ANTI-HEPATITIS B VIRUS ANTIBODIES AND USE THEREOF

Abstract

Antibodies (especially humanized antibodies) against the hepatitis B surface antigen (HBsAg), a nucleic acid molecule encoding same, a method for preparing same, and a pharmaceutical composition containing same. The anti-HBsAg antibodies have a higher binding affinity to HBsAg at a neutral pH than at an acidic pH, thereby significantly enhancing virus clearance efficiency and prolonging virus inhibition time. The antibodies and pharmaceutical composition may be used to prevent and/or treat HBV infections or diseases related to HBV infection (such as hepatitis B) for use in neutralizing the virulence of HBV in the body of a subject (such as a human) to reduce a serum level of HBV DNA and/or HBsAg in the body of the subject, or to activate a humoral immune response of a subject (such as a person infected with chronic HBV or a patient who has chronic hepatitis B) against HBV.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to HBsAg, wherein the antibody or antigen-binding fragment thereof binds to HBsAg with higher affinity at neutral pH than at acidic pH, and the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the following 3 CDRs: (i) HCDR1 with a sequence of X.sub.1X.sub.2YHX.sub.3N (SEQ ID NO: 26), wherein X.sub.1 is selected from Y or H, X.sub.2 is selected from G or R, X.sub.3 is selected from W or Y; (ii) HCDR2 with a sequence of YIX.sub.4X.sub.5DGSVX.sub.6YNPSLEN (SEQ ID NO: 27), wherein X.sub.4 is selected from S, N or H, X.sub.5 is selected from Y or H, X.sub.6 is selected from L, H or Q; and (iii) HCDR3 with a sequence of GFDH (SEQ ID NO: 13); and/or, (b) a light chain variable region (VL) comprising the following 3 CDRs: (iv) LCDR1 with a sequence of RSSQSLVHSYGDX.sub.7YLH (SEQ ID NO: 28), wherein X.sub.7 is selected from T or N; (v) LCDR2 with a sequence of KVSNRFS (SEQ ID NO: 15); and (vi) LCDR3 with a sequence of SQNTHX.sub.8PYT (SEQ ID NO: 29), wherein X.sub.8 is selected from V, L or H.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the following 3 CDRs: (i) HCDR1, consisting of a sequence selected from the following: SEQ ID NOs: 17, 21, 24; (ii) HCDR2, consisting of a sequence selected from: SEQ ID NOs: 12, 18, 20, 22; and (iii) HCDR3, consisting of a sequence shown in SEQ ID NO: 13; and/or, (b) a light chain variable region (VL) comprising the following 3 CDRs: (iv) LCDR1, consisting of a sequence selected from the following: SEQ ID NOs: 14, 25; (v) LCDR2, consisting of a sequence shown in SEQ ID NO: 15; and (vi) LCDR3, consisting of a sequence selected from the following: SEQ ID NOs: 16, 19, 23.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (1) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 23; (2) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 19; (3) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16; (4) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 24, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16; (5) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 23; or (6) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin (for example, a framework region contained in an amino acid sequence encoded by a human germline antibody gene), and the framework region optionally comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues; preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and/or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene; preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region contained in an amino acid sequence encoded by human heavy chain germline gene 4-28-02 (SEQ ID NO: 38), and a light chain framework region contained in an amino acid sequence encoded by human light chain germline gene 2D-28-01 (SEQ ID NO: 39), and the heavy chain framework region and/or the light chain framework region optionally comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues; preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH FR1 as shown in SEQ ID NO: 30, VH FR2 as shown in SEQ ID NO: 31, VH FR3 as shown in SEQ ID NO: 32, and VH FR4 shown in SEQ ID NO: 33; preferably, the VL of the antibody or antigen-binding fragment thereof comprises: VL FR1 as shown in SEQ ID NO: 34, VL FR2 as shown in SEQ ID NO: 35, VL FR3 as shown in SEQ ID NO: 36, and VL FR4 shown in SEQ ID NO: 37.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the following: (i) a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; (ii) a sequence with substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; or (iii) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; and (b) a light chain variable region (VL), which comprises an amino acid sequence selected from the following: (iv) a sequence shown in any one of SEQ ID NOs: 2, 4, 7, 9, 10; (v) a sequence with substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with a sequence shown in any one of SEQ ID NOs: 2, 4, 7, 9, 10; or (vi) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with a sequence shown in any one of SEQ ID NOs: 2, 4, 7, 9, 10; preferably, the substitution described in (ii) or (v) is a conservative substitution.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises: (1) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequence shown in SEQ ID NO: 4; (2) a VH with a sequence shown in SEQ ID NO: 5 and a VL with a sequence shown in SEQ ID NO: 2; (3) a VH with a sequence shown in SEQ ID NO: 6 and a VL with a sequence shown in SEQ ID NO: 7; (4) a VH with a sequence shown in SEQ ID NO: 8 and a VL with a sequence shown in SEQ ID NO: 9; (5) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequence shown in SEQ ID NO: 10; or (6) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequence shown in SEQ ID NO: 9.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin; preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (for example, IgG1, IgG2, IgG3 or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (for example, κ or λ); preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 41.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a variant of a human IgG1 heavy chain constant region, the variant has the following substitution as compared to a wild-type sequence from which it is derived: (i) M252Y, N286E, N434Y; or, (ii) K326D, L328Y; wherein the above-mentioned amino acid positions are positions according to the Kabat numbering system; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 42 or 43.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 40.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (2) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (3) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (4) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41; (5) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41; (6) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41; (7) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (8) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (9) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (10) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (11) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (12) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (13) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (14) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (15) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (16) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (17) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; or (18) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, Fab, Fab′, (Fab′).sub.2, Fv fragment, diabody, bispecific antibody, multispecific antibody, probody, chimeric antibody or humanized antibody; preferably, the antibody is a chimeric antibody or a humanized antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is able to specifically bind to HBsAg, neutralize a virulence of HBV, and/or reduce a serum level of HBV DNA and/or HBsAg in a subject.

13. An isolated nucleic acid molecule, which encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or its heavy chain variable region and/or light chain variable region.

14. A vector, which comprises the nucleic acid molecule according to claim 13; preferably, the vector is a cloning vector or an expression vector.

15. A host cell, which comprises the nucleic acid molecule according to claim 13 or the vector according to claim 14.

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which comprises culturing the host cell according to claim 15 under a condition that allows the expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

17. A pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier and/or excipient.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 17 in the manufacture of a medicament for the prevention and/or treatment of an HBV infection or HBV infection-associated disease (for example, hepatitis B) in a subject (for example, a human), for neutralizing a virulence of HBV in vitro or in a subject (for example, a human), for reducing a serum level of HBV DNA and/or HBsAg in a subject (for example, a human), and/or for activating a humoral immune response against HBV in a subject (for example, a person with chronic HBV infection or a patient with chronic hepatitis B).

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 17, for use in the prevention and/or treatment of an HBV infection or HBV infection-associated disease (for example, hepatitis B) in a subject (for example, a human), for use in neutralizing a virulence of HBV in vitro or in a subject (for example, a human), for use in reducing a serum level of HBV DNA and/or HBsAg in a subject (for example, a human), and/or for use in activating a humoral immune response against HBV in a subject (for example, a person with chronic HBV infection or a patient with chronic hepatitis B).

20. A method, which is used for the prevention and/or treatment of an HBV infection or HBV infection-associated disease (for example, hepatitis B) in a subject, for neutralizing a virulence of HBV in a subject (for example, a human), for reducing a serum level of HBV DNA and/or HBsAg in a subject (for example, a human), and/or for activating a humoral immune response against HBV in a subject (for example, a person with chronic HBV infection or a patient with chronic hepatitis B), the method comprises administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 17 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0177] FIG. 1 shows a schematic diagram of the working principle of an antibody with pH-dependent antigen-binding activity. Human plasma is neutral, with a pH of about 7.4, while the intracellular environment is acidic, with a pH of about 6.0. An antibody with pH-dependent antigen-binding activity can bind to an antigen in the plasma, the antigen-antibody complex is then internalized into the cell. The pH-dependent antibody will dissociate from the antigen in the acidic environment of the endosome. The antibody dissociated from the antigen will be captured by FcRn and circulated to the outside of the cell. In the extracelluar neutral environment, the FcRn releases the antibody, and the antibody returned to the plasma can bind to other antigen again, thereby realizing the cycle use of the antibody.

[0178] FIG. 2 shows the results of docking of Fab crystal structure based on the structural analysis of 162 to a short antigen mimic peptide, in which the blue structure is the short antigen peptide, and the red structure is part of the binding region of 162 antibody.

[0179] FIG. 3 shows a schematic diagram of the recombinant vector (pCGMT-scFv) encoding the scFv antibody, in which the scFv antibody has a structure of: NH.sub.2-VH-linker-VL-COOH.

[0180] FIGS. 4A to 4D show the ELISA results of the phage library displaying the pH-dependent scFv antibody derived from 162 and the antigen HBsAg. FIG. 4A: the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for the phage library derived from 162 after the third round of screening, the abscissa represents the phage antibody number, and the ordinate represents the OD value. The results show that these single clones all have strong antigen binding activity and have a significant decrease in binding activity at pH 6.0. FIG. 4B: the detection results of pH-dependent binding to HBsAg for the 13 single clones with high OD.sub.(450/630) value at pH 7.4 in the third round and showing the largest difference between OD.sub.(450/630) values at pH7.4 and pH 6.0, with 8 gradients and 3-fold dilution, in which the abscissa represents the dilution factor, and the ordinate represents the OD value. The results show that the pH-dependent antigen binding effect is better presented after the gradient dilution, in which the C32, C27, C26 and C19 show the better performance and C27 molecule has the best effect (the remaining 9 molecules are not shown). FIG. 4C: the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for the phage library derived from 162 after the fourth round of screening, the abscissa represents the phage antibody number, and the ordinate represents the OD value. FIG. 4D: the detection results of pH-dependent binding to HB sAg for the 8 single clones with high OD.sub.(450/630) value at pH 7.4 in the fourth round and showing the largest difference between OD.sub.(450/630) values at pH 7.4 and pH 6.0, with 8 gradients and 3-fold dilution, in which the abscissa represents the dilution factor, and the ordinate represents the OD value. The results show that the pH-dependent antigen binding effect is better presented after the gradient dilution, in which D3, D4 and D5 show the better performance, and D5 molecule has the best effect (the remaining 5 molecules are not shown).

[0181] FIG. 5 shows a summary of the mutation sites of C26, C27, C32, D3, D4 and D5.

[0182] FIG. 6A shows the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for the quantified cell supernatant obtained from the small scale eukaryotic transfection of C32, C27 and C26 in Example 3. FIG. 6B shows the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for the quantified cell supernatant obtained from the small scale eukaryotic transfection of D3, D4 and D5 in Example 3. The abscissa represents the antibody concentration (Log 10 ng/ml), and the ordinate represents the OD value. The results show that C32, C27, C26, D3, D4 and D5 all can maintain an antigen-binding activity equivalent to that of the parent antibody 162 at neutral pH, and all have a significant decrease in binding activity to antigen at pH 6.0.

[0183] FIG. 7 shows the working principle of scavenger antibody. The pH-dependent antigen binding activity plays a role in cells. Thus, if this first limiting factor of cell entry is not broken, the pH-dependent antigen-binding properties will not be applied subsequently, and the benefit of modification will be greatly reduced. A scavenger antibody obtained by further mutation of amino acids in the Fc region can enhance the binding to hFcRn receptor at neutral pH, or enhance the binding to FcγRs receptor. Tthe scavenger antibody is located outside the cell and acts as a “transport helper” for reciprocally transporting antigens into the cell, the antibody half-life can thus be extremely prolonged, and it can bind to antigen again, thereby improving the cell entry efficiency of antigens, and greatly improving the clearance efficiency.

[0184] FIGS. 8A to 8B show the protein gel results of pH-dependent antibodies and antibodies with DY modification. FIG. 8A: the picture of protein gel of pH-dependent antibodies, in which 162 is a positive control, and the results show that the expressed C26, D3, D4 and D5 antibodies are single-component. FIG. 8B: the picture of protein gel of antibodies with DY modification, in which 162 is a positive control, and the results show that the expressed antibodies C26 DY, D3 DY, D4 DY and D5 DY are single-component.

[0185] FIGS. 9A to 9D show the detection results of pH-dependent antibodies and antibodies with DY modification binding to HBsAg at pH 7.4 and pH 6.0 in Example 4, in which the abscissa represents the antibody concentration (Log 10 ng/ml) and the ordinate represents the OD value. The results show that D26, D3, D4 and D5 can maintain an antigen-binding activity equivalent to that of the parent (162) at the neutral pH, and have a significant decrease in antigen-binding activity under the condition of pH 6.0, and the corresponding antibodies with DY modification also can maintain an antigen-binding activity at the neutral pH and a pH-dependent antigen-binding activity comparable to those of the parent.

[0186] FIG. 10A shows the immunofluorescence experiment of mouse primary macrophages for the pH-dependent antibodies and antibodies with DY modification in Example 4, in which the green fluorescence represents hFcRn, the blue fluorescence represents nucleus, and the red fluorescence represents HBsAg. The results show that the DY modification enhances the phagocytosis of murine macrophages to the antigen-antibody complexes. FIG. 10B shows phagocytosis experiment based on human THP-1 phagocytic cells of the pH-dependent antibodies and the antibodies with DY modification in Example 4. The results show that the DY modification enhances the phagocytosis of human THP-1 phagocytic cells to the antigen-antibody complexes.

[0187] FIGS. 11A to 11B show the therapeutic effects of the C26 DY scavenger antibody and 162 in HBV transgenic mice after injection with a single dose of 5 mg/kg via tail vein in Example 4. FIG. 11C shows the therapeutic effects of the D3 DY, D3 DY, D4 DY and D5 DY in HBV transgenic mice after injection with a single dose of 5 mg/kg via tail vein in Example 4. FIG. 11A: the abscissa represents the number of days (d) after the injection of antibody, and the ordinate represents the HBsAg level in mouse serum after clearance (log 10 IU/ml). FIG. 11B shows: the changes in the concentration of antibody in mouse serum, in which the abscissa represents the number of days (d) after the injection of antibody, and the ordinate represents the antibody concentration (ng/ml). FIG. 11C: the abscissa represents the number of days (d) after the injection of antibody, and the ordinate represents the HBsAg level in mouse serum after clearance (log 10 IU/ml). The results show that the scavenger antibodies with DY modification C26 DY, D3 DY, D4 DY and D5 DY are stronger in antigen clearance ability by more than one order of magnitude than 162. This indicates that the scavenger antibodies with DY modification C26 DY, D3 DY, D4 DY and D5 DY could play the function of cyclically binding antigens and enhance the effect of antigen clearance at alow injection dose of 5 mg/kg.

SEQUENCE INFORMATION

[0188] Information of partial sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO Description  Sequence information  1 162 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYGYHWNWIRQFPGNKLE WIGYISYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  2 C27 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HVPYTFGGGTKLEIK  3 C26 D4 D5 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLE WIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  4 C26 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HHPYTFGGGTKLEIK  5 C27 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLE WIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  6 C32 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLE WIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  7 C32 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HLPYTFGGGTKLEIK  8 D3 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEW IGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYC ASGFDHWGQGTTLTVSS  9 D3 D5 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THVPYTFGGGTKLEIK 10 D4 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THLPYTFGGGTKLEIK 11 162 YGYHWN HCDR1 12 162 D3 D4 D5 YISYDGSVLYNPSLEN HCDR2 13 162 C26 C27 C32 GFDH D3 D4 D5 HCDR3 14 162 C26 C27 C32 RSSQSLVHSYGDTYLH LCDR1 15 162 C26 C27 C32 KVSNRFS D3 D4 D5 LCDR2 16 162 C27 D3 D5 SQNTHVPYT LCDR3 17 C26 C27 D4 D5 HGYHWN HCDR1 18 C26 YIHYDGSVLYNPSLEN HCDR2 19 C26 SQNTHHPYT LCDR3 20 C27 YINHDGSVQYNPSLEN HCDR2 21 C32 YRYHWN HCDR1 22 C32 YINYDGSVHYNPSLEN HCDR2 23 C32 D4 SQNTHLPYT LCDR3 24 D3 HGYHYN HCDR1 25 D3 D4 D5 RSSQSLVHSYGDNYLH LCDR1 26 General formula of X.sub.1X.sub.1YHX.sub.1N HCDR1 27 General formula of YIX.sub.4X.sub.5DGSVX.sub.6YNPSLEN HCDR2 28 General formula of RSSQSLVHSYGDX.sub.7YLH LCDR1 29 General formula of SQNTHX.sub.8PYT LCDR3 30 C26 C27 C32 D3 D4 EVQLQESGPGLVKPSQTLSLTCAVSGSSIT D5 HFR1 31 C26 C27 C32 D3 D4 WIRQFPGNKLEWIG D5 HFR2 32 C26 C27 C32 D3 D4 RVTITRDTSKNQFFLKLSSVTAEDTAKYYCAS D5 HFR3 33 C26 C27 C32 D3 D4 WGQGTTLTVSS D5 HFR4 34 C26 C27 C32 D3 D4 DVVMTQSPLSLPVTLGEPASISC D5 LFR1 35 C26 C27 C32 D3 D4 WYLQKPGQSPKLLIY D5 LFR2 36 C26 C27 C32 D3 D4 GVPDRFSGSGSGTDFTLKISRVETEDLGVYYC D5 LFR3 37 C26 C27 C32 D3 D4 FGGGTKLEIK D5 LFR4 38 4-28-02 QVQLQESGPGLVKPSQTLSLTCAVSGYSISSSNWWGW1RQPPGKGLE WIGYIYYSGSIYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYY CAR 39 2D-28-01 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQS PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 40 Human IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMTSRTPEVTCV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 Human κ light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDSAL constant region QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 42 Human IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region with V4 KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCV mutation VVDVSHEDPEVKFNWYVDGVEVHEAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK 43 Human IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region with DY KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV mutation VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNDAYPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 44 Primer 5′>GTTATTACTCGTGGCCCAGCCGGCCATGGCAGAGGTGCAGCTGC AGGAGTC <3′ 45 Primer 5′>CTCCAGCTTGTTCCCTGGGAACTGCCGGATCCAGTTSYRGTGGT RGYSGTRGGTGATGGAGCTACCAGA <3′ 46 Primer 5′>GTTCCCAGGGAACAAGCTGGAGTGGATTGGGYACMWCMRCYA CSACGGCAGCSWYCWSYACAATCCATCTCTCG <3′ 47 Primer 5′>GACTGTGAGAGTTGTGCCTTGGCCCCAGTGGTSGWRACCACTCG CACAGTA <3′ 48 Primer 5′>CCAGATCCGCCACCTCCACTCCCGCCTCCACCTGAGGAGACTGT GAGAGTTGTGCCTT <3′ 49 Primer 5′>GTGGAGGTGGCGGATCTGGAGGGGGTGGTAGCGATGTTGTGAT GACCCAATC <3′ 50 Primer 5′>CTTTGGAGACTGGCCTGGCTTCTGCAGGTACCAATGSWGGTRGK KGTCTCCATAGYKGTGRWS <3′ 51 Primer 5′>AGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAAC CGATTTTCTG <3′ 52 Primer 5′>TTTCCAGCTTGGTCCCCCCTCCGAAGKKGTRGKGRWSATGGKKG TKSTGAGAGCAGTAATAAAC <3′ 53 Primer 5′>TAGTCGACCAGGCCCCCGAGGCCTTTTATTTCCAGCTTGGTCCC CCCT <3′ 54 Signal peptide MGWSCIILFLVATATGVHS 55 Primer 5′- AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGA GTC 56 Primer 5′- GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGACGGTGG 57 Primer 5′- AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCA GTCTC 58 Primer 5′- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC

EXAMPLES

[0189] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention rather than limit the present invention.

[0190] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art know that the examples describe the present invention by way of example, and are not intended to limit the scope of protection sought to be protected by the present invention.

Example 1: Phage Screening of pH-Dependent Anti-HBsAg Antibodies

1.1 Determination of Mutation Sites for pH-Dependent Antibody Modification

[0191] The anti-HBV humanized antibody 162 (detailed in Chinese patent application 201610879693.5) developed in the laboratory was used as the parent antibody, and its variable region were modified for pH-dependent antigen binding. As shown in FIG. 1, the modified 162 could maintain the antigen-binding activity under neutral conditions, but its antigen-binding activity under acidic conditions was greatly reduced. The dissociated modified 162 could bind to intracellular FcRn so as to return to the plasma and bind to the antigen again, so that one molecule of the modified 162 with pH-dependent antigen binding ability could repeatedly bind and neutralize a plurality of molecules of antigen. Histidine was protonated under acidic conditions and was a key amino acid to bring the pH-dependent antigen binding properties. The 162 Fab had an analyzed crystal structure, the analyzed crystal structure was docked by simulation with an antigen short peptide, and part of the results was shown in FIG. 2, in which the blue structure represented the antigen short peptide, and the red structure represented part of the binding region of 162 antibody. According to the docking results, a total of 14 key amino acids for antigen and antibody binding were found. Considering that the simulated docking results had greater reference value, the amino acids on the interface and the amino acids on the both sides were selected for mutation, and 26 sites were determined.

1.2 Construction of Phage Library of pH-Dependent scFv Antbodies Derived from 162

[0192] Using the variable regions of the light and heavy chains of the 162 antibody as a template, the determined sites in the antibody variable region CDRs were mutated for pH-dependent modification, and the target fragments were amplified according to the primers in Table 2 to obtain the gene fragments coding the pH-dependent scFv antibodies derived from 162. PCR conditions were: 95° C., 5 min; 95° C., 30 s; 57° C., 30 s; 72° C., 30 s; 72° C., 10 min; for 25 amplification cycles; SOE-PCR reaction conditions were: 95° C., 5 min; 95° C., 30 s; 57° C., 30 s; 72° C., 30 s; 72° C., 10 min; for 5 amplification cycles. The amplified products were analyzed by agarose gel electrophoresis, and the amplification products were recovered/purified by using the DNA purification and recovery kit (TianGen, DP214-03), thereby obtaining the gene fragments H-K encoding the humanized scFv antibodies derived from 162. The structure of scFv antibodies was: NH.sub.2-VH-linker-VL-COOH, and the linker sequence could be (G.sub.4S).sub.3. Each of the gene fragments H-K was digested with SfiI, and then ligated to the vector pCGMT (from Scripps, Making chemistry selectable by linking it to infectivity) at a molar ratio of 10:1 (gene fragment:vector). The ligation products were transformed into competent Escherichia coli ER2738 by electroporation (electroporation conditions: 25 μF, 2.5 KV, 200 Ω). The transformed Escherichia coli was recovered in SOC medium for 45 min, and then 200 μL of bacterial solution was plated on LB plates (comprising 100 g/L ampicillin+tetracycline+2 g/mL glucose), and incubated by standing at 37° C. overnight. All colonies on the plates were the libraries that the mutation sites determined in the variable regions were randomly mutated into histidine, which were used for subsequent screening. Monoclonal colonies were picked out from the plates and sequenced to ensure the correctness of the sequences of recombinant vectors encoding the scFv antibodies. The schematic diagram of the recombinant vector (pCGMT-scFv) encoding the scFv antibody was shown in FIG. 3.

TABLE-US-00002 TABLE 2 Mutation primers for pH-dependent scFv antibodies derived from 162 Primer name Primer sequence VH-F SEQ ID NO: 44 HCDR1-R SEQ ID NO: 45 HCDR2-F SEQ ID NO: 46 HCDR3-R SEQ ID NO: 47 VH-R SEQ ID NO: 48 VK-F SEQ ID NO: 49 KCDR1-R SEQ ID NO: 50 KCDR2-F SEQ ID NO: 51 KCDR3-R SEQ ID NO: 52 VK-R SEQ ID NO: 53

1.3 Detection of Humanized scFv Antibodies

[0193] The library obtained in the previous step was screened for multiple rounds, and the positive monoclonal colonies obtained in the screening were cultured with 2×YT medium containing ampicillin (100 g/L) and glucose (2 g/mL) to reach an OD value of 0.6, and then added with M13KO7 for auxiliary super-infection. After 2 h, 100 g/L kanamycin was added and the super-infection was performed at 37° C. After 2 h, the culture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cell pellet was collected. The cell pellet was resuspended in a medium containing ampicillin and kanamycin (100 g/L), and cultured with shaking at 30° C. overnight. Subsequently, the culture was centrifuged at 12000 rpm for 10 min, the cells and supernatant were collected, and stored at 4° C. for testing.

[0194] An ELISA plate coated with HBsAg (200 ng/mL) antigen was used, and 100 μL of the supernatant to be tested was added to each well, and incubated at 37° C. for 1 h (two wells for each supernatant). Subsequently, the ELISA plate was washed once with PBST, and then the two wells of each supernatant were added with 120 μL of PBS with pH 7.4 and pH 6.0 respectively and incubated at 37° C. for 30 min. After washing with PBST of corresponding pH for 5 times, 100 μL , of anti M13-HRP diluted at 1:5000 was added, and incubated at 37° C. for 30 min. Subsequently, the ELISA plate was washed 5 times with PBST, and the substrate TMB solution was added. After 15 minutes of color development, the color reaction was terminated with H.sub.2SO.sub.4, and the reading was measured at OD450/630. The detection results of ELISA of the third round were shown in FIGS. 4A to 4D. The results showed that the phages displaying these scFv antibodies all had reactivity in ELISA detection and weakly bound to antigens at pH 6.0; six strains of pH-dependent phage antibodies with good effects were initially obtained, named C-26, C-27, C-32, D3, D4 and D5, respectively.

Example 2: Preparation of pH-Dependent Anti-HBsAg Antibodies

2.1 Construction of Recombinant Vector for Eukaryotic Expression

[0195] In the present invention, a large amount of antibody recombination needed to be carried out, so it was necessary to construct a set of light and heavy chain vectors that can efficiently recombine antibodies. In the present invention, the existing eukaryotic expression vector pTT5 in the laboratory was specially modified to construct a set of light and heavy chain recombinant vectors for double plasmid co-transfection. MGWSCIILFLVATATGVHS (SEQ ID NO: 54) was used as the signal peptide for the light and heavy chains. The sequences encoding the constant regions of the human antibody light and heavy chains were separately ligated to the downstream of signal peptide to construct a set of eukaryotic expression vectors pTT5-CH, pTT5-Cκ and pTT5-Cλ that facilitated antibody recombination.

[0196] The six scFv antibodies obtained in 1.3 were used to amplify the light and heavy chain variable region fragments with the primers in Table 3. The specific amplification reaction conditions were: 95° C., 5 min; 95° C., 30 s; 57° C., 30 s; 72° C., 30 s; 72° C., 10 min; for 25 amplification cycles. And the amplification products were recovered from the gel.

[0197] The laboratory-made Gibson assembly solution was used to ligate the above constructed eukaryotic expression vector with the recovered PCR product of antibody variable region gene (the primer carried a sequence homologous to the vector) to obtain the recombinant vectors VH+pTT5-CH (comprising the CH shown in SEQ ID NO: 40) and VH+pTT5-Cκ (comprising the CL shown in SEQ ID NO: 41). The recombinant vector was transformed into E. coli DH5α strain, plated on LB plate, and cultivated overnight in a 37° C. incubator. Monoclonal colonies were picked out from the plate and sequenced, and the sequencing results were subjected to sequence comparison using MEGA to confirm the correctness of its genes, and exclude the genes with wrong information.

TABLE-US-00003 TABLE 3 Primers for construction of eukaryotic expression vectors Primer name Primer sequence VH-F SEQ ID NO: 55 VH-R SEQ ID NO: 56 VK-F SEQ ID NO: 57 VK-R SEQ ID NO: 58

2.2 Small- and Large-Scale Expression of Antibody Genes

[0198] The constructed recombinant vectors VH+pTT5-CH and VH+pTT5-Cκ were co-transfected into HEK293 cells, and double plasmids for small-scale expression were co-transfected into a 24-well plate, 500 μL per well; if the cell supernatant of small-scale expression had antigenic activity, the transfection system was enlarged to 100 mL (determined by the amount of antibody used) of FreeStyle™ 293F suspension cells (the cell density was about 2×10.sup.6 cells/ml). The transfected cells were cultured in a shake flask in a 32° C., 5% CO.sub.2 incubator, and the supernatant was collected after 7 days of expression.

2.3 Antibody Purification

[0199] The cell expression supernatant was collected and purified with a Protein A column according to the manufacturer's instructions. The specific steps were as follows: the harvested cell culture supernatant was centrifuged at 8000 rpm for 10 min, the supernatant was retained, the pH value was adjusted to 8.4 with dry powder Na.sub.2HPO.sub.4, and then filtered with a filter membrane with 0.22 μm pore diameter. 10 mL of Sepharose 4B medium coupled with Protein A was loaded into column, it was connected to AKTA Explorer100 system, the pump A was connected to 0.2 M disodium hydrogen phosphate solution, and the pump B was connected to 0.2 M citric acid solution. Detection wavelength was UV 280 nm, flow rate was 5 mL/min, and the sample injection proportion of pumps A/B was adjusted. The column was first washed with 100% B (pH 2.3) to remove protein impurities, the pH was balanced with 10% B (pH 8.0), the signal at the detection wavelength returned to zero after it was stable, then the sample was loaded. After the flow through peak passed, 10% B was used for balance until the signal at the detection wavelength was reduced to zero and was stable, elution was performed using 70% B (pH 4.0), and the elution peak was collected. The elution peak sample was dialyzed into PBS buffer and subjected to assay of concentration and SDS-PAGE and HPLC analysis to determine the purity of IgG antibody.

Example 3: Property Analysis and Functional Evaluation of pH-Dependent Anti-HB sAg Antibodies

[0200] Through the method of Example 1, six strains of pH-dependent phage antibodies that bound to HBsAg were obtained by preliminary screening, named C26, C27, C32, D3, D4 and D5, respectively. Furthermore, the small-scale eukaryotic expression and purification of the 6 strains of phage antibodies were carried out by the method of Example 2. The VH and VL amino acid sequences of the 6 antibodies were shown in Table 4 below. In addition, the CDR sequences of the 6 antibodies were determined, and the CDR amino acid sequences of the heavy chain variable regions and the light chain variable regions thereof were shown in Table 5. The mutation sites that endowed C26, C27, C32, D3, D4 and D5 with pH-dependent antigen binding properties to HBsAg were summarized in FIG. 5.

TABLE-US-00004 TABLE 4 Amino acid sequences of C26/C27/C32/D3/D4/D5 light and  heavy chain variable regions Sequence  SEQ  name ID NO Amino acid sequence  C26 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSS C26 VK 4 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHHYTEGGGTKLEIK C27 VH 5 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL EWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSS C27 VK 2 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK C32 VH 6 EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKL EWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSS C32 VK 7 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIK D3 VH 8 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLE WIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKY YCASGFDHWGQGTTLTVSS D3 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK D4 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSS D4 VK 10 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIK D5 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSS D5 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG QSPKLLIYKVSNRESGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK

TABLE-US-00005 TABLE 5 CDR sequences of C26/C27/C32/D3/D4/D5  light and heavy chains C26 VH CDR1 HGYHWN SEQ ID NO: 17 VH CDR2 YIHYDGSVLYNPSLEN SEQ ID NO: 18 VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHHPYT SEQ ID NO: 19 C27 VH CDR1 HGYHWN SEQ ID NO: 17 VH CDR2 YINHDGSVQYNPSLEN SEQ ID NO: 20 VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHVPYT SEQ ID NO: 16 C32 VH CDR1 YRYHWN SEQ ID NO: 21 VH CDR2 YINYDGSVHYNPSLEN SEQ ID NO: 22 VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHLPYT SEQ ID NO: 23 D3 VH CDR1 HGYHYN SEQ ID NO:24 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHVPYT SEQ ID NO:16 D4 VH CDR1 HGYHWN SEQ ID NO:17 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHLPYT SEQ ID NO:23 D5 VH CDR1 HGYHWN SEQ ID NO:17 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO:15 VL CDR3 SQNTHVPYT SEQ ID NO:16

[0201] The inventors first performed small-scale transfection for the six monoclonal antibodies; after quantifying the supernatant after transfection, the pH-dependent antigen binding ability with HBsAg was detected by ELISA method, and the antibody concentration was uniformly diluted to 1111.11 ng/mL. Subsequently, 20% NBS was used to carry out a 3-fold concentration gradient dilution of the antibody concentration, for a total of 8 concentration gradients. Subsequently, the diluted antibody was added to a commercial HBsAg plate (purchased from Beijing Wantai), and incubated at 37° C. for 1 h (two wells per supernatant). Subsequently, the ELISA plate was washed once with PBST and spin-dried. Then, the two wells of each supernatant were added with 120 μL, of pH 7.4 and pH 6.0 PBS respectively and incubated at 37° C. for 30 min. It was then washed 5 times with PBST of corresponding pH and spin-dried. Subsequently, GAH-HRP-labeled secondary antibody was added, incubated for 30 min, the plate was washed 5 times with PBST, and spin-dried. The substrate TMB solution was added. After 15 minutes of color development, the color reaction was terminated with H.sub.2SO.sub.4, and the reading was measured at OD450/630.

[0202] The results were shown in FIGS. 6A to 6B. It could be seen from the results that the candidate molecules all could maintain an antigen-binding activity comparable to that of the parent antibody 162 at the neutral pH, and the antigen-binding activity was significantly reduced at pH 6.0. The EC50 results were summarized in Table 6.

TABLE-US-00006 TABLE 6 EC50 values of pH-dependent activity detection for C26, C27, C32, D3, D4 and D5 EC50 in pH 6.0 EC50 in pH 7.4 EC50(pH 6.0)/ antibody (ng/mL) (ng/mL) EC50(pH 7.4) C26 331.30 37.20 8.90 C27 949.60 491.30 1.93 C32 2255.00 1333 1.69 D3 473.10 35.43 13.35 D4 188.10 37.16 5.06 D5 60.04 21.04 2.85

Example 4: Construction and Functional Evaluation of Scavenger Antibody

[0203] The pH-dependent antibody needs to enter the cell to exert its pH-dependent antigen-binding activity. Therefore, if the first limiting factor of cell entry is not broken, the subsequent pH-dependent antigen-binding properties will have no chance to “play”. Therefore, in this example, the scavenger antibody was obtained by further mutation of amino acids in the Fc region, which could enhance the binding to hFcRn receptor at neutral pH, or enhance the binding to FcγRs receptor. As shown in FIG. 7, the scavenger antibody is located outside the cell and played the role of a “transportation helper” that reciprocally transported antigens into the cell, thereby extremely extending the antibody half-life, and it could bind to antigen again, thereby improving the efficiency of cell entry of antigen, and significantly improving the clearance efficiency.

[0204] The C26, D3, D4 and D5 were selected as the antibodies for subsequent evaluation, and subjected to Fc DY (K326D, L328Y) mutations to enhance the affinity with mFcγRII under neutral conditions (the modification of C26 Fc was commissioned to the General Biologicals, order number G122413) to obtain scavenger antibodies C26 DY, D3 DY, D4 DY and D5 DY that bound to mFcγRII. The above antibodies were subjected to large-scale eukaryotic expression and purification, and the specific steps were same as Examples 1.2 and 1.3.

[0205] FIGS. 8A to 8B showed the protein gel results of the original antibody and the modified antibodies. FIG. 8A: the picture of protein gel of the original antibody, in which the 162 was a positive control. The results showed that the expressed original antibody is single-component. FIG. 8B: the picture of protein gel of antibodies with DY modification, in which the 162 was a positive control. The results showed that the expressed antibodies with DY modification are single-component.

4.1 Evaluation of pH-Dependent Antigen-Binding Activity of Scavenger Antibodies Binding to mFcγRII

[0206] For the original antibody and the antibodies with DY modification after expression and purification, the inventors used the ELISA method to detect their pH-dependent antigen binding ability to HBsAg. First, a BCA protein quantification kit was used to determine the concentrations of the purified antibodies, and the antibodies were uniformly diluted to have a concentration of 1111.11 ng/mL. Subsequently, 20% NBS was used to carry out a 3-fold concentration gradient dilution for the antibody concentrations, for a total of 8 concentration gradients. Subsequently, the diluted antibody was added to a commercial HBsAg plate (purchased from Beijing Wantai) and incubated at 37° C. for 1 h (two wells per supernatant). Subsequently, the ELISA plate was washed once with PBST and spin-dried. Then the two wells of each supernatant were added with 120 μL of pH 7.4 PBS and pH 6.0 PBS respectively incubated at 37° C. for 30 min, washed 5 times with PBST of corresponding pH and spin-dried. Subsequently, GAH-HRP-labeled secondary antibody was added, and incubated for 30 min, the plate was washed 5 times with PBST, and spin-dried. And the substrate TMB solution was added. After 15 minutes of color development, the color reaction was terminated with H.sub.2SO.sub.4, and the reading was measured at OD450/630.

[0207] The results were shown in FIGS. 9A to 9D, in which the C26, D3, D4, D5 and their DY modification antibodies all had an antigen-binding activity equivalent to that of antibody 162, but showed a weak binding to antigen at pH 6.0, thereby exhibiting a good pH-dependent antigen-binding activity.

4.2 Verification of Function at Cellular Level for Scavenger Antibodies Binding mFcγRII

4.2.1 Labeling HBsAg with 488 Fluorescence

[0208] Take the labeling of 1 mg HBsAg as an example, the whole process was protected from light.

[0209] (1) 1 mL of 1 mg/mL HBsAg was dialyzed into borate buffer (PH 8.5, 500 mL), 4° C., 4 h;

[0210] (2) the molar ratio of HBsAg to 488 label was 1:5, and 0.1988 mg of 488 fluorescence was required after calculation;

[0211] (3) 10 mg/mL of 488 fluorescence solution was prepared with DMF and mixed well;

[0212] (4) 19.88 pL of 488 fluorescence was added to 1 mL of the dialyzed HBsAg, mixed well, and incubated at room temperature for 1 h;

[0213] (5) the incubation mixture was dialyzed into PBS at 4° C. overnight.

4.2.2 Immunofluorescence Experiment Based on Mouse Primary Macrophages

[0214] (1) 4 days before the experiment, 1.5 mL of 3% sodium thioglycolate solution was injected into the abdominal cavity of each mouse, without injecting into the intestine;

[0215] (2) two mice were executed and soaked in 75% alcohol for 3 minutes;

[0216] (3) the mouse was horizontally fixed on a foam board to expose the abdomen; the abdominal skin was cut with tissue scissors, the peritoneum was disinfected and incised to expose the abdominal cavity, the abdominal incision skin was pulled by two toothed forceps hold in the left hand and fixed, 1640 culture medium was pipetted by Pasteur pipette hold in the right hand for peritoneal lavage with 4 mL/time, for a total of two times. The pipette was used to gently and fully stir the abdominal cavity to make the lavage more fully and thoroughly. After fully stirring for about 2 minutes and standing for about 5 minutes to fully isolate the macrophages, the lavage solution was pipetted and transferred into a centrifuge tube;

[0217] (4) 4° C., 1100 g, 5 min;

[0218] (5) the supernatant was carefully discard, the cells were washed twice with 1640 medium, 4° C., 1100 g, 5 min, the supernatant was discarded, and the cells were resuspended in RPM1640;

[0219] (6) After counting the cells, the cell density was adjusted to 10.sup.6 cells/mL, the cells were cultured on a 24-well glass-bottom cell imaging culture plate, 250 μL/well, the medium was replaced after 2 h, and washing was carried out once with RPM1640, after the non-adherent cells were discarded, the cells were incubated overnight in a 37° C., CO.sub.2 incubator;

[0220] (7) the antibody and antigen labeled with the corresponding fluorescence were diluted in serum-free medium to: 800 ng/mL for antigen and 20 μg/mL for antibody;

[0221] (8) 125 μL of the antigen and 125 μL of the antibody were mixed uniformly, and then were allowed to stand for 1 hour in a 37° C., CO.sub.2 incubator;

[0222] (9) the cell supernatant in the cell imaging culture plate was discarded, the antigen-antibody complex was added, shaken evenly, and allowed to stand in a 37° C., CO.sub.2 incubator for 2 hours;

[0223] (10) the supernatant was discarded, and 1 mL of sterile PBS incubated at 37° C. in advance was used to “wash” the cell surface 3 to 5 times, and then totally removed by a pipette;

[0224] (11) the 1:2000 diluted Dio was added in an amount that immersed the cells, and allowed to stand at room temperature for 20 min;

[0225] (12) the supernatant was discarded, and 1 mL of sterile PBS incubated at 37° C. in advance was used to “wash” the cell surface 3 to 5 times, and then totally removed by a pipette;

[0226] (13) a live cell nuclear dye was added (2 drops were added to 1 mL of volume), allowed to stand at room temperature for 20 min, and placed in a high-content imager for imaging.

[0227] The results of the experiment were shown in FIG. 10A. It could be seen from the results that the DY modification enhanced the phagocytosis of mouse macrophages to the antigen-antibody complexes, leading to more antigen degradation.

4.2.3 Validation by Chemiluminescence Method Based on human THP-1 Phagocytic Cells

[0228] (1) Adherent THP-1 cells were coated on a plate at 2×10.sup.5/well, added with 1640 medium containing 10% serum, placed in a carbon dioxide incubator and cultured at 37° C. for 24 h;

[0229] (2) HBsAg was diluted with serum-free 1640 medium to 800 ng/mL, and the antibody to be tested with 20 ug/mL as the initial concentration was subjected to 2-fold gradient dilution, for a total of 11 gradients. 300 uL of the diluted HBsAg and 300 uL of the antibody to be tested were mixed at ratio of 1:1, and allowed to stand at 37° C. for 1 h;

[0230] (3) the THP-1 cell supernatant was discarded, 250 uL of the HBsAg-antibody mixture was added to the THP-1 cells, placed in a carbon dioxide incubator and cultured at 37° C. for 1 hour;

[0231] (4) the THP-1 cell supernatant was discarded, and washed 3 times with sterile PBS;

[0232] (5) 120 uL of DDM cell lysis solution was added to each well of THP-1 cells and allowed to stand and react at 4° C. for 1 hour;

[0233] (6) the supernatant of the lysate was subjected to detecting the concentration of HBsAg by using hepatitis B surface antigen quantitative detection kit (Beijing Wantai).

[0234] The results of the experiment were shown in FIG. 10B. It could be seen from the results that the DY modification enhanced the phagocytosis of human THP-1 phagocytic cells to the antigen-antibody complexes.

4.3 Determination of Therapeutic Effect of Scavenger Antibody Binding to mFcγRI in Animal Models

[0235] HBV transgenic mice were selected as animal models. The C26 DY, D3 DY, D4 DY and D5 DY scavenger antibodies and 162 were injected at a single dose of 5 mg/kg via tail vein (4 mice in each group) to the 6-8 weeks old HBV transgenic mice. By detecting the concentrations of HBsAg, antibody and HBV DNA in serum, the antigen clearance rates and antibody half-life of the scavenger antibodies in vivo were analyzed.

Quantitative Detection of HBsAg

[0236] (1) Preparation of reaction plate: the mouse monoclonal antibody HBs-45E9 was diluted with 20 mM PB buffer (Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer, pH 7.4) to 2 μg/mL, and 100 μL of coating solution was added to each well of a chemiluminescence plate, and the coating was carried out at 2-8° C. for 16-24 h, followed by another 2 hours at 37° C., the plate was washed once with PBST washing solution, and spin-dried. After washing, 200 μL of blocking solution was added to each well and the blocking was carried out at 37° C. for 2 h. Subsequently, the blocking solution was discarded, and the plate was placed in a drying room to dry, and stored at 2-8° C. for later use.

[0237] (2) Sample dilution: the collected mouse serum was diluted with a PBS solution containing 20% NB S (newborn bovine serum) at two gradients of 1:30 and 1:150 for subsequent quantitative detection.

[0238] (3) Sample denaturation treatment: 15 μL of the above-diluted serum sample was mixed well with 7.5 μL of denaturation buffer (15% SDS, dissolved in 20 mM PB7.4), and reacted at 37° C. for 1 h. Then, 90 μL of stop buffer (4% CHAPS, dissolved in 20 mM PB7.4) was added, and mixed well.

[0239] (4) Sample reaction: 100 μL of the above-mentioned denatured serum sample was added to a reaction plate, and reacted at 37° C. for 1 hour. Subsequently, the reaction plate was washed 5 times with PBST and spin-dried.

[0240] (5) Enzymatic label reaction: the HBs-A6A7-HRP reaction solution was added at 100 μL/well to a chemiluminescence plate, and reacted at 37° C. for 1 h. Then, the plate was washed 5 times with PBST and spin-dried.

[0241] (6) Luminescence reaction and measurement: a luminescence solution (100 μL/well) was added to the chemiluminescence plate, and light intensity measurement was performed.

[0242] (7) Calculation of HBsAg concentration in mouse serum sample: parallel experiments were performed using standard products, and a standard curve was drawn based on the measurement results of the standard products. Then, the light intensity measurement value of the mouse serum sample was substituted into the standard curve, and the concentration of HBsAg in the serum sample to be tested was calculated.

[0243] The results of the detection of HBsAg in the serum were shown in FIG. 11A and FIG. 11C. It could be seen from FIG. 11A that the scavenger antibody with DY modification C26 DY had an antibody clearance ability stronger more than one order of magnitude than that of 162, which was consistent with the detection results of antibody half-life in the serum (FIG. 11B). In the comparison of the concentrations of antibodies in the serum, the half-life of C26 DY was longer than that of 162 by nearly 12 days, which indicated that the scavenger antibody C26 DY had the function of circularly and reciprocally binding antigen, thereby increasing the duration time of antigen clearance. The experimental results in FIG. 11C showed that the antigen clearance ability of D3 DY, D4 DY and D5 DY was equivalent to that of C26 DY, and the duration time was longer than that of C26 DY, indicating that at a low injection dose of 5 mg/kg, the scavenger antibodies with DY modification D3 DY, D4 DY and D5 DY had a better function of circularly and reciprocally binding antigen, thereby performing better antigen clearance.

Example 5: Affinity Determination of 162 and C26

[0244] HBsAg was dissolved in sodium acetate (pH 4.5) at 5 μg/mL, and the chip coating program was run on the Biacore 3000 device to coat HBsAg on the CM5 chip. The coating volume of HBsAg was 2400 RU. The analyte was diluted 2-fold from 100 nM to prepare samples of 7 concentrations. The affinity determination program was run on the Biacore 3000 device, the flow rate was set to 50 μl/min, the binding time was set to 90 s, the dissociation time was set to 600 s, the temperature of sample chamber was set to 10° C., the regeneration solution was 50 mM NaOH, the regeneration flow rate was set to 50 μL/min, and the regeneration time was set to 60 s. The results were summarized in Table 7.

TABLE-US-00007 TABLE 7 Affinity determination of 162 and C26 KD(M) in KD(M) in KD(pH 6.0)/ Antibody pH 7.4 pH 6.0 KD(pH 7.4) 162 9.34E−10 C26 3.45E−09 9.82E−09 2.85

[0245] Although the specific embodiments of the present invention have been described in details, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are within the protection scope of the present invention. All of the present invention is given by the appended claims and any equivalents thereof.