs were incubated (1:5 diluted) on GNA pre-coated plates before addition of B cell supernatant containing antibodies (0.2 μg/mL). Next to a lysate of non-transfected 293T/17 cells, the RSV-F protein specific mAb D25 was used as a negative control. On the Y-axis the mean optical density (OD450 nm) is depicted and the standard deviation (SD) is included. The E1E2 sequences are indicated between brackets and the assay was performed in duplicate.
[0151]
[0152] Supernatants from B cell cultures containing antibodies were added to E2-his6-ST pre-coated wells. PBS treated wells and the RSV-F protein specific mAb D25 were used as negative controls. On the Y-axis the mean OD450 nm is depicted and the SD is included. The assay was performed in duplicate.
[0153]
[0154] A cell lysate from 293T/17 cells transfected with H77 derived E1E2 was denatured with DTT and SDS and added (diluted 1:5) to GNA pre-coated plates before the B cell supernatants containing antibodies were added. Native E1E2 lysate was used as positive control for antibody binding. The RSV-F protein specific mAb D25 was used as negative control. On the Y-axis the mean OD450 nm is depicted and the SD is included. The assay was performed in duplicate.
[0155]
EXAMPLES
Example 1
Materials and Methods
Cells and Plasmids
[0156] 293T/17 and Huh-7 cells were maintained in Dulbecco's modified essential medium (DMEM, Invitrogen) supplemented with 8% fetal bovine serum (FBS). To generate HCV pseudotyped particles (HCVpp) we transfected 293T/17 cells with 3 plasmids: i) pcDNA3.1 or pcDNA3.3 vector (Invitrogen) expressing E1E2 of isolate H77 (Genbank accession no AAB67037, with three amino acid changes; R564C, V566A, and G650E, Albecka, 2011 Journal of virology) or patient derived E1E2 sequences ii) the phCMV vector containing gag/pol and iii) the phCMV vector containing the Luciferase gene.
Isolation of cDNA Encoding E1E2 from Patients
[0157] HCV RNA from patients infected with different genotypes of HCV was isolated from stored patient plasma using the Boom extraction method (Boom et al. 1990) and cDNA was synthesized by random hexamer priming. The region containing the C-terminal part of Core (the signal sequence for ER targeting of E1), E1 and E2 was amplified with specific primers. Subsequently, the polymerase chain reaction (PCR) product was cloned into pcDNA3.3 using the TOPO PCR cloning kit (Invitrogen). All sequences were confirmed by Sanger sequencing.
Cloning of Published E1E2 Sequences
[0158] E1E2 sequences for the isolates UKN3A 1.28 (Genbank accession no. AY734984), UKN4.11.1 (Genbank accession no. AY7349986), UKN6.5.340 (Genbank accession no. AY736194) and UKN5.15.7 (Genbank accession no. EF427672) (Lavillette, 2005, Hepatology; Johansson, 2007, PNAS) were synthetized by GeneArt (Invitrogen). Subsequently, they were cloned into the pcDNA3.1 vector (Invitrogen).
Production of Soluble E2-his6-Sortase Tag
[0159] The sequences of the E2 ectodomain (corresponding to residues 384-717 of the polyprotein) from isolate H77 (sequence in
[0160] Generation of Immortalized B Cells
[0161] Human memory B cells were immortalized using the AIMSelect technology (Kwakkenbos et al. 2010 and Kwakkenbos et al. 2013). In brief, human CD27+IgG+ memory B cells were isolated from peripheral blood mononuclear cells of donors who spontaneously cleared a HCV infection. After stimulation with CD40L and interleukin (IL)-21, the cells were transduced with a retroviral vector containing the transgenes BCL6, Bcl-xL and the marker gene GFP. Transduced B cells were maintained in culture with irradiated CD40 Ligand expressing L-cells and recombinant mouse IL-21. The transduced B cells resemble germinal center B cells. They are characterized by the surface expression of the immunoglobulin (the B Cell Receptor (BCR)) and secrete immunoglobulin into the culture supernatant. HCV specific B cells were discovered by directly staining and FACS sorting of fluorescently labeled E2 specific B cells and/or by screening antibodies present in supernatant of B cells cultured in serial dilution for binding to 293T cells expressing E1 and E2.
Isolation of Cross-Binding Antibodies
[0162] To isolate B cells secreting E1E2 specific antibodies, two approaches were used.
i) Bcl6 and Bcl-xL transduced polyclonal B cells were seeded at 100 cells per well and maintained in culture for 2 to 3 weeks. The supernatants of B cell cultures were screened for binding to 293T cells expressing E1E2 genotype 1a isolate H77 by flow cytometry. Cultures that showed reactivity to E1E2 were sorted single cell using the FACSAria instrument (BD) in order to obtain monoclonal B cell cultures. To retrieve the positive monoclonal B cell cultures, culture supernatants were screened after 3 weeks for binding to 293T cells E1E2 of H77 by flow cytometry. Positive supernatants were tested for binding to cells transfected with E1E2 from different genotypes by flow cytometry. Subsequently, the antibody variable heavy (VH) and light (VL) chain sequences from these B cell clones were obtained.
ii) Bcl6 and Bcl-xL total transduced B cells were incubated with the soluble E2-his6-sortase tag (ST) from isolate AMS.2b.20876551.kloon21 (genotype 2b). B cells recognizing E2-his6-ST (detected with an anti-his antibody) were sorted by FACSAria and maintained in culture for 2 weeks. These E2 sorted B cells were further enriched for HCV E2 specificity by another round of single cell sorting using soluble E2-his6-ST from isolate H77 (genotype 1a). The latter sorting strategy results in selection of B cells recognizing E2 from genotypes 1 and 2. The sorted B cells were maintained in culture for 3 weeks before the culture supernatants were screened for E2-his6-ST binding by enzyme-linked immunosorbent assay (ELISA). Supernatants that showed reactivity were tested for binding to E1E2 cell lysates by ELISA and HCVpp neutralization. Finally, the VH and VL sequences from B cell clones that showed persistent broad reactivity were obtained.
Flow Cytometry
[0163] To test the binding of the B cell supernatants to the HCV envelope glycoproteins E1 and E2 or E2 only, 293T cells were transfected with an E1E2 expression plasmid using X-tremeGENE 9 (Roche). Two days after transfection, the cells were fixed with 4% paraformaldehyde (PFA) and frozen in FBS+10% DMSO at −80° C. Immediately after thawing, the transfected cells were permeabilized with Perm/wash Buffer (BD) for 15 min at 4° C. before being incubated with IgG containing B cell supernatants for 30 min at 4° C. After three wash steps with perm/wash buffer the cells were incubated with goat anti human IgG-PE (Southern biotech) for 30 min at 4° C. After washing, the cells were re-suspended in 4% PFA solution. Fluorescence was measured using Guava EasyCyte (Millipore). Non-transfected GFP expressing 293T/17 cells were used as control for non-E1E2 specific binding and unstained cells were used as negative control.
[0164] To determine whether the antibodies interfere with binding of CD81 to E1E2; E1E2 transfected cells were pre-incubated with the antibodies before the large extracellular loop (LEL) of CD81 (Sino Biological; 0.1 μg/mL) was added. Since the CD81 peptide is expressed on a mouse Fc tail, intracellular binding of CD81 was detected using an alexa647 conjugated anti-mouse Fc antibody (Jackson ImmunoResearch). After washing, the cells were re-suspended in 4% PFA solution. Fluorescence was measured using the FACS canto (BD Biosciences). The level of inhibition are calculated by dividing the percentage of Alexa 647 positive cells from each well by the mean percentage of Alexa 647 positive cells from wells where the cells were incubated without antibody. D25, a Respiratory Syncytial Virus F protein specific antibody was used as negative control.
E2 Specific Cell Sorting
[0165] To sort E2 specific B cells, cells were incubated with soluble E2-his (1 μg/mL) for 1 hour at 4° C. The cells were washed three times with Iscove's Modified Dulbecco's Media (IMDM) containing 8% FBS (wash buffer) before being incubated with anti his antibody Alexa 647 (Qiagen) for 30 min at 4° C. After washing the cells three times with wash buffer, the fluorescence was measured and the positive cells were sorted using FACSAria (BD). Cells from a HCV naive patient was used as negative control.
ELISA
[0166] To test the binding of antibodies and antibodies in B cell supernatants to soluble E2, plates were coated with E2-his6-ST (5 μg/mL) overnight at 4° C. After washing with Phosphate buffered saline (PBS), the plates were blocked with 1% fish skin gelatin (Sigma) for 1 hour at room temperature. After incubation the plates were washed 5 times with PBS containing 0.05% tween (PBS-T). To detect bound antibodies, the plates were incubated with HRP conjugated anti-human IgG (Jackson) for 1 hour at room temperature. After washing, bound antibodies were detected using 3,3′, 5,5′ tetramethyl benzidine (TMB) and the reaction was stopped using H.sub.2SO.sub.4. Optical density (OD) at 450 nm was measured with an EnVision Multilabel Reader (PerkinElmer). PBS coated wells were used as control for non-specific binding and D25 an RSV-F specific antibody was used as negative control.
[0167] To study antibody specificity across different HCV genotypes, 293T/17 cells were transfected with E1E2 expression plasmid using XtremeGENE 9 (Roche). Two days after transfection, the cells were lysed with 1% triton (Sigma). The cell lysate was clarified and frozen at −80° C. To prepare the ELISA plate, G. nivalis (GNA) lectin (Sigma) was coated at 5 μg/mL and after washing with PBS, the plate was blocked with 1% fish skin gelatin (Sigma) for 1 hour at room temperature. The plate was emptied before the E1E2 containing cell lysate (1:5 diluted in blocking buffer) was added for 1 hour at room temperature. After 5 washes with PBS-T, the antibody solutions or antibodies from B-cell supernatant were incubated for 1 hour at room temperature. The plate was washed 5 times with PBS-T and HRP-conjugated anti-human IgG antibody (Jackson) was incubated for 1 hour at room temperature. After washing, bound antibodies were detected using TMB substrate buffer and the reaction was stopped using H.sub.2SO.sub.4. OD450 nm was measured using an EnVision® Multilabel Reader (PerkinElmer). Lysate from non-transfected 293T/17 cells was used as control for non-E1E2 specific binding and D25 was used as negative control.
[0168] To determine if antibodies recognize continuous or discontinuous epitopes, E1 and E2 were denatured by incubating E1E2 cell lysate with 0.4% sodium dodecyl sulfate and 20 mM dithiotreitol at 75° C. After 15 min, the lysate was diluted 5 times before being added to GNA lectin coated wells. The subsequent steps of the assay were performed in a similar fashion as described above.
[0169] To determine which amino acids of E2 are crucial for antibody binding, E1E2 sequences of HCV isolate H77 were synthetized with single alanine mutations. The E2 mutants were designated X123Y, where 123 is the residue position, X indicates the amino acid in H77 sequence and Y indicates the replacing amino acid. For the positions N415, S424, L441, F442, Y443, Y485, Y527, W529, G530, D535 and W616, E1E2 sequences of HCV isolate H77 were synthetized with single alanine mutations by GeneArt (Invitrogen) and cloned into pcDNA3.1. For the positions T435, G436, A439, T526, R657 and D698, single alanine mutations were introduced in the E1E2 H77 sequence using primers coding the specific mutation (Biolegio) and by use of the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent) according the manufacturer's protocol. Subsequently, the PCR product was cloned into pcDNA3.3 using the TOPO PCR cloning kit (Invitrogen). All sequences were confirmed by Sanger sequencing. If the residue to change was an alanine, it was substituted by a glycine.
[0170] The 1:5 diluted lysate of 293T cells transfected with the different E1E2 alanine mutants, were added to GNA lectin coated plates before antibodies were added in a dilution range of 1000 to 0.2 ng/mL. The lysate preparation and ELISA were performed in a similar fashion as described before. Antibody binding to E1E2 alanine mutants was compared to the wild type E1E2. Antibody concentration to achieve 50% binding (EC50) in μg/mL was determined using non-linear regression analysis (Prism software). The relative binding of an antibody was calculated by dividing the EC50 obtained against the wild type vs the alanine protein mutant. The assay was performed in duplicate and repeated at least once.
Cloning and Production of Selected Antibodies
[0171] To obtain antibody VH and VL chain sequences total RNA was isolated with TriPure RNA extraction buffer (Roche) and cDNA was generated using SuperScript III reverse transcriptase (Invitrogen). The antibody VH and VL regions were amplified by PCR and sequences analyzed using the cognate forward and reverse primers. Subsequently the variable regions were cloned in frame between a secretion leader sequence and the human IgG1 and kappa constant regions into a pcDNA3.1 (Invitrogen) based vector. To produce recombinant antibodies the VH and VL vectors were transiently transfected into 293T/17 cells. Recombinant antibodies were purified from culture supernatant using a MabSelect Sure column on an AKTA Explorer 10s (GE healthcare).
HCV Neutralization Assay
[0172] To study the neutralization capacity of the newly discovered antibodies, HCVpp was produced in 293T/17 cells by co-transfecting plasmids (vector containing E1E2 sequence, phCMV-gag/pol and phCMV-Luciferase) in ratio 1:2:2. After 2 days, the culture supernatants containing HCVpp were harvested and incubated with serial antibody dilutions (from 50 μg/mL to 0.0008 μg/mL) for 1 hour at 37° C. The antibody/HCVpp mixture was added to Huh-7 cells and spin-inoculated for 45 minutes at 2000 g. After 3 days, the huh-7 cells were lysed using Bright Glo luciferase kit (Promega). The luciferase activity was measured using an EnVision Multilabel Reader (PerkinElmer).
[0173] To determine the background of the assay, 293T cells were transfected only with phCMV-gag/pol and phCMV-Luciferase. HCVpp supernatants that generated luciferase signals 10 times higher compared to the background were used. Of the relative light value of each well, the background was first subtracted. Then the percentage of neutralization was calculated by dividing the relative light units of each well by the mean of relative light units from wells incubated with HCVpp supernatant only. Antibody concentrations to achieve 50% neutralization (IC50) and antibody concentration to achieve 90% neutralization (IC90) in μg/mL were determined using nonlinear regression analysis. VSV-G (vesicular stomatitis virus G protein) pp was used as a positive control and D25 was used as a negative control. The assay was performed in triplicate.
SPR Analysis
[0174] Surface plasma resonance (SPR) analysis was performed on an IBIS MX96 SPR imaging system (IBIS Technologies BV) as described (Lokate et al. 2007). In short, one SPR analysis cycle consists of one (or more) injection steps in which analytes are flushed over a sensor that is coated with different ligands (i.e. antibodies). This is followed by a regeneration step in which any bound analyte is removed from the sensor. Multiple cycles can be performed in one experiment. Data was analyzed using Sprint software (version 1.6.8.0, IBIS Technologies BV).
[0175] To determine the affinity of the antibodies for ectodomain of HCV envelope glycoprotein E2, the SPR sensor was coated with anti-HCV antibodies on an amine-specific EasySpot gold-film gel-type SPR-chip (Ssens BV) by spotting them on the sensor surface using a continuous flow microspotter device (Wasatch Microfluidics) in coupling buffer (10 mM MES-NaOH, pH 4.5+0.05% Tween20). After spotting for 40 minutes the sensor was deactivated with 0.1 M ethanolamine, pH 8.5 and washed three times with system buffer (PBS+0.05% Tween20+0.05% NaN.sub.3). Before starting the analysis, the coupled sensor was briefly washed with regeneration buffer (10 mM glycine-HCl, pH 2), followed by three wash steps with system buffer. Then, the sensor was injected with a concentration series of soluble HCV E2-his (isolate H77 or isolate AMS.2b.20876551.kloon21) ranging from 0.1 to 1.6 μg/ml, diluted in system buffer and incubated for 10 minutes to measure binding kinetics. To measure complex dissociation the sensor was washed with system buffer and incubated for 15 minutes. K.sub.D was calculated as kd/ka. The assay was performed in triplicate and repeated in one separate experiment.
[0176] To determine if the antibodies recognize an identical region on E2, SPR assay was performed in a similar fashion as described above. After the injection of soluble E2-his6-ST (isolate H77), the chip was incubated with another antibody for 15 min to measure association, briefly washed with system buffer to remove unbound E2 and then incubated for another 15 min to measure complex dissociation.
Results
Isolation of E1E2 Specific Monoclonal Antibodies
[0177] PBMC (70E6 cells) were obtained from a donor who spontaneously cleared a HCV infection. CD27+IgG+ memory B cells were isolated (8.4E5 cells) and immortalized using the AIMSelect technology. Immortalized B cells secrete antibodies and express surface immunoglobulin.
[0178] To isolate B cells secreting E1E2 specific antibodies, two approaches were used. The first approach was based on screening of B cell supernatants for binding to E1E2 transfected cells. Immortalized polyclonal B cells (7.7E05 cells) were seeded at 100 cells per well and tested for binding to 293T cells expressing H77 derived E1E2 by flow cytometry, Eleven B cell cultures were selected for single cell sorting to obtain monoclonal B cell cultures. Monoclonal B cell cultures that again recognized H77 derived E1E2 expressed in 293T cells were isolated from 6 cultures and sequenced. Subsequently, the binding of these monoclonal B cell cultures to a panel of E1E2 derived from different genotypes 1a (H77), 1b (AMS.1b.20877857), 2b (AMS.2b.20876551.kloon21), 3a (UKN3A 1.28 and AMS.3a.21071213), 4 (UKN4.11.1), 4d (AMS.4d.20875969) was tested by ELISA. Two of the antibodies showed binding only to E1E2 derived from genotype 1a whereas the four other supernatants showed binding to E1E2 derived from genotypes 1, 2, 3 and 4. In total four different cross-genotype HCV E1E2 antibodies were isolated using this strategy.
[0179] In parallel, B cells directly recognizing fluorescent labelled E2 (AMS.2b.20876551.kloon21, genotype 2b) were sorted, cultured and in a second round of sorting, cells were retrieved that recognized soluble E2-his from isolate H77 (genotype 1a) in order to get for B cells specific for at least genotype1 and 2. Culture supernatants of these selected B cells (approximately 672 cells) were screened for E2 binding by ELISA and were tested for binding to E1E2 cell lysates of different genotypes by ELISA and for neutralization potency. VH VL sequencing of several clones revealed 2 distinct sequences. One antibody sequence was unique compared to the sequences obtained from the first screening strategy.
[0180] Together, the two screenings methods resulted in the selection of 5 unique E2 specific clones that show broad binding and neutralization.
[0181] The variable regions VH and VL of the five cross-genotype antibodies were cloned into a vector containing the human IgG1 and kappa constant regions to produce recombinant antibodies.
Binding Properties
[0182] To study binding properties of the selected antibodies AT12-007, AT12-009, AT12-010, AT12-011 and AT13-021, the recombinant antibodies were first tested for binding at 1 μg/mL to a panel of E1E2 derived from six different genotypes 1a (H77), 1b (AMS.1b.20877857), 2b (AMS.2b.20876551.kloon21), 3a (UKN3A 1.28 and AMS.3a.21071213), 4 (UKN4.11.1), 4d (AMS.4d.20875969), 5 (UKN5.15.7) and 6 (UKN6.5.340) by ELISA. An antibody was considered specific for E1E2 when the optical density (OD) at 450 nm was three times the standard deviation compared to the background OD on non-transfected cell lysate. All recombinant antibodies showed binding to the E1E2 from all genotypes tested (
[0183] Finally, as shown in Table 2, we determined the affinity (KD) of the antibodies for E2 from isolate H77 (genotype 1a) and isolate AMS.2b.20876551.kloon21 (genotype 2b). Of all antibodies tested AT12-011 showed the highest affinity for E2, namely 0.3 nM for genotype 1 and 0.2 nM for genotype 2b. AT12-009 bound E2 genotype 1 and E2 genotype 2b with similar affinities of 1.3 nM and 2.3 nM, respectively. AT12-010 bound E2 genotype 1 and E2 genotype 2b at 39.3 nM and 22.8 nM, respectively. In contrast to AT12-009 and AT12-011, AT12-007 and AT13-021 showed enhanced binding to E2 genotype 2 (3.8 nM and 2 nM for AT12-007 and AT13-021, respectively) compared to binding of AT12-007 (44 nM) and AT13-021 (14 nM) to E2 from genotype 1.
TABLE-US-00002 TABLE 2 Affinity of the antibodies for E2. The binding kinetics of the antibodies were measured for E2 from isolate H77 (genotype 1a) and isolate AMS.2b.20876551.clone21 (genotype 2b) by direct SPR. Means of KD are shown (+/− variance). E2 genotype 1a E2 genotype 2b Antibody K.sub.a (10.sup.−5 s.sup.−1 M.sup.−1) K.sub.d (10.sup.−5 S.sup.−1) K.sub.D (nM) K.sub.a (10.sup.−5 s.sup.−1 M.sup.−1) K.sub.d (10.sup.−5 s.sup.−1) K.sub.D (nM) AT12-007 4.6 (±0.9) 184 (±31) 44 (±16) 5.0 (±0.3) 19 (±0.6) 3.8 (±0.1) AT12-009 8.6 (±0.8) 11 (±1.6) 1.3 (±0.3) 8.9 (±1.6) 20 (±1.5) 2.3 (±0.5) AT12-010 3.6 (±0.5) 142 (±43) 39.3 (±6.4) 4.6 (±1.2) 98 (±4.2) 22.8 (±4.2) AT12-011 13.6 (±3.9) 3.9 (±1.7) 0.3 (±0.1) 8.8 (±3.4) 1.1 (±0.5) 0.2 (±0.1) AT13-021 2.9 (±0.6) 36 (±8.9) 14.2 (±6.2) 5.3 (±0.4) 10 (±0.3) 2.0 (±0.1)
Epitope Mapping
[0184] To identify the region of E2 recognized by the antibodies, SPR competition experiments were performed using E2-his from isolate H77. After the primary antibody was immobilized on chip and bound to E2, the secondary antibody was injected. When no signal was obtained with the secondary antibody this suggested that both antibodies recognized an identical epitope or at least an epitope in close proximity. As shown in table 3, AT12-007, AT12-009, AT12-010 and AT13-021 compete for binding. In contrast, AT12-011 did not compete for binding with any other antibody, which indicates that AT12-011 binds a different non-linear domain on E2 than antibodies AT12-007, AT12-009, AT12-010 and AT13-021.
[0185] To determine which amino acid residues in E2 are important for binding of our antibody panel, we generated a panel of H77 E2 alanine mutants, which have been shown to affect binding of known neutralizing antibodies. Antibody binding was determined by ELISA using E1E2 transfected 293T cell lysates. Table 4 presents the ratio of antibody binding to E2 mutants compared the binding to the wild-type protein. As could be expected from the competition results (table 3), AT12-007, AT12-009, AT12-010 and AT13-021 share an antigen binding domain on E2 that is at least composed of the residues F442A, Y527A, W529A, G530A, D535A, W616A since mutation of these residues resulted in ≦25% binding. Alanine substitutions of the residues S424A, T435A, G436A, L441A, Y443A and T526A, decreased (sometimes partially) binding of some or all antibodies. However, none of the alanine mutations affected binding of AT12-011. Suggesting that the binding domain of AT12-011 is completely different compared to the antibodies AR3, AR4A, AR5A, HC-1, HC-11, CBH-2, CBH-5, HC84, e20 and e137 as described in literature (Law et al. Nature Medicine, 2007; Giang et al., PNAS, 2012: Owsianka et al, Journal of general virology, 2008; Keck et al, Journal of virology, 2011: Keck et al, Plos pathogens, 2012; Perotti et al, Journal of virology, 2008; Mancini et al, plos one, 2009).
TABLE-US-00003 TABLE 4 Epitope mapping of the antibodies. The binding of antibodies to E2 alanine-mutants was tested by ELISA. Shown is the relative antibody binding to the mutant sequences compared the wild type sequence. When the EC50 could not be calculated because of very weak binding this is indicated by <10%. AT12-007 AT12-009 AT12-010 AT12-011 AT13-021 N415A 115% 104% 117% 82% 119% 5424A 34% 25% 16% 114% 17% T435A 84% 85% 47% 118% 21% G436A 35% 51% <10% 103% <10% A439G 67% 71% 45% 108% 53% L441A <10% 32% <10% 110% 7% F442A <10% 18% <10% 107% 9% Y443A <10% 64% <10% 105% 87% Y485A 114% 111% 107% 98% 114% T526A 31% 35% 29% 116% 27% Y527A 24% 23% 19% 121% 21% W529A <10% 18% <10% 120% 13% G530A <10% 6% <10% 108% <10% D535A 15% 5% 5% 118% <10% W616A <10% 20% <10% 125% <10% R657A 116% 104% 99% 100% 88% D698A 103% 98% 86% 87% 80%
Antibody Neutralization of HCVpp
[0186] To study neutralizing capacity of the antibodies, HCVpp from isolates H77 (gt 1a), AMS.1b.20877857 (gt 1b), AMS.2b.20876551.kloon21 (gt 2b), AMS.3a.21071213 (gt 3a), UKN4.11.1 (gt 4) and AMS.4d.20875969 (gt4d) were generated. The supernatants containing HCVpp were incubated with antibodies ranging from 50 μg/mL to 0.0008 μg/mL before being added on Huh-7 cells. 50% and 90% inhibitory concentrations (IC) neutralization values are shown in Table 5. Potency of the antibodies varied between genotypes. AT12-009 and AT13-021 neutralized all HCVpp within an IC50 range of 1 to 940 ng/ml, while AT12-007, AT12-010 and AT12-011 showed a more restricted neutralization. AT12-007 and AT12-010 neutralized all HCVpp tested except genotype 4d or 4a and 4d respectively. AT12-011 which showed the highest affinity of all antibodies neutralized HCVpp from genotype 1a, 1b and 2b.
TABLE-US-00004 TABLE 5 Neutralization of HCVpp expressing different genotypes. Antibody IC50 and IC90 inhibitory concentrations in μg/mL. AT12-007 AT12-009 AT12-010 AT12-011 AT13-021 Isolate Genotype IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 H77 1a 2.67 >50 0.07 7.85 1.06 >50 4.41 >50 0.30 9.98 AMS.1b.20877857.kloon2 1b 0.04 0.50 0.001 0.13 0.47 8.51 4.31 >50 0.09 5.67 AMS.2b.20876551.kloon21 2b 0.26 9.41 0.06 6.17 0.61 20.4 27.5 >50 0.12 8.84 AMS.3a.21071213.kloon26 3a 1.42 21.74 0.14 5.34 1.41 >50 >50 >50 0.25 12.3 UKN4 11.1 4a 1.87 >50 0.19 14.1 >50 >50 >50 >50 0.94 13.9 AMS.4d.20875969.kloon8 4d >50 >50 0.72 47.4 >50 >50 >50 >50 0.94 >50
Antibody Inhibition of CD81 Binding to E1E2 Protein
[0187] HCV E1E2 interacts with the Large Extracellular Loop (LEL) of CD81 on the target cell. To determine if the antibodies interfere with CD81 binding, we used a CD81 competition assay by flow cytometry. E1E2 transfected cells were pre-incubated with different concentrations of antibody before CD81-LEL was added and detected. The reduction in CD81 binding by the antibodies ranged from 10 μg/mL to 0.03 μg/mL (
Example 2
Materials and Methods
Cells and Plasmids
[0188] Freestyle™ 293-F cells were maintained in Freestyle Medium (Invitrogen) whereas 293T/17 and Huh-7 cells were maintained in Dulbecco's modified essential medium (DMEM, Invitrogen) supplemented with 8% fetal bovine serum (FBS). To generate HCV pseudotyped particles (HCVpp) we transfected 293T/17 cells with 3 plasmids: i) pcDNA3.1 or pcDNA3.3 vector (Invitrogen) expressing E1E2 of isolate H77 (Genbank accession no AAB67037, with three amino acid changes; R564C, V566A, and G650E, Albecka, 2011 Journal of virology) or patient derived E1E2 sequences ii) the phCMV vector containing gag/pol and iii) the phCMV vector containing the Luciferase gene. E1E2 sequences for the isolates UKN4.11.1 (Genbank accession no. AY7349986), UKN6.5.340 (Genbank accession no. AY736194) and UKN5.15.7 (Genbank accession no. EF427672) (Lavillette, 2005, Hepatology; Johansson, 2007, PNAS) were made as described in example 1 in paragraph “Cloning of published E1E2 sequences”. pcDNA3.3 E1E2 AMS.2b.20876551.kloon21 and pcDNA3.3 E1E2 AMS.3a.21071213 were made as described in Example 1 in paragraph “isolation of cDNA encoding E1E2 from patients”.
Production of Soluble E2-his6-Sortase Tag
[0189] Production of soluble E2-His6-sortase tag was performed as described in Example 1.
Generation of Immortalized B Cells
[0190] B cells were immortalized in a similar fashion as described in Example 1 The transduced B cells resemble germinal center B cells. They are characterized by the surface expression of the immunoglobulin (the B Cell Receptor (BCR)) and secrete immunoglobulin into the culture supernatant. HCV specific B cells were discovered by screening antibodies present in supernatant of B cells for HCVpp neutralization.
Isolation of HCV Neutralizing Antibodies
[0191] Immortalized B cells from donor 130 were seeded 1 cell per well using the FACS Aria III (BD). After 3 weeks of culture, B cell supernatants were tested for neutralization of HCVpp from isolate H77 (genotype 1a). B cell supernatants neutralizing 50% of HCVpp infection in two independent experiments were selected for further experiments. In order to determine the antibody binding epitope antibodies were tested in an ELISA using a cell lysate of Freestyle™ 293-F cells transfected with 12 different E1E2 alanine mutants. In addition, antibody titrations of B cell supernatants were tested for neutralization of HCVpp from isolate H77 (genotype 1a) in order to estimate the potency of the antibodies. Neutralizing B cell cultures and/or antibodies with unique binding properties were selected for sequencing and further experiments.
IgG ELISA
[0192] The concentration of antibodies in the B cell supernatant was determined using ELISA. After coating overnight with 5 μg/mL anti human IgG (Jackson), plates were blocked with 1% Fish skin gelatin. After at least 1 hour blocking, titration of purified antibodies or standard antibody (Jackson) were added. HRP conjugated anti-human IgG (Jackson) was used to detect human IgG antibodies. After washing, bound antibodies were detected using TMB and the reaction was stopped using H.sub.2SO.sub.4. OD at 450 nm was measured with an EnVision Multilabel Reader (PerkinElmer). Antibody concentrations were determined using nonlinear regression analysis. The mean of antibody concentration from three independent experiments was used.
ELISA
[0193] ELISA assays were performed in a similar fashion as described in Example 1. Cell lysates containing E1E2 alanine mutants were produced in a similar fashion as described in Example 1 with the following modifications. E1E2 sequences of HCV isolate H77 were synthetized with single alanine mutations. The E1 E2 mutants were designated XabcY, where abc is the residue position, X indicates the amino acid in H77 sequence and Y indicates the replacing amino acid. For the positions N415, S424, L441, F442, Y443, Y485, Y527, W529, G530, D535 and W616, E1E2 sequences of HCV isolate H77 were synthetized with single alanine mutations by GeneArt (Invitrogen) and cloned into pcDNA3.1. For the positions N209A, N325A, L413, G418, W420, N423, G436, N448, T526, Y527, N532, D533, T534, V538, P612, P664, P676, T435, G436, A439, T526, R657 and D698, single alanine mutations were introduced in the E1E2 H77 sequence using primers coding the specific mutation (Biolegio) and by use of the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent) according the manufacturer's protocol. Subsequently, the PCR product was cloned into pcDNA3.3 using the TOPO PCR cloning kit (Invitrogen). All sequences were confirmed by Sanger sequencing. If the residue to change was an alanine, it was substituted by a glycine. Freestyle™ 293-F cells were transfected with E1E2 expression plasmid using Polyethylenimine (Polysciences). Two days after transfection, the cells were lysed with 1% triton (Sigma). The cell lysate was clarified and frozen at −80° C.
HCV Neutralization Assay
[0194] HCV neutralization assays were performed in a similar fashion as described in Example 1 with the following modifications. The culture supernatants containing HCVpp were incubated with serial antibody dilutions (from 10 gig/mL to 0.04 μg/mL) or 3 fold serial dilution of B cell supernatants with starting concentration varying between 4 and 1 μg/mL for 1 hour at 37° C. The assay was performed at least in duplicates.
SPR Analysis
[0195] SPR affinity measurements were performed in a similar fashion as described in Example 1 with the following major modification. B cell supernatants (containing anti-HCV antibodies) or purified antibody controls are captured on a Gel-type anti-human-IgG-Fc SensEye SPR-chip (Ssens BV) and they were not as described in Example 1 spotted directly on the chip. Prior to capture, B cell supernatants are 1:1 diluted in system buffer (PBS+0.05% Tween20+0.02% sodium azide). After capture has completed, antibody—anti-human-IgG complexes are cross-linked using a Fix-It capture-crosslink kit (Ssens BV).
Results
Isolation of Neutralizing Antibodies
[0196] From donor 130, 40,000 memory IgG+CD27+ B cells were isolated and 24,000 cells were immortalized using the AIMSelect™ technology (as described in the Materials&Methods of Examples 1 and 2). Five thousand immortalized B cells from donor 130 were seeded 1 cell per well. After 2 separate HCVpp neutralization assays (isolate H77), 66 B cell supernatants that could neutralize HCV H77≧50% were selected and further tested in ELISA using cell lysates of E1E2 alanine mutants and the potency of these supernatants was retested in a HCVpp H77 neutralization assay. Thirteen monoclonal B cell cultures originally showed equal to better neutralization compared to AT12-009. When studying the binding pattern on the E1E2 alanine mutants, we found 6 clones that were not affected by any of the mutations and a relatively large panel that was epitope II binding only. By sequencing of these cultures, 4 different antibody sequences (AT15-009, AT15-011, AT15-012 and AT15-015) were identified. The heavy and light chain CDR and variable region sequences of these antibodies are depicted in Table 1.
Binding Properties
[0197] To study binding properties of the selected antibodies AT15-009, AT15-011, AT15-012 and AT15-015, B cell culture supernatant containing antibodies were first tested for binding to E1E2 from six different genotypes: 1a (H77), 2b (AMS.2b.20876551.kloon21), 3a (AMS.3a.21071213), 4 (UKN4.11.1), 4d (AMS.4d.20875969), 5 (UKN5.15.7) and 6 (UKN6.5.340) by ELISA at 0.2 μg/mL. An antibody was considered specific for E1E2 when the OD at 450 nm was three times the background compared to the OD of a lysate of non-transfected 293T/17 cells. AT15-012 recognized E1E2 from all genotypes (
[0198] We determined the affinity (K.sub.D) of the antibodies for E2 from isolate H77 (genotype 1a)(Table 6). AT15-012 showed the highest affinity for E2, namely 0.015 nM whereas we measured an affinity of 0.242 nM for AT15-009. As can be seen for antibodies AT12-009 and AT12-011, the K.sub.D values measured in this Example differ from the K.sub.D values measured in Example 1. This is due to differences in the experimental set up between the two Examples. In Example 1, purified antibodies are directly immobilized on the chip; in this experiment, non-purified antibodies are captured, directly from B cell supernatant, on an anti-human-IgG-Fc coated chip and then immobilized. In the latter setup, all antibodies are immobilized in the same orientation, and possibly with a higher antibody density. Antibody orientation and density influence the observed binding kinetics (Schasfoort et al., 2012), and this could be the cause for deviations in observed affinity between Example 1 and Example 2.
TABLE-US-00005 TABLE 6 Affinity of the antibodies AT15-009 and AT15-012 for E2. The binding kinetics of the antibodies was measured for E2 from isolate H77 (genotype 1a) by SPR. The K.sub.D value is the mean of K.sub.D from two independent experiments. KD (nM) Antibody KD (nM) Example 1 AT12-009 0.05 1.3 AT12-011 0.015 0.3 AT15-009 0.242 AT15-012 0.015
[0199] Since AT15-011 and AT15-015 do not bind soluble E2, their affinity could not be determined by SPR using soluble E2. We measured the binding of AT15-011 and AT15-015 to a lysate of 293T/17 cells transfected with E1E2 from H77 (genotype 1a) by ELISA. E1E2 was captured on a GNA lectin coated plate and antibodies were added (0.5 μg/mL to 0.00001 μg/mL). The 50% effective concentration (EC) was determined using non-linear regression analysis (Table 7). AT15-015 showed the highest binding for E1E2, namely 0.0009 gig/mL whereas we determined an EC50 of 0.0019 μg/mL for AT15-011.
TABLE-US-00006 TABLE 7 EC50 of the antibodies AT15-011 and AT15-015 for E1E2. The binding of the antibodies was measured using 293T/17 cell lysate containing E1E2 from isolate H77 (genotype 1a) ELISA. The assay was performed by in duplicate. EC50 (μg/mL) AT15-011 AT15-015 0.0019 0.0009
Neutralization Activity
[0200] To study the neutralizing capacity of the antibodies, HCVpp from isolates H77 (genotype 1a), AMS.3a.21071213 (genotype 3a), UKN4.11.1 (genotype 4) and AMS.4d.20875969 (genotype 4d) were generated. The B cell culture supernatants containing HCVpp were incubated with a dilution range of B cell supernatants before being added on Huh-7 cells. 50% inhibitory concentrations (IC) neutralization values are shown in Table 8. Potency of the antibodies varied between genotypes. AT15-009 and AT15-012 neutralized all HCVpp (genotype 1, 3 and 4) within an IC50 range of 1 to 0.1 μg/mL, while AT15-011 and AT15-015 showed a more restricted neutralization. AT15-011 and AT15-015 neutralized all HCVpp tested except genotype 4a and 4d using concentrations lower than 1.5 μg/mL and 1.65 μg/mL.
TABLE-US-00007 TABLE 8 Neutralization of HCVpp expressing different genotypes by the antibodies. Antibody IC50 in μg/mL. Isolate Genotype AT15-009 AT15-011 AT15-012 AT15-015 H77 1a 0.14 0.19 0.26 0.41 AMS.3a.21071213.kloon26 3a 0.71 1.48 0.90 1.65 UKN4 11.1 4a 1.33 >1.5 1.66 >1.65 AMS.4d. 20875969. kloon8 4d 2.26 >1.5 1.16 >1.65
Epitope Mapping
[0201] To determine which E2 or E1 amino acid residues are important for antibody binding, we generated a panel of H77 E2 alanine mutants, which have been shown to affect binding of known neutralizing antibodies as for instance HC-1, HC-11, CBH-2, CBH-5, e20, e137, HC33 antibodies, AR3 antibodies, AR4A, AR5A and HC84 antibodies family (Keck et al, Journal of virology, 2012; Keck et al, Journal of virology, 2011; Owsianka et al, Journal of general virology, 2008; Perotti et al, Journal of virology, 2008; Mancini et al, plos one, 2009; Law et al. Nature Medicine, 2007; Giang et al., PNAS, 2012, Keck et al, Plos pathogens, 2012). E1 alanine mutants were also generated. Antibody binding was determined by ELISA using a lysate of E1E2 transfected Freestyle™ 293-F cells. The data in Table 9 represents the ratio of antibody binding to E1 or E2 mutants compared to the binding of wild-type protein.
[0202] AT15-011 and AT15-015 share an antigen-binding domain on the E2 stem region that is at least composed of the residues R657A and D698A since mutation of these residues resulted in ≦25% binding. Although these residues are present in the soluble E2 protein, AT15-011 and AT15-015 do not bind soluble E2 suggesting that the antibody epitope comprises more residues in E2 and/or include residues in E1 and that the antibody epitope is present after the formation of the E1E2 complex. AT15-009 binding to E2 was (partially) decreased when amino acids F442A and W616A (≦50% binding) were mutated. For AT15-012, we could determine that the antigen binding domain is at least composed of G530 since mutation of this residue resulted in ≦50% binding.
TABLE-US-00008 TABLE 9 Epitope mapping of the antibodies. The binding of antibodies to E1 or E2 alanine-mutants was tested by ELISA. N209A and N325A are E1 alanine-mutants (the E1 sequence from HCV genotype 1a, strain H77 is shown in Figure 9). Shown is the relative antibody binding to the mutant sequences compared to the wild type sequence. When the EC50 could not be calculated because of very weak binding this is indicated by <10%. The assay was performed in duplicate. AT15-009 AT15-011 AT15-012 AT15-015 N209A 105% 70% 99% 59% N325A 103% 127% 94% 81% L413A 112% 171% 132% 166% N415A 116% 98% 101% 103% G418A 112% 148% 124% 124% W420A 102% 243% 111% 237% N423A 111% 158% 132% 125% S424A 106% 88% 67% 91% T435A 101% 169% 99% 136% G436A 93% 132% 91% 92% A439G 97% 137% 94% 122% L441A 57% 91% 83% 108% F442A 27% 86% 60% 91% Y443A 64% 78% 103% 93% N448A 113% 261% 129% 183% T526A 87% 140% 82% 122% Y527A 84% 86% 76% 100% W529A 82% 109% 77% 111% G530A 52% 53% 48% 71% N532A 118% 173% 125% 134% D533A 94% 116% 82% 73% T534A 93% 117% 95% 104% D535A 55% 80% 52% 96% V538A 126% 111% 103% 99% P612A 94% 132% 81% 116% W616A 29% 87% 71% 105% R657A 123% 2% 138% <10% P664A 101% 167% 83% 119% P676A 102% 67% 92% 78% D698A 116% <10% 117% 6%
Example 3
Material and Methods
Production of the Antibodies
[0203] Production of the antibodies AT12-007, AT12-009, AT12-010 and AT13-021 was performed as described in Example 1.
ELISA
[0204] Cell lysates containing E1E2 alanine mutants were produced in a similar fashion as described in Example 2.
[0205] ELISA assays were performed in a similar fashion as described in Example 2.
Results
Epitope Mapping
[0206] In Example 1, we used alanine mutants of eighteen E2 residues to determine residues important for antibody binding of AT12-007, AT12-009, AT12-010, AT12-011 and AT13-021. To determine if additional E2 amino acid residues are important for antibody binding of AT12-007, AT12-009, AT12-010, AT12-011 and AT13-021, we generated a new panel of H77 E2 alanine mutants, which have been shown to affect binding of known neutralizing antibodies as for instance HC33 antibodies, AR3 antibodies, CBH-5, e20 and e137 (Keck et al, Journal of virology, 2012; Law et al. Nature Medicine, 2007; Owsianka et al, Journal of general virology, 2008; Perotti et al, Journal of virology, 2008; Mancini et al, plos one, 2009). Antibody binding was determined by ELISA using a lysate of E1E2 transfected Freestyle™ 293-F cells. The data in Table 10 represents the ratio of antibody binding to E2 mutants compared to the binding of wild-type protein. In Example 1, we showed that AT12-007, AT12-009, AT12-010 and AT13-021 share an antigen binding domain on E2 that is at least composed of the residues F442, Y527, W529, G530, D535, W616. Of these 4 antibodies, only AT12-010 showed ≦25% binding to W420A, suggesting that W420 is part of the epitope of AT12-010. In contrast to AT12-007, AT12-009, AT12-010 and AT13-021, none of the alanine mutations affected binding of AT12-011 in Example 1 and Example 2. This suggests that the binding domain of AT12-011 is completely different compared to known antibodies like, but not limited to, the HC33 antibodies, AR3 antibodies, CBH-5, e20 and e137.
TABLE-US-00009 TABLE 10 Extention of epitope mapping of the antibodies from Example 1. Binding of antibodies to E2 alanine-mutants was tested by ELISA. Shown is the relative antibody binding to the mutant sequences compared to the wild type sequence. When the EC50 could not be calculated because of very weak binding this is indicated by <10%. The assaywas performed in duplicate. ND: not done. AT12-009 AT12-011 AT12-007 AT12-010 AT13-021 N209A 83% 130% ND ND ND N325A 87% 114% ND ND ND L413A 147% 86% 187% 217% 185% G418A 128% 63% 130% 203% 131% W420A 120% 152% 87% <10% 103% N423A 118% 181% ND ND ND N448A 127% 179% ND ND ND T526A 57% 150% ND ND ND Y527A 52% 125% ND ND ND N532A 114% 145% ND ND ND D533A 71% 133% ND ND ND T534A 100% 76% ND ND ND V538A 93% 121% ND ND ND P612A 67% 110% ND ND ND P664A 73% 131% ND ND ND P676A 81% 154% ND ND ND
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