Antibodies that potently neutralize hepatitis B virus and uses thereof

Abstract

The present invention relates to antibodies, and antigen binding fragments thereof that bind to the antigenic loop region of hepatitis B surface antigen (HBsAg) and potently neutralize infection of both hepatitis B virus (HBV) and hepatitis delta virus (HDV). The invention also relates to epitopes to which the antibodies and antigen binding fragments bind, as well as to nucleic acids that encode and cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in the diagnosis, prophylaxis and treatment of hepatitis B and hepatitis D.

Claims

1. An isolated antibody, or an antigen-binding fragment thereof, that binds to the antigenic loop region of HBsAg and neutralizes infection with hepatitis B virus and hepatitis delta virus, wherein the antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences comprising: (i) SEQ ID NOs: 34-38 and 58, respectively; or (ii) SEQ ID NOs: 34-37, 39 and 58, respectively; or (iii) SEQ ID NOs: 34, 66, 36-38, and 58, respectively; or (iv) SEQ ID NOs: 34, 66, 36, 37, 39, and 58, respectively.

2. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences comprising SEQ ID NOs: 34-38 and 58, respectively.

3. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences comprising SEQ ID NOs: 34-37, 39 and 58, respectively.

4. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences comprising SEQ ID NOs: 34, 66, 36-38, and 58, respectively.

5. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences comprising SEQ ID NOs: 34, 66, 36-37, 39, and 58, respectively.

6. The antibody, or antigen-binding fragment thereof, of claim 1, comprising a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), wherein V.sub.H and V.sub.L comprise amino acid sequences having at least 95% identity to SEQ ID NOs: 41 and 59, respectively.

7. The antibody, or antigen-binding fragment thereof, of claim 1, comprising a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), wherein V.sub.H and V.sub.L comprise amino acid sequences having at least 95% identity to SEQ ID NOs: 41 and 65, respectively.

8. The antibody, or antigen-binding fragment of claim 1, comprising a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), wherein V.sub.H and V.sub.L comprise amino acid sequences having at least 95% identity to SEQ ID NOs: 67 and 59, respectively.

9. The antibody, or antigen-binding fragment thereof, of claim 1, comprising a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), wherein V.sub.H and V.sub.L comprise amino acid sequences having at least 95% identity to SEQ ID NOs: 67 and 65, respectively.

10. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody, or the antigen-binding fragment thereof, comprises a Fc moiety.

11. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody, or antigen-binding fragment thereof, is human.

12. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a purified antibody, a single chain antibody, a Fab, a Fab, a F(ab)2, a Fv, or a scFv.

13. A nucleic acid molecule comprising a polynucleotide encoding the antibody, or antigen-binding fragment thereof, of claim 1.

14. The nucleic acid molecule of claim 13, wherein the polynucleotide is codon-optimized for expression in a host cell.

15. A vector comprising the nucleic acid molecule according to claim 13.

16. A cell comprising the nucleic acid of claim 13.

17. A pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of claim 1, and a pharmaceutically acceptable excipient, diluent, or carrier.

18. A kit, comprising the antibody, or antigen-binding fragment thereof, of claim 1, and one or more of: (i) a polymerase inhibitor; (ii) an interferon; or (iii) a checkpoint inhibitor.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

(2) FIG. 1 shows for Example 1 the binding of HBC34 monoclonal antibody to three different HBsAg serotypes (adw, ady, and ayw) as measured by ELISA.

(3) FIG. 2 shows for Example 2 the ability of various anti-HB antibodies, namely HBV immunoglobulins (HBIG), HBC34, and further monoclonal antibodies against PreS1 (18/7) or HBsAg to neutralize HBV infection of HepaRG cell in vitro. Each antibody was tested at three different concentrations, namely 5 g/ml, 0.5 g/ml and 0.05 g/ml, except for HBIG, which was tested at 5000 g/ml, 500 g/ml and 50 g/ml.

(4) FIG. 3 shows for Example 2 the staining of HBcAg in HepaRG cells infected in the presence of three different concentrations (5 g/ml, 0.5 g/ml or 0.05 g/ml) of HBC34 monoclonal antibody and, as a reference, the nuclear staining.

(5) FIG. 4 shows for Example 2 the neutralization activity of different concentrations of HBC34 on infectious HDV. At a concentration of 0.12 g/ml HBC34 no HDV-positive cells were detectable, indicating potent neutralization of HDV. HBIG, in contrast, did not neutralize HDV (tested in 1:1000 dilution, i.e. 50 g/ml).

(6) FIG. 5 shows for Example 3 the amino acid sequences of the antigenic loop of HBsAg of the 10 HBV genotypes A, B. C. D, E, F, G, H, I and J. Those sequences comprise the epitope recognized by the HBC34 antibody (highlighted in grey). The sequence on the top (HBV-D J02203) was used to design the peptide library in Example 5, FIG. 7.

(7) FIG. 6 shows for Example 3 the binding, as determined by cytofluorimetric analysis, of the human monoclonal antibody HBC34 and a control antibody (both at 5 g/ml) to permeabilized Hep2 cells transiently transfected with plasmids expressing the different HBsAg genotypes A, B, C, D, E, F, G, H, I and J as indicated in the Figure.

(8) FIG. 7 shows for Example 4 the amino acid sequences of the antigenic loop of the 19 HBsAg mutants tested. Circled are the residues of the HBsAg mutants that were weakly (dotted circle) or not bound by HBC34 antibody.

(9) FIGS. 8A and 8B show for Example 4 the binding of the human monoclonal antibody HBC34 and two other HBsAg-specific antibodies (Ab5 and Ab6) all tested at 5 g/ml on Hep2 cells transfected with plasmids expressing the different HBsAg genotype D mutants as indicated in the Figure (WT: HBsAg Genotype D, Genbank accession no. FJ899792).

(10) FIG. 9 shows for Example 5 the binding of HBC34 to a library of 650 linear and looped peptides as determined using the Pepscan technology as well as the sequences of the four peptides bound by HBC34. Residues indicate as 1 are cysteines that were introduced to allow the chemical linking to scaffolds in order to reconstruct conformational epitopes. If other cysteines besides the newly introduced cysteines are present, they are replaced by alanine (underlined alanine residues).

(11) FIG. 10 shows for Example 5 a western blot staining by Ab4 and HBC34 on HBV viral particles under reducing conditions. Ab4 is a comparative antibody, which is also reactive against the antigenic loop.

(12) FIG. 11 shows for Example 6 the levels of HBV viremia in humanized uPA/SCID mice inoculated with 510.sup.7 copies of HBV genome equivalents (genotype D), which received from three weeks post-infection treatment with either HBC34 (at 1 mg/kg administered i.p. twice per week), a control antibody (control AB) or entecavir (ETV; administered orally at 1 g/ml) for 6 weeks. In the spreading phase of HBV infection (weeks 3 to week 6 p.i. (post infection)) viremia increased >2 log in the group which received the control antibody, while HBV titers decreased in mice treated with HBC34 or entecavir.

(13) FIG. 12 shows for Example 6 staining of hepatocytes for the presence of HBsAg (intrahepatical analysis) in mice of Example 6 at the end of the experiment (week 9). Nearly all hepatocytes stained HBsAg positive in mice which received the control antibody, while spreading was efficiently blocked by both, treatment with entecavir and treatment with HBC34 (ca. 1-5% HBsAg-positive cells).

(14) FIG. 13 shows for Example 6 cccDNA measurements, which did not differ significantly between mice that were sacrificed 3 weeks post HBV infection (3 week hbv; i.e. no treatment) and mice that were treated from week 3 to week 9 post-infection with HBC34 or entecavir. In contrast, the estimated amount of cccDNA/cell increased up to 2 logs in the group receiving the control antibody when sacrificed 9 weeks post-infection.

(15) FIG. 14 shows for Example 6 levels of circulating HBsAg at baseline (BL), week 3 of treatment (week 6 post-infection) and week 6 of treatment (week 9 post-infection). Levels of circulating HBsAg decreased >1 log (and below the limit of detection) in mice receiving HBC34, but not in mice treated with entecavir, while HBsAg levels increased >2 logs (reaching levels of 5000-10000 IU/ml) in the control group.

(16) FIG. 15 shows for Example 7 HBV titer (left panel) and levels of circulating HBsAg (right panel) in a chronic hepatitis B setting at baseline (BL), week 3 of treatment (week 15 post-infection, week 3) and week 6 of treatment (week 18 post-infection, week 6). HBV titer and levels of circulating HBsAg were decreased in mice receiving HBC34 at week 3 and week 6 of treatment. Individual curves represent individual animals. Control antibody: dotted lines, HBC34: continuous lines.

(17) FIG. 16 shows for Example 8 HBV titer (left panel) and HDV titer (right panel) in uPA/SCID mice repopulated with primary human hepatocytes and co-infected with a patient-derived serum containing HDV-RNA and HBV-DNA. Five weeks after infection treatment with HBC34 or a control antibody (control) was started. HBV titer (left panel) and HDV titer (right panel) are shown at baseline (week3, week5BL), at week 3 of treatment (week 8 post-infectionweek 8) and at week 6 of treatment (week 11 post-infectionweek 11). Individual curves represent individual animals. Control antibody: dotted lines, HBC34: continuous lines.

(18) FIG. 17 (part 1) shows for Example 9 a schematics of the antigenic loop of HBV-s-Antigen, the epitope of HBC34 is highlighted in grey. To map the epitope of HBC34 a library of 1520 different peptides was tested as determined using the Pepscan technology. FIG. 17 (part 2-5) show for Example 9 the magnitude of binding (ELISA intensities) of HBC34 to 16 different sets of peptides. HBC34 is binding to a conformational epitope as demonstrated by the binding to peptides of sets 13-16 composed of combinatorial CLIPS constructs, representing two parts of a discontinuous epitope (part 2) and to peptides of sets 9-12 composed of looped peptides (part 3). No binding of HBC34 is observed with sets of linear peptides 1-4 (part 4) and 5-8 (part 5) further supporting the notion that HBC34 binds to a discontinuous conformational epitope.

(19) FIG. 18 shows for Example 9 the binding of HBC34 to a peptide library composed of 812 discontinuous or looped T3 CLIPS peptides. In order to fine tune the epitope mapping described in FIG. 17, 3 sets of peptides (dubbed RN1, RN2 and RN3) were generated based on the previous sets (FIG. 17) by means of full substitution analysis. FIG. 18 shows binding of HBC34 to sets 1-3 where residues at the boxed positions were substituted with by one of 13 amino acids selected from series AEFGHKLPQRSVY_, where _ stands for residue deletion. The original residue at the permutated position is indicated on the left of each panel as well as either (i) in the horizontally boxed sequence (when the original amino acid is part of the permutation series) or (ii) below the sequence (when the original amino acid is not part of the permutation series).

(20) FIG. 19 shows for Example 10 the effect of a combination therapy with HBC34 (1 mg/kg i.p twice a week) and Lamivudine (supplemented at 0.4 mg/ml in drinking water) in reducing the levels of HBV viremia (panel A) and circulating HBV-s-Antigen (panel B) in humanized uPA/SCID mice inoculated with 210.sup.9 copies of HBV genome equivalents (genotype D), which received for 4 weeks starting from 8 weeks post-infection, treatment with either either a control antibody (control), HBC34 alone (HBC34), lamividune alone (Lamivudine) or a combination treatment (HBC34 and lamivudine). Combination therapy caused higher reduction of viremia compared with either drugs alone.

(21) FIG. 20 shows for Examples 11 and 12 the alignments of the two sets of VH (panel A) and VL (panel B) sequences generated to obtain the antibody variants 1-32. CDRs (defined according to IMGT) are highlighted in grey.

(22) FIG. 21 shows for Example 11 the binding of 18 engineered HBC34 variants (obtained by combining the mutated VH and VL sequences as indicated in columns 2 and 3) to HBsAg (adw subtype) as determined by ELISA. In columns 4, loss of binding is indicated with , strongly reduced binding is indicated with +/, reduced binding is indicated with +/, binding similar or equal to the original antibody is indicated with +.

(23) FIG. 22 shows for Example 11 binding of 8 different engineered variants of HBC34 to HBsAg (adw) as determine in direct antigen-based ELISA assay. These 8 variants were select among the 18 HBC34 mutants described in FIG. 21; in order to better characterize the affinity for HBsAg the 8 antibodies were titrated and compared with the parental antibody sequence.

(24) FIG. 23 shows for Example 11 the summary of the characteristics of the 8 antibodies described in FIG. 22. EC50 were determined by fitting the curves in FIG. 22 using Graphpad prism. Productivity was determined by (ELISA) quantification of secreted IgG in the supernatant of a 300 ml transfection of 293 Expi cells with each of the 8 variants as well as the parental antibody.

(25) FIG. 24 shows for Examples 11 and 12 a table summarizing the characteristics of 15 variants, among which are 12 additional engineered variants of HBC34, designed based on the results of the previous set by introducing additional mutations in the frameworks (panel A). Binding curves were obtained by titrating the antibodies in antigen-based ELISA assay and EC50 were calculated by fitting the curves with Graphpad prism. Productivities were calculated based on quantification of the IgG secreted in the supernatant of a 30 ml transfection of 293 Expi cells with each of the 15 variants and the parental antibody. Fold-changes were plotted and are shown in panel B.

(26) FIG. 25 shows for Examples 11 and 12 the binding, as determined by cytofluorimetric analysis, of HBC34 and variants 6, 7, 19, 23 and 24. All antibodies were titrated starting from 5 g/ml and bound to permeabilized Hep2 cells transiently transfected with plasmids expressing the different HBsAg genotypes A, B, C, D, E, F, G, H, I and J.

EXAMPLES

(27) In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Identification and Characterization of Human Monoclonal Antibody HBC34

(28) A human monoclonal antibody was isolated in a similar manner as described in Traggiai E. et al., 2004, Nat Med 10(8): 871-5 from a human patient. The antibody was characterized by determining the nucleotide and amino acid sequences of its variable regions (Tables 2 and 3) and the complementarity determining regions (CRDs) therein and termed HBC34. Accordingly, HBC34 is an IgG1-type fully human monoclonal antibody having the CDR, VH and VL sequences as shown above in Tables 2 and 3.

(29) Next, it was determined to which of the three HBsAg serotypes adw, ady, and ayw the human monoclonal antibody HBC34 binds to. Interestingly, HBC34 binds with high affinity to three HBsAg serotypes (adw, ady and ayw) with similar and low EC50 values, as measured by ELISA (FIG. 1).

(30) Protective titers of HBV antibodies are expressed in International Units (IU) which allows standardization over different assays. In 1977, an International Reference Preparation for anti-HBs immunoglobulin (W1042) was established. The plasma used in the preparation of this standard was derived from individuals who had been naturally infected with hepatitis B virus (Barker, L. F., D. Lorenz, S. C. Rastogi, J. S. Finlayson, and E. B. Seligmann. 1977. Study of a proposed international reference preparation for antihepatitis B immunoglobulin. WHO Expert Committee on Biological Standardization technical report series. WHO Expert Committee on Biological Standardisation 29th Report BS 77.1 164. Geneva, Switzerland, World Health Organization, 1977; World Health Organization: Anti-hepatitis B immunoglobulin. WHO Tech Rep Ser 1978; 626:18). The activity of HBC34, as measured diagnostically with an immunoassay (Abbott Architect diagnostic immunoassay), is 5000 IU/mg. As a comparison the activity of HBIG is 1 IU/mg.

Example 2: Antibody HBC34 Potently Neutralizes Infectious HBV and HDV

(31) The first object of Example 2 was to determine whether HBC34 neutralizes infectious HBV and to compare the neutralization activity of HBC34 to that of other anti-HB antibodies. To this end, differentiated HepaRG cells were incubated with a fixed amount of HBV in the presence or absence of antibodies (HBC34, 18/7, Ab2, Ab3 and HBIG) in medium supplemented with 4% PEG 8000 (Sigma-Aldrich) for 16 hours at 37 C. At the end of the incubation, the cells were washed and further cultivated. Medium was changed every 3 days. Infection was detected by measuring in enzyme-linked immunosorbent assay (ELISA) the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant from day 7 to 11 post-infection and by detecting HBcAg staining in an immunofluorescence assay.

(32) As shown in FIGS. 2 and 3, HBC34 neutralized completely HBV infection when tested at 5 and 0.5 g/ml, whereas comparative human monoclonal anti-HB antibodies Ab2 and Ab3, which are also binding to HBsAg, did not result in complete neutralization. This indicates that not all antibodies binding to HBsAg are able to neutralize HBV infection (e.g. Ab2 and Ab3). Of note, HBIG neutralized HBV infection only when tested at 5000 and 500 g/ml, i.e. with a 1000 fold lower potency as compared to HBC34. 18/7 is a murine monoclonal antibody against the pre-S1 region of HBsAg.

(33) The second object of Example 2 was to determine the neutralizing activity of HBC34 against HDV on differentiated HepaRg cells. Sera from HDV carriers were used as HDV infection inoculum. Delta antigen immunofluorescence staining was used as a readout. As shown in FIG. 4, HBC34 completely blocked HDV infection when tested at 0.12 g/ml. As a comparison, HBIG were also tested and were ineffective (tested at 1/1000, i.e. 50 g/ml).

Example 3: Antibody HBC34 Recognizes all 10 HBV Genotypes A, B, C, D, E, F, G, H, I, and J

(34) HBC34 was tested for its ability to recognize the 10 HBV genotypes A, B, C, D, E, F, G, H, I, and J (as shown in FIG. 5) by flow cytometry analysis. In particular, human epithelial cells (Hep2 cells) were transfected with plasmids expressing each of the HBsAg of the 10 HBV genotypes A, B, C, D, E, F, G, H, I, and J (as shown in FIG. 5). Human monoclonal antibody HBC34 (5 g/ml) and a control antibody (5 g/ml) were used for staining of transiently transfected permeabilized cells. Two days after transfection, Hep2 cells were collected, fixed and permeabilized with saponin for immunostaining with HBC34 or a control Ab. Binding of antibodies to transfected cells was analysed using a Becton Dickinson FACSCanto2 (BD Biosciences) with FlowJo software (TreeStar). As shown in FIG. 6 HBC34 recognized all 10 HBV HBsAg genotypes with similar patterns of staining.

Example 4: Antibody HBC34 Recognizes all Functional HBsAg Mutants

(35) HBC34 was tested for its ability to bind to the 19 different HBsAg genotype D mutants (based on HBsAg Genotype D, Genbank accession no. FJ899792, as shown in FIG. 7) HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R1221, HBsAg T123N, HBsAg T123N/C124R, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg N146A (see SEQ ID NO's 16-33 for the amino acid sequences of the antigenic loop regions of those mutants) by flow cytometry analysis. In particular, human epithelial cells (Hep2 cells) were transfected with plasmids expressing the different HBsAg mutants and analyzed as in Example 3. 5 g/ml of human monoclonal antibody HBC34 and two other HBsAg-specific antibodies (Ab5 and Ab6) were used for testing the binding of HBC34 to the transfected Hep2 cells.

(36) As shown in FIGS. 8A-8B, HBC34 was found to bind to 18 of the 19 HBsAg mutants. HBC34 binding, but not Ab5 and Ab6 binding, was completely abolished only in the mutant HBsAg T123N/C124R, i.e. when residues 123 and 124 were both mutated. Of note, the mutation of these two residues (i.e. T123 and C124) into alanine was shown to be associated with a loss of HBV infectivity, which is most likely due to the loss of the disulphide bridge formed by C124 that could result in a conformational change in the antigenic loop (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). Thus, human monoclonal antibody HBC34 binds to 18 HBsAg mutants.

Example 5: Antibody HBC34 Binds to a Conserved Conformational Epitope in the Antigenic Loop

(37) The epitope recognized by HBC34 was identified by using a library of 650 linear and looped peptides (CLIPS Discontinuous Epitope Mapping technology from Pepscan, Lelystad, The Netherlands) designed to cover the entire antigenic loop region of the HBsAg. The linear and CLIPS peptides are synthesized based on standard Fmoc-chemistry. The looped peptides are synthesized on chemical scaffolds in order to reconstruct conformational epitopes, using the Chemically Linked Peptides on Scaffolds, CLIPS, technology as described in Timmerman et al., 2007, Journal of Molecular Recognition 20: 283-99. For example the single looped peptides are synthesized containing two cysteines and the size of the loop is varied by introducing the cysteine residues at variable spacing. If other cysteines besides the newly introduced cysteines are present, they are replaced by alanine. The side-chains of the multiple cysteines in the peptides are coupled to CLIPS templates by reacting onto credit-card format polypropylene PEPSCAN cards (455 peptide formats/card) with a 0.5 mM solution of CLIPS template such as 1,3-bis (bromomethyl)benzene in ammonium bicarbonate. The binding of antibody to each peptide is tested in a PEPSCAN-based ELISA. The 455-well credit card format polypropylene cards containing the covalently linked peptides are incubated with the test antibody (at 1 g/ml) in blocking solution. After washing the peroxidase substrate 2,2-azino-di-3-ehylbenzthiazoline sulfonate (ABTS) and 2 l of 3% H.sub.2O.sub.2 are added. After one hour, the color development is measured with a a charge coupled device (CCD)-camera and an image processing system. The raw data are optical values and range from 0 to 3000 (a log scale similar to 1 to 3 of a standard 96-well plate ELISA reader).

(38) As shown in FIG. 9, HBC34 was found to recognize a double looped peptide having an amino acid sequence according to SEQ ID NO 52:

(39) TABLE-US-00017 XGSSTTSTGPCRTCMTXPSDGNATAIPIPSSWX
wherein the residues coded as X were substituted with Cysteines and the underlined residues were substituted from C to A (SEQ ID NO 52).

(40) Three additional peptides were recognized with a lower signal: (a) a linear 15-mer peptide having an amino acid sequence according to SEQ ID NO 53:

(41) TABLE-US-00018 (SEQ ID NO 53) TSTGPCRTCMTTAQG, (b) another linear peptide having an amino acid sequence according to SEQ ID NO 54:

(42) TABLE-US-00019 (SEQ ID NO 54) GMLPVCPLIPGSSTTSTGPCRTCMTT, and (c) a double looped peptide having an amino acid sequence according to SEQ ID NO 55:

(43) TABLE-US-00020 XSMYPSASATKPSDGNXTGPCRTCMTTAQGTSX wherein the residues coded as X were substituted with Cysteines and the underlined residues were substituted from C to A (SEQ ID NO 55).

(44) This analysis indicated that the core epitope of HBC34 is formed by a conformational epitope formed by an amino acid sequence according to SEQ ID NO: 56:

(45) TABLE-US-00021 (SEQ ID NO 56 PCRTCMTTAQG;
amino acids 120-130 of the S domain of HBsAg (HBV-D J02203).

(46) Moreover, as shown in FIG. 10 the human monoclonal antibody HBC34 does not react at all in a western blot on HBV viral particles under reducing conditions.

(47) These results confirm that the epitope of HBsAg, to which HBC34 binds to, is a conformational epitope.

(48) These results are consistent with what observed in Example 4 where HBC34 binding was lost in the presence of the T123N/C124R mutations.

(49) The region of HBsAg, which comprises the conformational epitope, to which HBC34 binds to, is polymorphic in the different HBV genotypes. In the following generic sequence of the epitope region of HBsAg the residues mutated in the different genotypes are indicated with an X:

(50) TABLE-US-00022 PCX.sub.1TCX.sub.2X.sub.3X.sub.4AQG,
wherein X.sub.1 is preferably R or K, X.sub.2 is preferably M or T, X.sub.3 is preferably T or I, and X.sub.4 is preferably T, P or L
(SEQ ID NO: 57).

(51) Moreover, an additional comparison of the above sequence to the 18 HBsAg mutants, to which the human monoclonal antibody HBC34 binds to, indicates that HBC34 binds to an epitope formed by an amino acid sequence according to SEQ ID NO: 2:

(52) TABLE-US-00023 X.sub.1 X.sub.2 X.sub.3 TC X.sub.4 X.sub.5 X.sub.6A X.sub.7G
wherein X.sub.1 is P, T or S, X.sub.2 is C or S, X.sub.3 is R, K, D or I, X.sub.4 is M or T, X.sub.5 is T, A or I, X.sub.6 is T, P or L, and X.sub.7 is Q, H or L.

Example 6: Administration of HBC34 Starting 3 Weeks Post HBV Infection Prevents Viral Spreading in Humanized uPA Mice

(53) The object of Example 6 was to investigate whether human monoclonal antibody HBC34 is able to prevent spreading of HBV. In other words, it was the aim to investigate the capacity of the entry inhibitor HBC34 antibody to inhibit infection of the human hepatocytes in vivo by treating mice after the initial infection establishment. To this end, nave uPA/SCID mice repopulated with primary human hepatocytes (see Petersen et al. Nature Biotechnology, 2008, 26:335-341) were used. These mice were engrafted with cryopreserved human hepatocytes by intrasplenic injection. Eight weeks later successful repopulation of human hepatocytes in the host liver was determined measuring human serum albumin, which is exclusively expressed by transplanted human hepatocytes. Mice with appropriate human albumin levels were inoculated i.p. with 510.sup.7 copies of HBV genome equivalents (genotype D, HBeAg positive) to permit viral entry. Three weeks after infection, the treatment protocol started whereby mice received either HBC34 treatment (at 1 mg/kg administered i.p. twice per week), a control antibody or entecavir (ETV; administered orally at 1 pig/ml in water, Baraclude Solution, Bristol-Myers Squibb) for 6 weeks. Liver specimens removed at sacrifice were snap-frozen in liquid nitrogen for immunofluorescence analysis.

(54) HBV DNA was extracted from serum samples using the QiAmp MinElute Virus spin kit (Qiagen, Hilden, Germany). HBV-specific primers and hybridization probes were used to determine HBV DNA viremia and cccDNA loads quantitatively as described previously (Volz T et al., Gastroenterology 2007; 133: 843-852). DNA and RNA were extracted from liver specimens using the Master Pure DNA purification kit (Epicentre, Biozym, Germany) and RNeasy RNA purification kit (Qiagen, Hilden, Germany). Intrahepatic HBV DNA values were normalized for cellular DNA contents using the beta-globin gene kit (Roche DNA control Kit; Roche Diagnostics). Levels of rcDNA were estimated by subtracting cccDNA amounts from total HBV DNA. Viral RNAs and genomic RNAs were reverse transcribed using oligo-dT primers and the Transcriptor Kit (Roche Applied Science) and quantified by using primers specific for total viral RNAs. HBV RNA levels were normalized to human specific GAPDH RNA. HBsAg quantification from blood samples was performed using the Abbott Architect platform (quant. HBsAg kit, Abbott, Ireland, Diagnostic Division), as recommended by the manufacturer. Cryostat sections of chimeric mouse livers were immunostained using humanspecific cytokeratin-18 monoclonal (Dako, Glostrup, Denmark) to stain human hepatocytes. For the detection of the HBV core antigen (HBcAg), the polyclonal rabbit anti-HBcAg was used. Specific signals were visualized by employing the Alexa-labeled secondary antibodies (Invitrogen, Darmstadt, Germany) or TSA-Fluorescein (HBcAg) System (Perkin Elmer, Jgesheim, Germany), while nuclear staining was obtained with Hoechst 33342 (Invitrogen). Stained sections were analyzed by fluorescence microscope.

(55) The median baseline level of HBV DNA at the beginning of the treatment was 210.sup.6 DNA copies/ml. In the spreading phase of HBV infection (weeks 3 to week 6 p.i. (post infection)) viremia increased >2 log in the group which received the control antibody, while HBV titers decreased in mice treated with HBC34 or entecavir (FIG. 11).

(56) Mice were also analyzed intrahepatically at the end of the experiment (i.e. week 9) by staining hepatocytes for the presence of HBcAg. Nearly all hepatocytes stained HBcAg positive in mice which received the control antibody, while spreading was efficiently blocked by both, treatment with entecavir and treatment with HBC34 (ca. 1-5% HBcAg-positive cells). These results indicate that HBC34 can efficiently block viral spreading during the ramp-up phase of HBV infection (FIG. 12).

(57) In line with the histological and serological data, cccDNA measurements showed that intrahepatic cccDNA loads did not differ significantly between mice that were sacrificed 3 weeks post HBV infection and mice that were treated from week 3 to week 9 post-infection. In comparison, the estimated amount of cccDNA/cell increased up to 2 logs in the control group sacrificed 9 weeks post-infection, suggesting that newly formed rcDNAs could not be efficiently converted into cccDNA in treated mice (FIG. 13). The same tendency was also found by measuring other intrahepatic viral parameters, such as the levels of relaxed circular DNA (rcDNA) and HBV RNA transcripts.

(58) Moreover, levels of blood circulating HBsAg were measured at baseline (BL), week 3 of treatment (week 6 post-infection) and week 6 of treatment (week 9 post-infection). It is of note that the levels of circulating HBsAg decreased >1 log (and below the limit of detection) in mice receiving HBC34, but not in mice treated with entecavir, while HBsAg levels increased >2 logs (reaching levels of 5000-10000 IU/ml) in the control group (FIG. 14). The measurement of HBsAg was not influenced by the presence of the HBC34 antibody as determined in a spike-in experiment where the addition of HBC34 antibody to HBsAg positive mouse sera did not alter the expected measurement using the Abbott Architect diagnostic immunoassay.

(59) These results indicate that HBC34 can block HBV viral spread and promote the clearance of HBsAg.

Example 7: Administration of HBC34 in Chronically HBV Infected Humanized uPA Mice Promoted HBV and HBsAg Clearance

(60) To mimic the hepatitis B chronic setting, nave humanized uPA/SCID mice were infected with HBV and after 12 weeks post infection, a median level of HBV DNA of 210.sup.9 copies/ml and a level of HBsAg of 10000 IU/ml was reached. These levels are as high as the levels that are commonly observed in human patients with chronic HBV infection.

(61) Thereafter, the mice were treated starting from week 12 post-infection either with HBC34 or with a control antibody for 6 weeks (1 mg/kg i.p. twice per week). As shown in FIG. 15 HBV titer and levels of blood circulating HBsAg were decreased in mice receiving HBC34 for 3 weeks (week 15 post-infection) and 6 weeks (week 18 post-infection). Thus, HBC34 promoted a clear reduction of both HBV viremia and HBsAg levels after 6 weeks of treatment. HBeAg and human albumin levels were not altered in HBC34-treated mice, which indicates the absence of liver toxicity.

Example 8: Administration of HBC34 Blocks HDV Infection In Vivo

(62) Nave humanized uPA/SCID mice were co-infected with a patient-derived serum containing HDV-RNA and HBV-DNA. Five weeks after infection (when HBV titers reached levels between 10.sup.7 to 10.sup.9, and HDV RNA reached levels between 10.sup.3 to 10.sup.6 copies/mi) mice were treated with HBC34 or a control antibody for 6 weeks (1 mg/kg i.p. twice per week).

(63) HBV DNA viremia was measured as described in Examples 6 and 7. HDV viremia was determined via reverse transcription of viral RNA (extracted from serum samples using the QiAmp MinElute Virus Spin Kit, Qiagen, Venlo, Netherlands) and quantitative RT-PCR using the ABI Fast 1-Step Virus Master (Applied Biosystems, Carlsbad, USA), HDV specific primers and probes on a ABI Viia7 (Applied Biosystems, Carlsbad, USA).

(64) As shown in FIG. 16, HBC34 efficiently blocked HDV viral spread both 3 weeks and 6 weeks after treatment (weeks 8 and 11, respectively). Similarly to what was observed in HBV chronically infected mice, HBC34 promoted a HBV viral DNA titers reduction of 2 logs (FIG. 13).

Example 9: Fine Epitope Mapping of the HBC34 Discontinuous Epitope

(65) In order to further refine the epitope recognized by HBC34 antibody described in Example 5 a new library of 1520 peptides composed of 16 different sets was generated: Set 1 (dubbed LIN15): Linear 15-mer peptides derived from the target sequence (SEQ ID NO: 5: QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one residue. Native Cys residues are protected by an acetamidomethyl group (also referred to as Acm; denoted as 2 in the respective amino acid sequences). Set 2 (dubbed LIN22): Linear 22-mer peptides derived from the target sequence (SEQ ID NO: 5: QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one residue. Native Cys residues are protected by Acm (denoted 2). Set 3 (dubbed LIN30): Linear 30-mer peptides derived from the target sequence (SEQ ID NO: 5: QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one residue. Native Cys residues are protected by Acm (denoted 2). Set 4 (dubbed LIN15.AA): Peptides of set 1, but with residues on positions 9 and 10 replaced by Ala. When a native Ala occurred on either position, it was replaced by Gly. Set 5 (dubbed LIN22.AA): Peptides of set 2, but with residues on positions 12 and 13 replaced by Ala. When a native Ala occurred on either position, it was replaced by Gly. Set 6 (dubbed LIN30.AA): Peptides of set 3, but with residues on positions 16 and 17 replaced by Ala. When a native Ala occurred on either position, it was replaced by Gly. Set 7 (dubbed CYS.A): Combinatorial peptides of length 27. On positions 1-11 and 17-27 are linear sequences, which contain pairing Cys residues. These 11-mer sequences joined via GGSGG (SEQ ID NO: 79) linker. Cys residues, which do not participate in disulfide bridge formation are protected by Acm (denoted 2). Set 8 (dubbed as CYS.B): Linear 22-mer sequences, which contain two Cys forming a disulfide bridge. Cys residues, which do not participate in disulfide bridge formation are protected by Acm (denoted 2). Set 9 (dubbed LOOP12): Constrained peptides of length 12. On positions 2-11 are 10-mer sequences derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 12 are Cys residues, which are joined by mP2 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 10 (dubbed as LOOP15): Constrained peptides of length 15. On positions 2-14 are 13-mer sequences derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 15 are Cys residues, which are joined by mP2 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 11 (dubbed LOOP21): Constrained peptides of length 21. On positions 2-20 are 19-mer sequences derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 21 are Cys residues, which are joined by mP2 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 12 (dubbed LOOP31): Constrained peptides of length 31. On positions 2-30 are 29-mer sequences derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 31 are Cys residues, which are joined by mP2 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 13 (dubbed MAT.A): Combinatorial peptides of length 25. On positions 2-12 and 14-24 are 11-mer peptides derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1, 13 and 25 are Cys residues, which are joined by T3 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 14 (dubbed MAT.B): Combinatorial peptides of length 28. On positions 2-12 and 14-27 are 11-mer and 14-mer peptides respectively. On positions 1, 13 and 28 are Cys residues, which are joined by T3 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 15 (dubbed MAT.C): Combinatorial peptides of length 28. On positions 2-15 and 17-27 are 14-mer and 11-mer peptides respectively. On positions 1, 16 and 28 are Cys residues, which are joined by T3 CLIPS. Native Cys residues are protected by Acm (denoted 2). Set 16 (dubbed MAT.D): Combinatorial peptides of length 31. On positions 2-15 and 17-30 are 14-mer peptides derived from the target sequence of Antigenic Loop of HBV-S-Ag. On positions 1, 16 and 31 are Cys residues, which are joined by T3 CLIPS. Native Cys residues are protected by Acm (denoted 2).

(66) When tested under high stringency conditions antibody HBC34 did not bind any peptide present on the arrays. When tested under low stringency conditions (5 g/ml in 0.1% Pepscan buffer and preconditioning containing a combination of horse serum and ovalbumin) the antibody bound constrained and combinatorial peptidesbinding to peptides from set 14 and set 16 was somewhat lower as compared to set 13 and set 15. No binding was recorded on linear epitope mimics. Data are shown in FIG. 17. These data show that antibody HBC34 recognizes a conformational discontinuous epitope composed of peptide stretches .sub.18TGPCRTC.sub.24 (SEQ ID NO: 80) and .sub.45GNCTCIP.sub.51 (SEQ ID NO: 81), where peptide stretch .sub.18TGPCRTC.sub.24 (SEQ ID NO: 80) is the dominant part of the epitope (FIG. 17).

(67) To fine map the epitope of antibody HBC34 by means of full substitution analysis based on the results described above, 812 discontinuous or looped T3 CLIPS peptides composed of three different set were synthesized: Set 1 (dubbed RN1; Discontinuous T3 CLIPS): Epitope mutant series derived from the discontinuous mimic C2IPIPSSWAFGCSTTSTGP2RT2C (SEQ ID NO: 82). For each position of this sequence, a substitution analysis was performed. In other words, for each position of the peptide sequence, variants were made in which the original amino acid at such position was replaced by one of the 13 amino acids selected from the group consisting of alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), lysine (K), leucine (L), proline (P), glutamine (Q), arginine (R), serine (S), valine (V), tyrosine (Y), and ; where _ stands for residue deletion. Native Cys residues are protected by Acm (denoted 2). Set 2 (dubbed RN2; Discontinuous T3 CLIPS): Epitope mutant series derived from the discontinuous mimic CGN2T2IPIPSSWAFCSTTSTGP2RT2C (SEQ ID NO: 83). For each position of this sequence, a substitution analysis was performed in the same manner as for Set 1 (i.e., GN2T residues were not mutated). Native Cys residues are protected by Acm (denoted 2). Set 3 (dubbed RN3; Loop T3 CLIPS): Epitope mutant series derived from the looped mimic CGGGCSTTSTGP2RT2C_(SEQ ID NO: 84). For positions 6-16 of this sequence, a substitution analysis was performed in the same manner as for set 1. Native Cys residues are protected by Acm (denoted 2).

(68) HBC34 antibody was tested in the PEPSCAN-based ELISA at 20 g/ml on the peptide array pre-conditioned with 0.1% SQ (Pepscan buffer containing 0.1% of a combination of horse serum and ovalbumin). The peptide arrays were incubated with HBC34 antibody solution (overnight at 4 C.). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SBA) for one hour at 25 C. After washing, the peroxidase substrate 2,2-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 l/ml of 3 percent H.sub.2O.sub.2 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD)camera and an image processing system.

(69) As expected, when tested under low stringency conditions antibody HBC34 bound peptides from all sets. Results of the experiment show that residues .sub.120PCR.sub.122 and C.sub.124 are critical for the binding, while only certain replacements of residues I150, I152, W156 and F158 notably decrease the binding (FIG. 18). Moreover, data recorded on all three arrays coherently have shown that replacements of any residue within region .sub.114STTSTGPCRTC.sub.124 (SEQ ID NO: 85) with E would decrease the binding, while inverse replacements with R or Y increase binding. However, the same was not observed for region .sub.145GNCTCIPIPSSWAFC.sub.159 (SEQ ID NO: 86).

(70) Taken together these results suggest that antibody HBC34 recognizes a discontinuous epitope with residues .sub.120PCR.sub.122 and C.sub.124 being crucial for the binding. Presence of residues .sub.145GNCTCIPIPSSWAF.sub.158 (SEQ ID NO: 87) was shown to provide structural context for establishing and stabilizing epitope-paratope interactions. This conclusion arose from the observation that discontinuous epitope mimics (when .sub.145GNCTCIPIPSSWAF.sub.158 (SEQ ID NO: 87) and .sub.114STTSTGPCRTC.sub.124 (SEQ ID NO: 85) are present in one mimic) are more tolerant to the replacements than sequence .sub.14STTSTGPCRTC.sub.124 (SEQ ID NO: 85) alone (set 3, RN3). Additionally, P151 which fixes the torsion angles thereby providing conformational rigidity was shown to impact the binding especially when replaced by G known for inversed properties. Replacement G145P similarly impacts binding of HBC34. It was repeatedly observed that R/Y replacements improve the binding to any position within motif .sub.114STTSTGPCRTC.sub.124 (SEQ ID NO: 85), but not within motif .sub.145GNCTCIPIPSSWAF.sub.158 (SEQ ID NO: 87), while E replacements decrease binding. This observation may suggest that residues .sub.114STTSTGPCRTC.sub.124 (SEQ ID NO: 85) bind to a negatively charged paratope within the antibody HBC34 (or close to a cluster of negative charges) and improvement of the binding as well as the decreased result from electrostatic forces and rather characterize the paratope features than those of the epitope.

Example 10: Increased Reduction of HBsAg in Chronically HBV Infected Humanized uPA Mice Treated with a Combination of HBC34 and Lamivudine

(71) In a further study, the efficacy of a combination therapy including the antibody HBC34 was investigated. For combination with HBC34, a polymerase inhibitor, namely lamivudine, was selected.

(72) To mimic the chronic hepatitis B setting, nave humanized uPA/SCID mice were infected with HBV and after 8 weeks post infection, a median level of HBV DNA of 210.sup.9 copies/ml and a level of HBsAg of 9000 IU/ml was reached. These levels are as high as the levels that are commonly observed in human patients with chronic HBV infection.

(73) Thereafter, mice were treated starting from week 8 post-infection either with antibody HBC34 alone, with the polymerase inhibitors lamivudine alone, with a combination of HBC34 and lamivudine, or with a control antibody for 4 weeks (HBC34 at 1 mg/kg i.p. twice per week; lamivudine supplemented in drinking water at 0.4 mg/ml).

(74) HBV viremia and HBsAG levels in serum were assessed in treatment week 0 (before treatment), treatment week 2, treatment week 4, and treatment week 6, or in treatment week 0 (before treatment), treatment week 3 and treatment week 6. Results are shown in FIG. 19 A (HBV viremia) and B (HBsAG).

(75) As shown in FIG. 19 treatment with HBC34, lamivudine or both drugs in combination caused mean 0.7 log, 1.3 log and 2.4 log reduction of viremia (A), respectively. Notably, HBsAg (B) dropped 1.3 log (mean BL=15,600 IU/ml) in mice receiving HBC34 alone and 2.6 log (mean BL=2,600 IU/ml) in the combination group, whereas no significant HBsAg reduction (0.2 log; mean BL=9,000 IU/ml) was detected in mice treated with lamivudine alone.

(76) In summary, the combination of HBC34 and lamivudine clearly achieved the strongest effect. Interestingly, such a strong effect of the combination of HBC34 and lamivudine was observed, even if lamivudine alone was not effective. In view thereof, the observed strong effect of the combination of HBC34 and lamivudine is clearly an unexpected synergistic effect.

(77) In summary, the surprisingly strong HBsAg reduction achieved in combination therapy proves that HBC34 antibody can be used, e.g. in chronic settings, in combination with polymerase inhibitors to accelerate HBsAg clearance both in HBV mono-infected and HBV/HDV co-infected patients.

Example 11: Sequence Engineering of HBC34 Antibody: CDR3 of VH and VL

(78) A first series of HBC34 mutants was generated with mutations in the CDR3 of VH and VL by mutating (i) residue W107 of the VH CDR3 into either A or F, (ii) residue M115 of the VH CDR3 into I or L, and/or (iii) residue W107 of the VL CDR3 into either A or F. A total of 18 HBC34 variants were produced by combining the un-mutated VH or VL of HBC34 (hereafter referred as WT, wild type or parental antibody) with different combinations of VH and VL mutants as illustrated in FIG. 20 and FIG. 21.

(79) The produced HBC34 antibody variants were tested by ELISA for binding to HBsAg adw antigen, similarly as in Example 1. Results are shown in FIG. 21.

(80) Of note, the mutation of W107 of the VH CDR3 into A (in HBC34-V5, HBC34-V8, HBC34-V9, HBC34-V10, HBC34-V12 and HBC34-V17 variants, FIG. 21) completely abolished HBC34 binding to HBsAg. This indicates that W107 is a key residue in the HBC34 paratope for antigen recognition. The mutation of W107 of the VH CDR3 into F (an amino acid with similar aromatic characteristic as W) partially affected HBC34 binding (HBC34-V1), indicating that W cannot be mutated without compromising HBC34 binding affinity to HBsAg.

(81) The mutation of M115 of the VH CDR3 into L did not affect HBC34 binding (HBC34-V13), while the mutation into I (HBC34-V11) partially reduced HBC34 binding, indicating that M115 could be substituted by L, but not by I, without compromising HBC34 binding. Consistently with the results obtained with the single mutation W107A, the double mutation W107A and M115A (HBC34-V10) completely abolished HBC34 binding.

(82) The mutation of W107 of the VL CDR3 into F did not affect HBC34 binding (HBC34-V7), while the mutation into A (HBC34-V15) partially reduced HBC34 binding, indicating that W107 of the VL CDR3 could be substituted by F, but not by A, without compromising HBC34 binding. As expected the combination of the mutations W107F in the CDR3 of the VH and W107A in the CDR3 of the VL completely abolished binding (HBC34-v2). The combination of the mutations M 15L in VH CDR3 and W107F in VL CDR3s (HBC34-V6), of the mutations M1151 in the VH CDR3 with W107F in the VL CDR3 (HBC34-v4) and of the mutations W107F in the VH CDR3 and W107F in the VL CDR3, partially affected HBC34 binding to HBsAg, indicating that the combination of these two mutations, but not the individual mutations, is not compatible to retain the binding affinity of the parental HBC34 antibody.

(83) In a next step, six of the 18 HBC34 variants described above were selected for further characterization (HBC34-V1, V3, V4, V6, V7, V11 and V13) in order (i) to confirm the initial results; (ii) to measure the binding affinity by ELISA (i.e. to determine the EC50 of binding); and (iii) to assess the productivity of these HBC34 variants from transiently transfected 293-Expi cells (FIG. 22 and FIG. 23).

(84) As observed in the first experiment, HBC34-V3 variant (carrying the double mutation W107F of the VH CDR3 and W107F of the VL CDR3) bound to HBsAg (adw serotype) with an EC50 9 fold higher as compared to the parental HBC34 antibody. In addition, HBC34-V3 is produced at a concentration almost 5 times lower as compared to HBC34. HBC34-V11 and HBC34-V13 variants carrying the single mutation M1151 and M115L, respectively, bound to HBsAg with EC50 identical or slightly superior to that of HBC34. However, both variants were produced less efficiently than HBC34 (0.6 and 0.3 lower productivity when compared to HBC34). These results indicate that HBC34-V11, and even more HBC34-V13 variants, bind with high affinity to HBsAg but are produced less efficiently in mammalian cells. Similarly, HBC34-V1 variant carrying the single mutation W107F in the VH CDR3 bound to HBsAg comparably with HBC34 (1.6-fold higher EC50), but was produced 4-fold less efficiently (i.e. 0.25) as compared to HBC34. The combination of W107 of the VL CDR3 with either W107, M1151 or M115L of the VH CDR3 (HBC34-V3, HBC34-V4 and HBC34-V6) reduced both binding affinity (1.6-9.0 fold higher EC50) and productivity (0.20-0.35 lower antibody concentration in the culture supernatants). Surprisingly, the single mutation W107F of the VL CDR3 (HBC34-V7) bound to HBsAg similarly to HBC34 and was produced even more efficiently (up to 1.7) than HBC34, reaching the remarkably high concentration in the culture supernatant of 533 g/ml (FIG. 23).

Example 12: Sequence Engineering of HBC34 Antibody: Framework Regions

(85) Twelve additional HBC34 variants were produced (HBC34-V19 to HBC34-V30; FIG. 24A) in which several mutations were introduced in the framework regions (FRs) of both VH and VL that corresponded to the residues found in the HBC34 unmutated common ancestor (HBC34-UCA) (FIG. 20) and combined with the VH CDR3 mutation M115L and with the VL CDR3 mutation W107F.

(86) Results are shown in FIG. 24. The introduction of 9 mutations in the FRs of VL (HBC34-27, HBC34-V28, HBC34-V29 and HBC34-V30) in the presence of the W107 mutation in the VL CDR3, combined with the WT, M115L reduced significantly HBC34 binding to HBsAg, thus indicating an important role for the mutated residues in VL (FIG. 24A-B). HBC34 variants, wherein the same VL variant described above (i.e. W107F/FR1234-GL) was combined with the VH carrying the M115L mutation and additional 9 mutations in FRs, did not bind to HBsAg, indicating that mutations in both VH and VL contribute essentially to HBsAg binding.

(87) Importantly, the removal of only one of the 9 mutations introduced in the FRs of VL (i.e. K66Y) in HBC34-V23 and HBC34-V24 increased significantly the binding (100 fold lower EC50) to HBsAg as compared to the corresponding variants carrying the Y66K mutation (HBC34-V27 and HBC34-V28). Similarly, the removal of the K66Y mutation in HBC34-V25 and HBC34-V26 restored HBsAg binding as compared to the corresponding non-binding variants carrying the Y66K mutation (HBC34-V27 and HBC34-V28).

(88) Of these, the HBC34-V23 variant retained high affinity binding (1.5 higher EC50 as compared to HBC34) and was produced similarly to the parental HBC34 antibody. Of note, the HBC34-V24 variant differing for only one amino acid from HBC34-V23 variant (i.e. M115L in VH), bound to HBsAg with a EC50 similar to that of HBC34-V23 but was not produced efficiently (only 0.14 productivity as compared to HBC34). These results indicate that, while not affecting significantly the binding of HBC34 variants to HBsAg, the presence of L at position 115 has a negative impact on the productivity of HBC34 variants carrying this mutation. Indeed, on average all HBC34 variants carrying the M115L mutation (HBC34-V6, HBC34-V13, HBC34-V19, HBC34-V20, HBC34-V21, HBC34-V22, HBC34-V24, HBC34-V25, HBC34-V26, HBC34-V28, HBC34-V29 and HBC34-V30) have a mean productivity which is 0.3 as compared to that of the parental HBC34 antibody.

(89) Remarkably, the introduction of 5 or 9 mutations in the FRs of VH in the presence of the M115L mutation in VH CDR3 (HBC34-V19 and HBC34-V20 variants, respectively) did not decrease appreciably the binding to HBsAg, suggesting that the mutated residues do not have an important role in the high affinity antigen recognition by HBC34 antibody. The introduction of the W107F mutation on the backbone of HBC34V19 and HBC34-V20 variants in HBC34-V21 and HBC34-V22 reduced the binding to HBsAg of 20-30. Interestingly, the same mutation (i.e. W107 in the VL CDR3) did not affect the binding of other variants not carrying the same 5 or 9 mutations in the FRs of the VH, a result which might indicate that residues in the VH FRs have a cooperative role (e.g. by stabilizing a certain conformation of the variable region scaffold) in binding to HBsAg with residue 107 of the VH.

(90) Finally and consistently with the results of Example 11 shown in FIG. 23, the HMB34-V7 antibody carrying the single mutation W107 in the VL CDR3 showed a comparable binding to HBsAg (i.e. 1.4) as compared to HBC34 and was produced more efficiently (1.2) than HBC34 (on average in the two experiments performed HBC34-V7 was produced 1.5, i.e. 50%, more efficiently than HBC34 antibody. This result suggests that the W107F mutation in VL CDR3, while not affecting appreciably the binding affinity to HBsAg, has a positive impact on HBC34 antibody productivity.

(91) Finally, HBC34 and the variants HBC34-V6, HBC34-V7, HBC34-V19, HBC34-V23 and HBC34-V24 were tested for their ability to recognize the 10 HBV genotypes A, B, C, D, E, F, G, H, I, and J (as shown in FIG. 25) by flow cytometry analysis. In particular, human epithelial cells (Hep2 cells) were transfected with plasmids expressing each of the HBsAg of the 10 HBV genotypes A, B, C, D, E, F, G, H, I, and J. All antibodies were tested at multiple concentrations (8 serial dilutions from 5000 ng/ml to 7 ng/ml) for staining of transiently transfected permeabilized cells. Two days after transfection, Hep2 cells were collected, fixed and permeabilized with saponin for immunostaining with HBC34 and the five selected variants. Binding of antibodies to transfected cells was analysed using a Becton Dickinson FACSCanto2 (BD Biosciences) with FlowJo software (TreeStar). As shown in FIG. 25, HBC34 and all of the five variants tested recognized all 10 HBV HBsAg genotypes at a similar level.

(92) TABLE-US-00024 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks 1 X.sub.1 X.sub.2 X.sub.3 TC X.sub.4 X.sub.5 X.sub.6A X.sub.7G epitope wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6 and X.sub.7 may be any amino acid 2 X.sub.1 X.sub.2 X.sub.3 TC X.sub.4 X.sub.5 X.sub.6A X.sub.7G wherein X.sub.1 is P, T or S, X.sub.2 is C or S, X.sub.3 is R, K, D or I, X.sub.4 is M or T, X.sub.5 is T, A or I, X.sub.6 is T, P or L, and X.sub.7 is Q, H or L. 3 MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLG S domain of HBsAg GTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFI (GenBank acc. no. LLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQ J02203) GTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARF SWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILS PFLPLLPIFFCLWVYI 4 MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFL S domain of HBsAg GGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFL (GenBank acc. no. FILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTGTGPCRTCTTPA FJ899792) QGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASAR FSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTL SPFLPLLPIFFCLWVYI 5 QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTK J02203 (D, ayw3) PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 6 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTK FJ899792 (D, adw2) PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 7 QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCCCTK AM282986 (A) PSDGNCTCIPIPSSWAFAKYLWEWASVRFSW 8 QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKP D23678 (B1) TDGNCTCIPIPSSWAFAKYLWEWASVRFSW 9 QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKP AB117758 (C1) SDGNCTCIPIPSSWAFARFLWEWASVRFSW 10 QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCCSKP AB205192 (E) SDGNCTCIPIPSSWAFGKFLWEWASARFSW 11 QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCSKP X69798 (F4) SDGNCTCIPIPSSWALGKYLWEWASARFSW 12 QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTK AF160501 (G) PSDGNCTCIPIPSSWAFAKYLWEWASVRFSW 13 QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCTKP AY090454 (H) SDGNCTCIPIPSSWAFGKYLWEWASARFSW 14 QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTK AF241409 (I) PSDGNCTCIPIPSSWAFAKYLWEWASARFSW 15 QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCCCTKP AB486012 (J) SDGNCTCIPIPSSWAFAKFLWEWASVRFSW 16 CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCT HBsAg KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Y100C/P120T 17 QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTK HBsAg P120T PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 18 QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTK HBsAg P120T/S143L PLDGNCTCIPIPSSWAFGKFLWEWASARFSW 19 QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCCCTKP HBsAg C121S SDGNCTCIPIPSSWAFGKFLWEWASARFSW 20 QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTSMYPSCCCTK HBsAg R122D PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 21 QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCCTKP HBsAg R122I SDGNCTCIPIPSSWAFGKFLWEWASARFSW 22 QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCCCTK HBsAg T123N PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 23 QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTSMYPSCCCTK HBsAg Q129H PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 24 QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTSMYPSCCCTKP HBsAg Q129L SDGNCTCIPIPSSWAFGKFLWEWASARFSW 25 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSHYPSCCCTK HBsAg M133H PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 26 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSLYPSCCCTKP HBsAg M133L SDGNCTCIPIPSSWAFGKFLWEWASARFSW 27 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSTYPSCCCTKP HBsAg M133T SDGNCTCIPIPSSWAFGKFLWEWASARFSW 28 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTE HBsAg K141E PSDGNCTCIPIPSSWAFGKFLWEWASARFSW 29 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTK HBsAg P142S SSDGNCTCIPIPSSWAFGKFLWEWASARFSW 30 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTK HBsAg S143K PKDGNCTCIPIPSSWAFGKFLWEWASARFSW 31 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTK HBsAg D144A PSAGNCTCIPIPSSWAFGKFLWEWASARFSW 32 QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTK HBsAg G145R PSDRNCTCIPIPSSWAFGKFLWEWASARFSW 33 QGMLPVCPLIPCSSTTGTGPCRTCTTPAQGTSMYPSCCCTK HBsAg N146A PSDGACTCIPIPSSWAFGKFLWEWASARFSW 34 GRIFRSFY CDRH1 aa 35 NQDGSEK CDRH2 aa 36 AAWSGNSGGMDV CDRH3 aa 37 KLGNKN CDRL1 aa 38 EVK CDRL2 aa 39 VIYEVKYRP CDRL2 long aa 40 QTWDSTTVV CDRL3 aa 41 ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVRQA VH aa PGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAKNSLFL QMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTTVSVSS 42 SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPG VL aa QSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQAMDEA AYFCQTWDSTTVVFGGGTRLTVL 43 GGACGCATCTTTAGAAGTTTTTAC CDRH1 nuc 44 ATAAACCAAGATGGAAGTGAGAAA CDRH2 nuc 45 GCGGCTTGGAGCGGCAATAGTGGGGGTATGGACGTC CDRH3 nuc 46 AAATTGGGGAATAAAAAT CDRL1 nuc 47 GAGGTTAAA CDRL2 nuc 48 gtcatctatGAGGTTAAAtaccgcccc CDRL2 long nuc 49 CAGACGTGGGACAGCACCACTGTGGTG CDRL3 nuc 50 GAACTGCAGCTGGTGGAGTCTGGGGGAGGCTGGGTCC VH nuc AGCCGGGGGGGTCCCAGAGACTGTCCTGTGCAGCCTC TGGACGCATCTTTAGAAGTTTTTACATGAGCTGGGTCCG CCAGGCCCCAGGGAAGGGGCTGGAGTGGGTGGCCAC TATAAACCAAGATGGAAGTGAGAAATTATATGTGGACTC TGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCA AGAACTCACTATTTCTGCAAATGAACAACCTGAGAGTCG AGGACACGGCCGTTTATTACTGCGCGGCTTGGAGCGG CAATAGTGGGGGTATGGACGTCTGGGGCCAGGGGACC ACGGTCTCCGTCTCCTCA 51 TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCC VL nuc CCAGGACAGACAGTCAGCATCCCCTGCTCTGGAGATAA ATTGGGGAATAAAAATGTTTGCTGGTTTCAGCATAAGCC AGGCCAGTCCCCTGTGTTGGTCATCTATGAGGTTAAATA CCGCCCCTCGGGGATTCCTGAGCGATTCTCTGGCTCCA ACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACC CAGGCTATGGATGAGGCTGCCTATTTCTGTCAGACGTG GGACAGCACCACTGTGGTGTTCGGCGGAGGGACCAGG CTGACCGTCCTA 52 XGSSTTSTGPCRTCMTXPSDGNATAIPIPSSWX peptide wherein the residues coded as Xwere substituted with Cysteines 53 TSTGPCRTCMTTAQG peptide 54 GMLPVCPLIPGSSTTSTGPCRTCMTT peptide 55 XSMYPSASATKPSDGNXTGPCRTCMTTAQGTSX peptide wherein the residues coded as Xwere substituted with Cysteines 56 PCRTCMTTAQG amino acids 120-130 of the S domain of HBsAg (HBV-D J02203 57 PCX.sub.1TCX.sub.2X.sub.3X.sub.4AQG, epitope wherein X.sub.1 is preferably R or K, X.sub.2 is preferably M or T, X.sub.3 is preferably T or I, and X.sub.4 is preferably T, P or L 58 QTFDSTTVV CDRL3 v7 and CDRL3 v23 (aa) 59 SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPGQ VL v7 SPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQAMDEAAY FCQTFDSTTVVFGGGTRLTVL 60 AAGCTGGGGAACAAAAAT CDRL1 v7 and CDRL1 v23 (nuc) 61 GAGGTGAAA CDRL2 v7 and CDRL2 v23 nuc 62 GTCATCTACGAGGTGAAATATCGGCCT CDRL2 long v7 and CDRL2 long v23 nuc 63 CAGACATTCGATTCCACCACAGTGGTC CDRL3 v7 and CDRL3 v23 nuc 64 TCTTACGAGCTGACACAGCCACCTAGCGTGTCCGTCTCT VL v7 nuc CCAGGACAGACCGTGTCCATCCCTTGCTCTGGCGACAA GCTGGGGAACAAAAATGTCTGTTGGTTCCAGCACAAGC CAGGGCAGAGTCCCGTGCTGGTCATCTACGAGGTGAAA TATCGGCCTTCAGGAATTCCAGAACGGTTCAGCGGATCA AACAGCGGCAATACTGCAACCCTGACAATTAGCGGGAC CCAGGCCATGGACGAAGCCGCTTATTTCTGCCAGACATT CGATTCCACCACAGTGGTCTTTGGCGGGGGAACTAGGC TGACCGTGCTG 65 SYELTQPPSVSVSPGQTASITCSGDKLGNKNACWYQQKPG VL v23 aa QSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQAMDEA DYYCQTFDSTTVVFGGGTKLTVL 66 INQDGSEK HBC34wt CDRH2 aa 67 EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQAP HBC34 v31, HBC34 GKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQ v32 and HBC34 v33 MNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTTVTVSS VH 68 GAGGTGCAGCTGGTGGAATCCGGCGGGGGACTGGTGC HBC34 v31, HBC34 AGCCTGGCGGCTCACTGAGACTGAGCTGTGCAGCTTCT v32 and HBC34 v33 GGAAGAATCTTCAGATCTTTTTACATGAGTTGGGTGAGA VH (nuc) CAGGCTCCTGGGAAGGGACTGGAGTGGGTCGCAAACA TCAATCAGGACGGATCAGAAAAGCTGTATGTGGATAGC GTCAAAGGCAGGTTCACTATTTCCCGCGACAACGCCAAA AATTCTCTGTTTCTGCAGATGAACAATCTGCGGGTGGAG GATACCGCTGTCTACTATTGTGCAGCCTGGTCTGGCAAC AGTGGAGGCATGGACGTGTGGGGACAGGGAACCACAG TGACAGTCAGCTCC 69 TCTTACGAGCTGACACAGCCCCCTAGCGTGTCCGTCTCT VL v23 nuc CCAGGCCAGACAGCATCCATCACTTGCTCTGGCGACAA GCTGGGGAACAAAAATGCCTGTTGGTATCAGCAGAAGC CAGGGCAGAGTCCCGTGCTGGTCATCTACGAGGTGAAA TATCGGCCTTCAGGAATTCCAGAAAGATTCAGTGGATCA AACAGCGGCAATACTGCTACCCTGACAATTAGCGGGAC CCAGGCCATGGACGAAGCTGATTACTATTGCCAGACATT CGATTCCACCACAGTGGTCTTTGGCGGGGGAACTAAGC TGACCGTGCTG 70 GAACTGCAGCTGGTCGAATCAGGAGGAGGGTGGGTCC HBC34 wt VH AGCCCGGAGGGAGCCAGAGACTGTCTTGTGCCGCATCA codon optimized GGGAGGATCTTCAGGAGCTTCTACATGTCCTGGGTGCG CCAGGCACCAGGCAAGGGACTGGAGTGGGTCGCCACC ATCAACCAGGACGGATCTGAAAAGCTGTATGTGGATAGT GTCAAAGGCCGGTTCACAATTAGCAGAGACAACGCTAA AAATTCTCTGTTTCTGCAGATGAACAATCTGCGAGTGGA GGATACCGCCGTCTACTATTGCGCCGCTTGGTCTGGCAA CAGCGGCGGGATGGATGTCTGGGGGCAGGGCACAACA GTGAGCGTCTCTTCC 71 TCATACGAACTGACTCAGCCTCCCTCCGTCTCCGTCTCAC HBC34 wt VL codon CTGGACAGACCGTCTCAATCCCCTGCTCCGGCGAT optimized AAACTGGGCAACAAGAACGTGTGCTGGTTCCAGCACAA ACCCGGACAGAGTCCTGTGCTGGTCATCTACGAGGTCA AGTATCGGCCAAGCGGCATTCCCGAAAGATTCAGCGGC TCCAACTCTGGGAATACCGCAACACTGACTATCTCTGGA ACCCAGGCAATGGACGAGGCAGCTTACTTTTGCCAGACT TGGGATTCAACTACTGTCGTGTTCGGCGGCGGAACTAG ACTGACTGTCCTG 72 GGGAGGATCTTCAGGAGCTTCTAC HBC34 wt CDRH1 codon optimized 73 ATCAACCAGGACGGATCTGAAAAG HBC34 wt CDRH2 codon optimized 74 GCCGCTTGGTCTGGCAACAGCGGCGGGATGGATGTC HBC34 wt CDRH3 codon optimized 75 AAACTGGGCAACAAGAAC HBC34 wt CDRL1 codon optimized 76 GAGGTCAAG HBC34 wt CDRL2 codon optimized 77 GTCATCTACGAGGTCAAGTATCGGCCA HBC34 wt CDRL2 long codon optimized 78 CAGACTTGGGATTCAACTACTGTCGTG HBC34 wt CDRL3 codon optimized 79 GGSGG linker 80 TGPCRTC epitope 81 GNCTCIP epitope 82 CCIPIPSSWAFGCSTTSTGPCRTCC discontinuous wherein in particular thy cysteines at positions 2, 21, and 24 epitope mimic are coupled to acetamidomethyl. 83 CGNCTCIPIPSSWAFCSTTSTGPCRTCC discontinuous wherein in particular thy cysteines at positions 4, 6, 24, and epitope mimic 27 are coupled to acetamidomethyl. 84 CGGGCSTTSTGPCRTCC looped epitope wherein in particular thy cysteines at positions 13 and 16 are mimic coupled to acetamidomethyl. 85 STTSTGPCRTC epitope 86 GNCTCIPIPSSWAFC epitope 87 GNCTCIPIPSSWAF epitope 88 PCRXC epitope