NOVEL ANTI-HEPATITIS B VIRUS ANTIBODY AND USES THEREOF

Abstract

Disclosed are antibodies to anti-hepatitis B surface antigen (HBsAg) (especially humanized antibodies), nucleic acid molecules encoding same, methods for preparing same, and pharmaceutical compositions containing same. The antibodies have higher affinity for HBsAg at neutral pH than at acidic pH, thereby significantly enhancing the virus clearance efficiency and prolonging the virus inhibition time. The antibodies and the pharmaceutical compositions can be used for preventing and/or treating HBV infections or diseases related to HBV infections (e.g., hepatitis B), for neutralizing the virulence of HBV in a subject (e.g., a human), for reducing the serum level of HBV DNA and/or HBsAg in the body of the subject, or for activating the humoral immune response of the subject (e.g., a chronic HBV infected or chronic hepatitis B patient) 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 GGSIX.sub.1X.sub.2NFW (SEQ ID NO: 50), wherein X.sub.1 is selected from T or H, and X.sub.2 is selected from S or H; (ii) HCDR2 with a sequence of X.sub.3GX.sub.4X.sub.5X.sub.6X.sub.7T (SEQ ID NO: 51), wherein X.sub.3 is selected from S or H, X.sub.4 is selected from P, S or Y, X.sub.5 is selected from G or D, X.sub.6 is selected from T or H, and X.sub.7 is selected From Y or H; and (iii) HCDR3 with a sequence of ARSHX.sub.8YGX.sub.9X.sub.10DYAFDF (SEQ ID NO: 52), wherein X.sub.8 is selected from D or H, X.sub.9 is selected from S or H, and X.sub.10 is selected from H or N; and/or, (b) a light chain variable region (VL) comprising the following 3 CDRs: (iv) LCDR1 with a sequence of QDIX.sub.11X.sub.12S (SEQ ID NO: 53), wherein X.sub.11 is selected from S or H, and X.sub.12 is selected from S, Y or H; (v) LCDR2 with a sequence of YAN (SEQ ID NO: 12); and (vi) LCDR3 with a sequence of QQYHX.sub.13LPLT (SEQ ID NO: 54), wherein X.sub.13 is selected from S or Y.

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: 8, 14, 17, 20; (ii) HCDR2, consisting of a sequence selected from: SEQ ID NOs: 9, 15, 21, 18; and (iii) HCDR3, consisting of a sequence selected from the following: SEQ ID NOs: 10, 16, 22; 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: 11, 19, 23; (v) LCDR2, consisting of a sequence shown in SEQ TD NO: 12; and (vi) LCDR3, consisting of a sequence shown in SEQ ID NO: 13.

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: 8, HCDR2 shown in SEQ ID NO: 9, HCDR3 shown in SEQ ID NO: 10; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 11, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13; (2) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ TD NO: 11, LCDR2 shown in SEQ TD NO: 12, LCDR3 shown in SEQ ID NO: 13; (3) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 20, HCDR2 shown in SEQ ID NO: 21, HCDR3 shown in SEQ ID NO: 22; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ TD NO: 23, LCDR2 shown in SEQ TD NO: 12, LCDR3 shown in SEQ ID NO: 13; or, (4) 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: 10; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13.

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 IGHV4-4*08, and a light chain framework region contained in an amino acid sequence encoded by IGKV1-39*01, 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: a VH FR1 as shown in SEQ ID NO: 24, a VH FR2 as shown in SEQ ID NO: 25, a VH FR3 as shown in SEQ ID NO: 26, and a VH FR4 as shown in SEQ ID NO: 27; preferably, the VL of the antibody or antigen-binding fragment thereof comprises: a VL FR1 as shown in SEQ ID NO: 28, a VL FR2 as shown in SEQ ID NO: 29, a VL FR3 as shown in SEQ ID NO: 30, and a VL FR4 shown in SEQ ID NO: 31.

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: 1, 3, 4 and 6; (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: 1, 3, 4, 6; 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: 1, 3, 4, 6; 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, 5, and 7; (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, 5, 7; 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, 5, 7; 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: 1 and a VL with a sequence shown in SEQ ID NO: 2; (2) a VH with a sequence shown in SEQ TD NO: 3 and a VL with a sequence shown in SEQ ID NO: 2; (3) a VH with a sequence shown in SEQ ID NO: 4 and a VL with a sequence shown in SEQ ID NO: 5; or (4) a VH with a sequence shown in SEQ ID NO: 6 and a VL with a sequence shown in SEQ ID NO: 7.

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 TD NO: 58.

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: 47 or 48.

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: 57.

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: 1 and a CH shown in SEQ ID NO: 57, and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 58; (2) a heavy chain comprising a VH shown in SEQ ID NO: 1 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: 58; (3) a heavy chain comprising a VH shown in SEQ ID NO: 1 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: 58; (4) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 57, and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 58; (5) 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: 2 and a CL shown in SEQ TD NO: 58; (6) 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: 2 and a CL shown in SEQ ID NO: 58; (7) a heavy chain comprising a VH shown in SEQ ID NO: 4 and a CH shown in SEQ ID NO: 57, and a light chain comprising a VL shown in SEQ ID NO: 5 and a CL shown in SEQ ID NO: 58; (8) a heavy chain comprising a VH shown in SEQ ID NO: 4 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO: 5 and a CL shown in SEQ ID NO: 58; (9) a heavy chain comprising a VH shown in SEQ ID NO: 4 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 5 and a CL shown in SEQ ID NO: 58; (10) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 57, and a light chain comprising a VL shown in SEQ ID NO: 7 and a CL shown in SEQ TD NO: 58; (11) 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: 58; or (12) 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: 58.

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 11, 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

[0172] 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.

[0173] FIG. 2 shows a simulation of the three-dimensional structure of M1D variable region. The rod structure represents the CDR region, the blue represents the VK CDR, the green represents the VH CDR, and the red represents the surface amino acids. A total of 25 surface amino acids can be found in the CDR region.

[0174] FIG. 3 shows a schematic diagram of the recombinant vector (pCGMT-scFv) encoding the scFv antibody, in which the structure of the scFv antibody is: NH.sub.2—VH-linker-VL-COOH.

[0175] FIGS. 4A to 4C show the ELISA results of the phage library displaying the pH-dependent scFv antibody derived from M1D 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 of the pH-dependent scFv antibody derived from M1D after the third round of screening, wherein the abscissa represents the phage antibody number or dilution factor, 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 6 single clones with high OD.sub.(450/630) value at pH 7.4 in the third round and showing the largest difference in 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, the ordinate represents the OD value. The results show that the pH-dependent antigen binding effect is better presented after gradient dilution, among which A31-73 shows the best effect. FIG. 4C: the preliminary results of pH-dependent binding detection of scFv antibody phage to HBsAg for the 24 excellent candidates of phage-scFv after the fourth round of screening with 4 gradients and 5-fold dilution, in which the abscissa represents the dilution factor, the ordinate represents the OD value. The results show that A41-8, A42-12, and A42-23 are better than A31-73, while A44-1 and A44-20 are equivalent to A31-73.

[0176] FIG. 5 shows a summary of the mutation sites of A31-73, A42-13, A42-23 and A41-8.

[0177] FIG. 6 shows the HPLC results of A31-73, A42-12, A42-23 and A41-8 in Example 2, in which MID-C/S is a positive control, referring to different batches of M1D antibodies, and it can be seen from the results that the four antibodies are single-component.

[0178] FIG. 7 shows the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for A31-73, A42-12, A42-23 and A41-8 in Example 3, in which the abscissa represents the antibody concentration (Log 10 ng/ml), the ordinate represents the OD value. The results show that the four antibodies all can maintain the equivalent antigen-binding activity at neutral pH of the parent antibody, and all have a significant decrease in the antigen-binding activity under the condition of pH 6.0, in which A31-73 is better than A42-12, A42-23 and A41-8.

[0179] FIGS. 8A to 8B show the results of the therapeutic effect of A31-71, A41-8, A42-12 and the parental M1D in HBV transgenic female mice in Example 3. FIG. 8A: the clearance effect of HBsAg in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the level of HBsAg in the mouse serum after clearance (log 10 U/ml). FIG. 8B: the change of the antibody concentration in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the antibody concentration (Log 10 ng/ml). The results show that the HBsAg level of MID rebounds back to the baseline after about 10 days, while our modified antibodies rebound back to the baseline after about 15 days; regarding the antibody concentration in the serum, M1D has the lowest antibody concentration on the day 10, while the three modified antibodies still can be detectable on the day 15, indicating that the pH-dependent modified antibodies show an ability of clearing HBsAg comparable to that of the parent, and they have an antigen inhibition time longer than that of the parent, that is, their clearance effect can be exerted for a longer time.

[0180] FIG. 9 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. As shown in FIG. 9, the 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.

[0181] FIG. 10 shows the HPLC results of two molecules A31-73 and A41-8 in Example 4, in which the red and the green represent irrelevant positive controls. It can be seen from the results that all antibodies are single-component.

[0182] FIG. 11 shows the detection results of binding to HBsAg at pH 7.4 and pH 6.0 for A31-73 V4 and A41-8 V4 in Example 4, in which the abscissa represents the antibody concentration (Log 10 ng/ml), and the ordinate represents OD value. The results show that both molecules can maintain the antigen-binding activity and pH-dependent antigen-binding activity at neutral pH of the parent antibody, and the pH-dependent antigen-binding activity is slightly enhanced.

[0183] FIGS. 12A to 12B show the immunofluorescence results on MDCK cell of A31-73 V4 and A31-73 (FIG. 12A), and A41-8 V4 and A41-8 (FIG. 12B) in Example 4. The green fluorescence represents hFcRn, the blue fluorescence represents the nucleus, and the red fluorescence represents HBsAg. The results show that the antigen fluorescence intensities in the cells treated with A31-73 V4 and A41-8 V4 variants are significantly higher than those of the cells treated with A31-73 and A41-8, indicating that the scavenger antibody with V4 modification can efficiently transport more antigens into the cells. It is confirmed that the V4 mutation in the scavenger antibody can enhance the binding of antibody to hFcRn under neutral conditions. After the antigen-antibody complex enters the cell, the antigen-antibody complex is dissociated under the intracellular pH of only about 6.0, and the antibody returns to the cell surface to perform the antigen-binding function again. The enhancement of scavenger antibody's ability to bind to hFcRn under neutral conditions can improve the subsequent pH-dependent antigen-binding properties.

[0184] FIGS. 13A to 13C show the therapeutic effects of the two scavenger antibodies A31-73 V4 and A41-8 V4, M1D and negative control 16G12 by single injection via tail vein at dose of 20 mg/kg in hFcRn transgenic mice with stable HBV virus titer in Example 4. FIG. 13A: the clearance effect of HBsAg in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the clearance level of HBsAg in mouse serum (log 10 IU/ml). FIG. 13B: the clearance effect of HBV DNA in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the clearance level of HBV DNA in mouse serum (log 10 IU/ml). FIG. 13C: change in the antibody concentration in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the antibody concentration (Log 10 ng/ml). The results show that the scavenger antibodies have an antigen clearance ability comparable to that of MID, but have a longer inhibition time. After M1D exerts the maximum antigen clearance effect, the antigen quickly begins to rebound, and M1D starts to fail on the day 14, and the HBsAg in serum returns to the baseline level (based on the negative control 16G12). However, after the two scavenger antibodies exert their maximum clearance effect, they can still maintain a low antigen level for a long time, and rebound slowly. In the A41-8 V4 treatment group, HBsAg rebounds back to the baseline in about 30 days. A31-73 V4 showed a better performance, after the expected end of the experiment, HBsAg still does not return to the baseline level, which is consistent with the detection results of antibody half-life in serum.

[0185] FIG. 14 shows the protein gel results of A31-73 DY in Example 4, and M1D is a positive control. It can be seen from the results that all antibodies are single-component.

[0186] FIG. 15 shows the detection results of A31-73 DY binding to HBsAg at pH 7.4 and pH 6.0 in Example 4, wherein the abscissa represents the antibody concentration (Log 10 ng/ml), and the ordinate represents the OD value. The results show that A31-73 DY can maintain the antigen-binding activity and pH-dependent antigen-binding activity at neutral pH of the parent antibody, and the pH-dependent antigen-binding activity is slightly enhanced.

[0187] FIG. 16 shows the results of immunofluorescence experiment for A31-73 DY and A31-73 on murine primary macrophages in Example 4, wherein the green fluorescence represents hFcRn, the blue fluorescence represents the nucleus, and the red fluorescence represents HBsAg. The results show that the DY modification enhances the phagocytosis of antigen-antibody complexes by murine macrophages.

[0188] FIGS. 17A to 17C show the therapeutic effects of A31-73 DY scavenger antibody and MID in Example 4 after single injection via tail vein at dose of 5 mg/kg in HBV transgenic mice. FIG. 17A: the clearance effect of HBsAg in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), the ordinate represents the level of HBsAg in mouse serum after clearance (log 10 IU/ml). FIG. 17B: the clearance effect of HBV DNA in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the clearance level of HBV DNA in mouse serum (log 10 IU/ml). FIG. 17C: change in the antibody concentration in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the antibody concentration (ng/ml). The results show that the scavenger antibody A31-73 DY with DY modification is more than an order of magnitude stronger than M1D in antigen clearance. A31-73 DY can not only quickly clear HBsAg in HBV transgenic mice, but also effectively reduce the HBV DNA titer in mice, which is consistent with the detection results of antibody half-life in serum. Comparing the antibody concentration in serum, the half-life of A31-73 DY is longer than that of M1D by nearly 8 days, which shows that the scavenger antibody A31-73 DY has the function of reciprocally binding antigen, thereby increasing the time of antigen clearance.

[0189] FIGS. 18A to 18C show the therapeutic effect of scavenger antibody A31-73 DY and negative control 16G12 in Example 4 after the injection of scavenger antibody at a dose of 5 mg/kg for 5 times in an HBV mouse model which is obtained by transfection by adeno-associated virus. FIG. 18A: a schematic diagram of this animal experiment, the number represents the number of days, the red arrow represents the time point of blood collection, and the white arrow represents the time point of administration. FIG. 18B: the clearance effect of HBsAg in mouse serum, wherein the abscissa represents the number of days after antibody injection (d), and the ordinate represents the clearance level of HBsAg in mouse serum (log 10 IU/ml). FIG. 18C: relative titers of Anti-HBs in mouse serum, wherein the abscissa represents the name of antibody, and the ordinate represents the relative titer of Anti-HBs (log 10 OD450/OD630). The results show that the scavenger antibody A31-73 DY breaks through the limitation of traditional antibodies and achieves the treatment at low-dose and low-frequency, the HBsAg titer in mice is long-term inhibited at a level of below 100 IU/mL, and the humoral immune response is activated in muse after treatment, allowing the production of Anti-HBs.

SEQUENCE INFORMATION

[0190] The 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 A31-73 QVQLQESGPGLVKPSETLSLTCTVSGGSIHSNFWSWIRQPPGKGLE VH WIGYISGPGHHTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARSHDYGHHDYAFDFWGQGTTVTVSS 2 A31-73 A42-12 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPKL VK LIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYH YLPLTFGGGTKVEIK 3 A42-12 VH QVQLQESGPGLVKPSETLSLTCTVSGGSIHHNFWSWIRQPPGKGLE WIGYIHGPGHYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARSHDYGSNDYAFDFWGQGTTVTVSS 4 A42-23 VH QVQLQESGPGLVKPSETLSLTCTVSGGSITHNFWSWIRQPPGKGLE WIGYISGYDTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARSHDYGHHDYAFDFWGQGTTVTVSS 5 A42-23 VK DIQMTQSPSSLSASVGDRVTITCRATQDIHHSLNWYQQKPGKAPK LLIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFATYYCQQY HYLPLTFGGGTKVEIK 6 A41-8 VH QVQLQESGPGLVKPSETLSLTCTVSGGSITSNFWSWIRQPPGKGLE WIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARSHHYGSNDYAFDFWGQGTTVTVSS 7 A41-8 VK DIQMTQSPSSLSASVGDRVTITCRATQDISYSLNWYQQKPGKAPK WYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFATYYCQQY HYLPLTFGGGTKVEIK 8 A31-73 GGSIHSNFW HCDR1 9 A31-73 SGPGHHT HCDR2 10 A31-73 A42-23 ARSHDYGHHDYAFDF HCDR3 11 MID A31-73 QDISSS A42-12 LCDR1 12 MID A31-73 YAN A42-12 A42-23 A41-8 LCDR2 13 A31-73 A42-12 QQYHYLPLT A42-23 A41-8 LCDR3 14 A42-12 GGSIHHNFW HCDR1 15 A42-12 HGPGHYT HCDR2 16 MID A42-12 ARSHDYGSNDYAFDF HCDR3 17 A42-23 GGSITHNFW HCDR1 18 A42-23 SGYDTYT HCDR2 19 A42-23 QDIHHS LCDR1 20 MID A41-8 GGSITSNFW HCDR1 21 MID A41-8 SGSGTYT HCDR2 22 A41-8 ARSHHYGSNDYAFDF HCDR3 23 A41-8 QDISYS LCDR1 24 MID QVQLQESGPGLVKPSETLSLTCTVS HFR1 25 MID SWIRQPPGKGLEWIGYI HFR2 26 MID DYNPSLKSRVTTSVDTSKNQFSLKLSSVTAADTAVYYC HFR3 27 MID WGQGTTVTVSS HFR4 28 M1D DIQMTQSPSSLSASVGDRVTITCRAT LFR1 29 M1D LNWYQQKPGKAPKLLIY LFR2 30 M1D RLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFATYYC LFR3 31 M1D FGGGTKVEIK LFR4 32 Primer 5′>GTTATTACTCGTGGCCCAGCCGGCCATGGCACAGGTGCAGC TGCAGGAG<3′ 33 Primer 5′>CACTCCAGACCCTTCCCTGGGGGCTGGCGGATCCAGCTCCA GAAGTTGYKGKKGATGYKGYSGYSAGAGAC<3′ 34 Primer 5′>CCAGGGAAGGGTCTGGAGTGGATTGGGTATATCYMTGGTY MTSRTMMTYATMMCGACTACAACCCCTC<3′ 35 Primer 5′>CCCTTGGCCCCAGAAATCAAAAGCGTRGTSGTKGYKGYSGT RGTSGTGCGATCTCGCACAGTAATAC<3′ 36 Primer 5′>CCTCCACTCCCGCCTCCACCTGAAGAGACGGTGACGGTGGT CCCTTGGCCCCAGAAAT<3′ 37 Primer 5′>TGGAGGCGGGAGTGGAGGTGGCGGATCTGGAGGGGGTGGT AGCGACATACAGATGACGCAG<3′ 38 Primer 5′>TGCTGATACCAATTTAAAGAACTGCTAATGTCSTGAGTTGCC CGGCAAGTG<3′ 39 Primer 5′>TCTTTAAATTGGTATCAGCAAAAACCGGGGAAAGCCCC<3′ 40 Primer 5′>CACCTTGGTCCCTCCGCCGAAAGTGAGGKGTAAACTATGGT RCTGTTGACAGTAATAAGT<3′ 41 Primer 5′>TAGTCGACCAGGCCCCCGAGGCCTTTGATTTCCACCTTGGTC CCTCCGCC<3′ 42 Primer 5′- AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAG GAGTC 43 Primer 5′- GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGACGGTGG 44 Primer 5′- AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACG CAGTCTC 45 Primer 5′- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC 46 M1D QQYHSLPLT LCDR3 47 Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA heavy chain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSN constant region TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYTS with V4 mutation RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHEAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTTSKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDTAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HYHYTQKSLSLSPGK 48 Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA heavy chain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constant region TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS with DY mutation RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNDAYPAPTEKTT SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 49 Signal peptide MGWSCIILFLVATATGVHS 50 General formula of GGSIX.sub.1X.sub.2NFW HCDR1 51 General formula of X.sub.3GX.sub.4X.sub.5X.sub.6X.sub.7T HCDR2 52 General formula of ARSHX.sub.8YGX.sub.9X.sub.10DYAFDF HCDR3 53 General formula of QDIX.sub.11X.sub.12S LCDR1 54 General formula of QQYHX.sub.13LPLT LCDR3 55 IGHV4-4*08 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE WIGYIYTSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV YYCAR 56 IGKV1-39*01 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL LIYAASSLQSGVPSRFSGSGSGTDFTLTTSSLQPEDFATYYCQQSYS TP 57 Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA heavy chain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constant region TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 58 Human κ light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDS chain constant ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT region HQGLSSPVTKSFNRGEC

EXAMPLES

[0191] 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.

[0192] 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 claimed by the present invention.

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

[0193] 1.1 Determination of Mutation Site for pH-Dependent Antibody Modification

[0194] The anti-HBV humanized antibody M1D (described in detail in Chinese Patent Application 201810307136.5) previously developed in the laboratory was used as the parent antibody, its variable regions were modified for pH-dependent antigen binding. As shown in FIG. 1, the modified M1D could maintain the antigen-binding activity under neutral conditions, but had a significant decrease in antigen-binding activity under acidic conditions; the dissociated modified M1D could bind to intracellular FcRn so as to return to the plasma and bind to antigen again, so that one molecule of M1D after pH-dependent antigen binding modification could repeatedly bind to and neutralize a plurality of molecules of antigens. Histidine could be protonated under acidic conditions and was a key amino acid to bring pH-dependent antigen binding properties. The three-dimensional structure of the variable regions of M1D was simulated (the three-dimensional structure of the variable regions was simulated according to the amino acid sequences of the variable regions) and shown in FIG. 2. The stick-shaped structure represented the CDR region, the blue represented the VK CDR, the green represented the VH CDR, and the red represented the surface amino acids. A total of 25 surface amino acids were found in the CDR regions, combined with the experience gained from the amino acids disclosed in the literatures, it was determined that there were 24 random replacement sites for histidine.

[0195] 1.2 Phage Library Construction of pH-Dependent scFv Antibodies Derived from M1D

[0196] Using the variable regions of the light and heavy chains of the M1D antibody as a template, the 24 sites determined in the antibody variable region CDRs were replaced with histidines, 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 MID. 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 pH-dependent scFv antibodies derived from M1D. 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, 200Ω). 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 Mutant primers for pH-dependent scFv antibodies derived from M1D Primer name Sequence VH-F SEQ ID NO: 32 HCDR1-R SEQ ID NO: 33 HCDR2-F SEQ ID NO: 34 HCDR3-R SEQ ID NO: 35 VH-R SEQ ID NO: 36 VK-F SEQ ID NO: 37 KCDR1-R SEQ ID NO: 38 KCDR2-F SEQ ID NO: 39 KCDR3-R SEQ ID NO: 40 VK-R SEQ ID NO: 41

[0197] 1.3 Screening of pH-Dependent scFv Antibodies

[0198] 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 comprising ampicillin (100 g/L) and glucose (2 g/mL) to reach an OD value of 0.6, and then M13KO7 was added for auxiliary super-infection. After 2 h, 100 g/L of kanamycin was added and 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 comprising ampicillin and kanamycin (100 g/L), and cultured under 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.

[0199] The ELISA plate coated with HBsAg (200 ng/mL) antigen was taken, 100 μL of the supernatant to be tested was added to each well, and incubation was performed at 37° C. for 1 h (two wells for each supernatant). Subsequently, the ELISA plate was washed once with PBST, and then 120 μL of pH 7.4 PBS and pH 6.0 PBS were added to the two wells of each supernatant respectively and incubated at 37° C. for 30 min. Washing was performed 5 times with PBST of corresponding pH, 100 μL of anti M13-HRP diluted 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 OD.sub.450/620. The results of the third round of ELISA testing were shown in FIGS. 4A to 4C. The results of FIGS. 4A to 4C showed that the phages displaying these scFv antibodies all had reactivity in ELISA detection and could weakly bind to the antigen at pH 6.0. 4 strains of pH-dependent phage antibodies with good effects were initially obtained, named A31-73, A42-13, A42-23 and A41-8.

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

[0200] 2.1 Construction of Recombinant Vector for Eukaryotic Expression

[0201] 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 TD NO: 49) 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.

[0202] The four 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.

[0203] 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 heavy chain constant region shown in SEQ ID NO: 57) and VH+pTT5-Cκ (comprising the light chain constant region shown in SEQ ID NO: 58). The recombinant vector was transformed into E. coli DH5a 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 Primer sequence VH-F SEQ ID NO: 42 VH-R SEQ ID NO: 43 VK-F SEQ ID NO: 44 VK-R SEQ ID NO: 45

[0204] 2.2 Small- and Large-Scale Expression of Antibody Genes

[0205] 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.

[0206] 2.3 Antibody Purification

[0207] 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. The HPLC results of A31-73, A42-12, A42-23 and A41-8 were shown in FIG. 6, and the four antibodies are single-component.

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

[0208] The 4 strains of phage antibodies with property of pH-dependent binding to HBsAg that were obtained through the preliminary screening by the method of Example 1 were named as A31-73, A42-13, A42-23 and A41-8, respectively. Furthermore, the four strains of phage antibodies were subjected to the eukaryotic expression and purification by the method of Example 2. The VH and VL amino acid sequences of the four antibodies were shown in the table below. In addition, the CDR sequences of the four antibodies were determined, and the amino acid sequences of the CDRs of the heavy chain variable regions and the light chain variable regions were shown in Table 5. The mutation sites that endowed A31-73, A42-13, A42-23 and A41-8 with the property of pH-dependent antigen-binding to HBsAg were summarized in Table 5.

TABLE-US-00004 TABLE 4 Amino acid sequences of A31-73/A42-12/A42-23/A41-8 light and heavy chain variable regions Sequence SEQ ID name NO Amino acid sequence A31-71 1 QVQLQESGPGLVKPSETLSLTCTVSGGSILISNFWSWIRQPPG VH KGLEWIGYISGPGFIFITDYNPSLKSRVTISVDTSKNQFSLKLSS VTAADTAVYYCARSHDYGHHDYAFDFWGQGTTVTVSS A31-73 VK 2 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGK APKLLIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYHYLPLTFGGGTKVEIK A42-12 VH 3 QVQLQESGPGLVKPSETLSLTCTVSGGSIHHNFWSWIRQPPG KGLEWIGYIFIGPGHYTDYNPSLKSRVTISVDTSKNQFSLKLSS VTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS A42-12 VK 2 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGK APKLLIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYHYLPLTFGGGTKVEIK A42-23 VH 4 QVQLQESGPGLVKPSETLSLTCTVSGGSITHNFWSWIEtQPPG KGLEWIGYISGYDTYTDYNPSLKSRVTISVDTSKNQFSLKLSS VTAADTAVYYCARSHDYGHHDYAFDFWGQGTTVTVSS A42-23 VK 5 DIQMTQSPSSLSASVGDRVTITCRATQDIFIHSLNAVYQQKPGK APKLLIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYHYLPLTFGGGTKVEIK A41-8 6 QVQLQESGPGLVKPSETLSLTCTVSGGSITSNFWSWIFtQPPGK VH GLEWIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSV TAADTAVYYCARSHHYGSNDYAFDFWGQGTTVTVSS A41-8 7 DIQMTQSPSSLSASVGDRVTITCRATQDISYSLNWYQQKPGK VK APKLLIYYANRLQSGVP.SRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYHYLPLTFGGGTKVEIK

TABLE-US-00005 TABLE 5 Sequence of A31-73/A42-12/A42-23/A41-8 light and heavy chain CDRs A31-73 VH CDR1 GGSHASNFW SEQ ID NO: 8 VH CDR2 SGPGHHT SEQ ID NO: 9 VH CDR3 ARSFIDYGHHDYAFDF SEQ ID NO: 10 VL CDR1 QDISSS SEQ ID NO: 11 VL CDR2 YAN SEQ ID NO: 12 VL CDR3 QQYHYLPLT SEQ ID NO: 13 A42-12 VH CDR1 GGSIFIFINFW SEQ ID NO: 14 VH CDR2 HGPGHYT SEQ ID NO: 15 VH CDR3 ARSHDYGSNDYAFDF SEQ ID NO: 16 VL CDR1 QDISSS SEQ ID NO: 11 VL CDR2 YAN SEQ ID NO: 12 VL CDR3 QQYHYLPLT SEQ ID NO: 13 A42-23 VH CDR1 GGSITHNFW SEQ ID NO: 17 VH CDR2 SGYDTYT SEQ ID NO: 18 VH CDR3 ARSHDYGFIFIDYAFDF SEQ ID NO: 10 VL CDR1 QDIHHS SEQ ID NO: 19 VL CDR2 YAN SEQ ID NO: 12 VL CDR3 QQYHYLPLT SEQ ID NO: 13 A41-8 VH CDR1 GGSITSNFW SEQ ID NO: 20 VH CDR2 SGSGTYT SEQ ID NO: 21 VH CDR3 ARSHHYGSNDYAFDF SEQ ID NO: 22 VL CDR1 QDISYS SEQ ID NO: 23 VL CDR2 YAN SEQ ID NO: 12 VL CDR3 QQYHYLPLT SEQ ID NO: 13

[0209] 3.1 Determination of Antigen-Binding Activity of pH-Dependent Anti-HBsAg Antibodies at Different pH

[0210] The present inventors tested the ability of pH-dependent antigen binding to HBsAg for A31-73, A42-12, A42-23 and A41-8 respectively by ELISA method. First, BCA protein quantification kit was used to determine the concentration of the purified antibody, and the antibody concentrations were unified to 1111.11 ng/mL by dilution. Subsequently, 20% NBS was used to perform the gradient dilution of antibody concentration by 3-fold gradient dilution, and a total of 8 concentrations were obtained by the gradient dilution. 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 120 μL of PBS with pH 7.4 and pH 6.0 were added to the two wells of each supernatant and incubated at 37° C. for 30 min. The plate was 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. 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.

[0211] The results were shown in FIG. 7. At pH 7.4, all of A31-73, A42-12, A42-23 and A41-8 had an antigen-binding activity comparable to that of M1D, but at pH 6.0, their antigen-binding activities were significantly reduced. The EC50 results were summarized in Table 6.

TABLE-US-00006 TABLE 6 EC50 for pH-dependent activity detection of A31-73, A42-12, A42-23 and A41-8 EC50 in pH 6.0 EC50 in pH 7.4 EC50(pH 6.0)/ Antibody (ng/mL) (ng/mL) EC50(pH 7.4) A31-73 14046.00 17.32 810.97 A42-12 4604.00 15.13 304.30 A42-23 345.00 12.68 27.21 A41-8 31.13 15.78 1.97

[0212] 3.2 Determination of Therapeutic Effect of pH-Dependent Anti-HBsAg Antibodies in Animal Model

[0213] HBV transgenic mice were used to evaluate the ability of the above-mentioned pH-dependent anti-HBsAg antibodies to eliminate HBV virus in animals. The pH-dependent anti-HBsAg antibodies and the parental antibody M1D were administered to HBV transgenic mice (presented by Professor Chen Peizhe, National Taiwan University) at a dose of 10 mg/kg via tail vein injection, with 4 to 5 HBV transgenic mice per group. Subsequently, blood samples were collected from the mice through retro-orbital venous plexus, and the changes in HBsAg and antibody levels in mouse serum were detected.

[0214] 3.2.1 Quantitative Detection of HBsAg

[0215] (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, 100 μL of coating solution was added to each well of a chemiluminescence plate to perform coating 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 to perform blocking at 37° C. for 2 h. Subsequently, the blocking solution was discarded, and the plate was dried in a drying room, and stored at 2-8° C. for later use.

[0216] (2) Sample dilution: the collected mouse serum was diluted with a PBS solution comprising 20% NBS (newborn bovine serum) to form two gradients of 1:30 and 1:150 for subsequent quantitative detection.

[0217] (3) Sample denaturation treatment: 15 μL of the above-diluted serum sample was fully mixed 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.

[0218] (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.

[0219] (5) Enzymatic label reaction: HBs-A6A7-HRP reaction solution was added to the chemiluminescence plate at 100 μL/well, and reacted at 37° C. for 1 h. Then, the plate was washed 5 times with PBST and spin-dried.

[0220] (6) Luminescence reaction and measurement: a luminescence solution was added (100 μL/well) to the chemiluminescence plate, and light intensity detection was performed.

[0221] (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.

[0222] 3.2.2 Quantitative Detection of Antibody in Serum

[0223] (1) Sample dilution: the serum samples were diluted with 20% NBS into 3 gradients: 1000 times, 10000 times, 100000 times; the corresponding control antibody was diluted with 20% NBS to 20 ng/mL in the first well, and then 3 times diluted into 6 gradients,

[0224] (3) Sample addition: 100 μL/well of the sample diluted in the previous step and corresponding antibody diluent were added to a commercial HBsAb plate (purchased from Beijing Wantai), and incubated 37° C. for 1 h.

[0225] (4) Washing: the plate was washed 5 times with 300 μL/well of PBST by a plate washer, and spin-dried;

[0226] (5) Adding enzyme-labeled secondary antibody: 100 μL/well of GAH-HRP-labeled secondary antibody diluent was added, and incubated at 37° C. for 30 min;

[0227] (6) Washing: the plate was washed 5 times with 300 μL/well of PBST by a plate washer, and spin-dried.

[0228] (7) Color development: 100 μL/well of color development solution, 37° C., 10-15 min;

[0229] (8) Stop and read: 50 μL/well of stop solution was added, the reading value was measured at OD450/630 within 10 minutes by a microplate reader.

[0230] (9) Calculation of antibody concentration in mouse serum sample: a standard curve was drawn based on the measurement results of corresponding standard antibodies. Then, the light intensity measurement value of the mouse serum sample was substituted into the standard curve, and the antibody concentration in the serum sample to be tested was calculated.

[0231] The detection results of HBsAg and antibody levels in the serum of mice after antibody treatment were shown in FIGS. 8A to 8B. The results showed that the HBsAg level of the parent antibody M1D rebounded back to the baseline in about 10 days, while the HBsAg level of pH-dependent antibodies rebounded back to the baseline in about 15 days; comparing the antibody concentrations in serum, M1D showed the lowest antibody concentration on the 10.sup.th day, while the three pH-dependent antibodies still can be detectable on the 15.sup.th day, indicating that the pH-dependent antibodies had an antigen inhibition time longer than that of the parent antibody while maintaining the ability to eliminate HBsAg which is equivalent to that of the parent antibody, that was, the clearance effect of pH-dependent antibodies could be exerted for a longer time. The clearance results of HBsAg by A31-73, A42-12 and A41-8 were shown in FIG. 8A, and the results of serum concentrations of A31-73, A42-12 and A41-8 were shown in FIG. 8B. The results of animal treatment effects indicated that A31-73, A42-12 and A41-8 had a potential to treat HBV infection or a disease associated with HBV infection (for example, hepatitis B).

Example 4: Construction and Functional Evaluation of Scavenger Antibody

[0232] 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. 9, 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.

[0233] 4.1 Scavenger Antibody Capable of Binding to hFcRn

[0234] 4.1.1 Construction. Expression and Purification of Scavenger Antibody Capable of Binding to hFcRn

[0235] The A31-73 and A41-8 with the best performance in HBV transgenic mice in Example 3 were subjected to V4 (M252Y, N286E, N434Y) mutation in Fc region (this work was commissioned to General Biology, order number: G120460) to enhance the affinity to hFcRn under neutral conditions. Two scavenger antibodies A31-73 V4 and A41-8 V4 were obtained, and the heavy chain constant region after mutation was shown in SEQ ID NO:47. The above two antibodies were subjected to large-scale eukaryotic expression and purification, which specific operation steps were shown in Examples 2.2 and 2.3. The HPLC results of A31-73 V4 and A41-8 V4 were shown in FIG. 10, and the results indicated that the antibodies are single-component.

[0236] 4.1.2 In Vitro Activity Detection of Scavenger Antibody Capable of Binding to hFcRn

[0237] 4.1.2.1 Determination of Antigen Binding Activity of Scavenger Antibody Capable of Binding to hFcRn at Different pH

[0238] The antigen binding activities of A31-73 V4 and A41-8 V4 at pH 7.4 and pH 6.0 were detected respectively, specific operation steps of which were shown in Example 3.1. The results were shown in FIG. 11. The results showed that both A31-73 V4 and A41-8 V4 could maintain an antigen-binding activity equivalent to that of the parent at neutral pH, and the antigen-binding activity at acidic pH was further weakened, indicating that the pH-dependent antigen binding activities of A31-73 V4 and A41-8 V4 were enhanced to a certain extent.

[0239] 4.1.2.2 Function Verification at Cellular Level of Scavenger Antibody Capable of Binding to hFgRn

[0240] 4.1.2.2.1 Labeling HBsAg with 488 Fluorescence

[0241] The labeling of 1 mg of HBsAg was taken as an example, and the whole process was protected from light.

[0242] (1) 1 mL of 1 mg/mL HBsAg was dialyzed into borate buffer (PH 8.5, 500 mL) at 4° C. for 4 h;

[0243] (2) The molar ratio of HBsAg to 488 label was 1:5, and 0.1988 mg of 488 fluorescence was required after calculation;

[0244] (3) 10 mg/mL of 488 fluorescence solution was prepared with DMF and mixed well;

[0245] (4) 19.88 μL of 488 fluorescence was added to 1 mL of the dialyzed HBsAg, mixed well, and incubated at room temperature for 1 h;

[0246] (5) The incubation mixture was dialyzed into PBS at 4° C. overnight.

[0247] 4.1.2.2.2 Immunofluorescence Experiment Based on MDCK Cells

[0248] (all steps involving the fluorescence labelled sample were operated in a dark environment)

[0249] (1) Cell plating: the cells at a density of 2×10.sup.5 cells/mL were cultured in a 24-well glass-bottom culture plate for cell imaging overnight at 250 μL/well;

[0250] (2) the antibody and antigen labeled with the corresponding fluorescence were diluted in serum-free medium: 800 ng/mL for antigen, and 2 μg/mL for antibody;

[0251] (3) 125 μL each of the antigen and antibody were mixed uniformly, and then allowed to stand for 1 hour in a 37° C., CO.sub.2 incubator;

[0252] (4) 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 CO.sub.2 incubator at 37° C. for 2 hours;

[0253] (5) the supernatant was discarded, 1 mL of sterile PBS incubated at 37° C. in advance was used to “wash” the cell surface 3 to 5 times, and removed by pipetting;

[0254] (6) 1 mL of 10% paraformaldehyde was added, and allowed to stand for 0.5 h in a 37° C., CO.sub.2 incubator protected from light;

[0255] (7) the paraformaldehyde was discarded, 1 mL of sterile PBS incubated at 37° C. in advance was used to “wash” the cell surface 3 to 5 times, and removed by pipetting;

[0256] (8) 0.5 mL of DAPI (diluted at 1:1000 with sterile PBS) was added, and allowed to stand for 15 minutes in a 37° C., CO.sub.2 incubator protected from light;

[0257] (9) the DAPI diluent was discarded, 1 mL of sterile PBS incubated at 37° C. in advance was used to “wash” the cell surface for 3-5 times, and removed by pipetting, and 0.5 mL of PBS was added, and placed in a high-content imager for imaging.

[0258] The experimental results were shown in FIGS. 12A to 12B. The results showed that the antigen fluorescence intensity in cells treated with A31-73 V4 and A41-8 V4 was significantly higher than that of cells treated with A31-73 and A41-8, indicating that the above scavenger antibodies can efficiently transport more antigens into the cells. This result confirmed that the V4 mutation in the scavenger antibody could enhance the binding of the antibody to hFcRn under neutral conditions. After the antigen-antibody complex entered into the cells, the intracellular pH was only about 6.0, the antibody was dissociated from the antigen-antibody complex and returned to the cell surface to perform the antigen-binding function again. The enhancement of ability to bind to hFcRn under neutral conditions for scavenger antibody could open the “convenience door” for subsequent pH-dependent antigen binding properties.

[0259] 4.1.3 Determination of Therapeutic Effect of Scavenger Antibodies Capable of Binding to hFcRn in Animal Models

[0260] The V4 modification was aimed at the hFcRn receptor. The receptor of HBV transgenic mice was mFcRn, which could not be used to evaluate the effect of modification. Therefore, the model of hFcRn transgenic mice infected with rAAV-HBV adr was adopted. The hFcRn transgenic mice aged 6 to 8 weeks (purchased from Biosaitu) were subjected to single injection of an appropriate dose of rAAV-HBV adr (purchased from Beijing Wujiahe Molecular Medicine Institute Co., Ltd.) via tail vein. The HBV virus titer in the mice were monitored for four consecutive weeks. After the virus titer was stable, the hFcRn transgene mice with stable HBV virus titer (4 in each group) were injected via tail vein with two scavenger antibodies, M1D and negative control 16G12 respectively, at a single dose of 20 mg/kg, and assisted with intraperitoneal injection of anti-CD4 antibody to suppress humoral immune response. Then the concentrations of HBsAg, antibody and HBV DNA in serum were measured to analyze the in vivo antigen clearance rate and antibody half-life of the scavenger antibodies.

[0261] 4.1.3.1 Quantitative Detection of HBsAg

[0262] The specific steps were shown in Example 3.2.1, and the detection results of A31-73 V4 and A41-8 V4 were shown in FIG. 13A.

[0263] 4.1.3.2 Quantitative Detection of Antibodies in Serum

[0264] The specific steps were shown in Example 3.2.2, and the detection results of A31-73 V4 and A41-8 V4 were shown in FIG. 13B.

[0265] 4.1.3.3 Quantitative Detection of HBV DNA

[0266] The quantitative detection of HBV DNA was carried out in accordance with the instructions of the HBV DNA real-time fluorescence quantitative detection kit (the kit was purchased from Beijing Jinmaige Biotechnology Co., Ltd.).

[0267] In this example, the virus clearance abilities of A31-73 V4, A41-8 V4 and the reference antibody M1D in animals were determined. The experimental results were shown in FIG. 13C.

[0268] The results of FIGS. 13A to 13C showed that scavenger antibodies A31-73 V4 and A41-8 V4 had antigen clearance capabilities comparable to that of M1D, but had a longer inhibition time. After M1D exerted the maximum antigen clearance effect, the antigen rebound started quickly, and M1D started to fail on the 14.sup.th day and the HBsAg in serum returned to the baseline level (based on the negative control 16G12). However, the 2 scavenger antibodies could still maintain a lower antigen level for a long-term after exerting the maximum clearance effect, and rebound slowly. In the A41-8 V4 treatment group, HBsAg rebounded back to the baseline in about 30 days. In the A31-73 V4 treatment group, it did not return to the baseline level after the expected end of the experiment. These was consistent with the detection results of antibody half-life in serum. Such technical effects are remarkable and unexpected.

[0269] 4.2 Scavenger Antibody Capable of Binding to mFcγRII

[0270] 4.2.1 Construction. Expression and Purification of Scavenger Antibody Capable of Binding to mFcγRII

[0271] The A31-73 and A41-8 with the best performance in HBV transgenic mice in Example 3 were subjected to DY (K326D, L328Y) mutation on Fc region (this work was commissioned to General Biology, order number: G120460) to obtain A31-73 DY and A41-8 DY respectively, the heavy chain constant region after mutation was shown in SEQ ID NO:48. The antibodies was subjected to large-scale eukaryotic expression and purification, the specific operation steps of which were shown in Examples 2.2 and 2.3. The protein gel results of A31-73 DY were shown in FIG. 14. The results showed that the antibody are single-component.

[0272] 4.2.2 In Vitro Activity Detection of Scavenger Antibody Capable of Binding to mFcγRII

[0273] 4.2.2.1 Determination of antigen binding activity of scavenger antibodies capable of binding to mFcγRII at different pH

[0274] The antigen binding activities of A31-73 DY at pH 7.4 and pH 6.0 were detected respectively, specific operation steps of which are shown in Example 3.1. The results were shown in FIG. 15. The A31-73 DY could maintain an antigen-binding activity and pH-dependent antigen-binding activity equivalent to those of the parent antibody at neutral pH, and the antigen-binding activity at acidic pH was further weakened, indicating the pH-dependent antigen-binding activity of A31-73 DY was enhanced to a certain extent.

[0275] 4.2.2.2 Functional Verification at Cellular Level of Scavenger Antibodies Capable of Binding to mFcγRII

[0276] 4.2.2.2.1 Labeling HBsAg with 488 Fluorescence

[0277] Specific steps were shown in 4.1.2.2.1.

[0278] 4.2.2.2.2 Immunofluorescence Experiment Based on Mouse Primary Macrophages

[0279] (1) 4 days before the experiment, 1.5 mL of 3% sodium thioglycolate solution was injected into the abdominal cavity of each mouse, but not injected into the intestine;

[0280] (2) two mice were executed and soaked in 75% alcohol for 3 minutes;

[0281] (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;

[0282] (4) 4° C., 1100 g, 5 min;

[0283] (5) the supernatant was carefully discarded, the cells were washed twice with 1640 medium, centrifuged at 4° C., 1100 g for 5 min, the supernatant was discarded, and the cells were resuspended in RPM1640;

[0284] (6) after counting the cells, the cells were adjusted to have a density of 10.sup.6 cells/mL, cultured on a 24-well glass-bottom cell imaging culture plate, 250 μL/well, the medium was changed after 2 hours, the washing was performed once with RPM1640, the non-adherent cells were discarded, and then incubation was performed overnight in a 37° C., CO.sub.2 incubator;

[0285] (7) the antibody and antigen labeled with corresponding fluorescence were diluted in serum-free medium to: 800 ng/mL for antigen, and 20 μg/mL for antibody;

[0286] (8) 125 μL each of the antigen and antibody were mixed uniformly, and then allowed to stand for 1 hour in a 37° C., CO.sub.2 incubator;

[0287] (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 CO.sub.2 incubator at 37° C. for 2 hours;

[0288] (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 removed by pipetting;

[0289] (11) Dio was diluted at 1:2000, added at an amount to submerge the cells, and allowed to stand at room temperature for 20 min;

[0290] (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 removed by pipetting;

[0291] (13) the live cell nuclear dye (2 drops were added to 1 mL volume) was added, allowed to stand at room temperature for 20 min, and placed in a high-content imager for imaging.

[0292] The results of the experiment were shown in FIG. 16. It could be seen from the results that the DY modification enhanced the phagocytosis of antigen-antibody complex by mouse macrophages, leading to more antigen degradation.

[0293] 4.2.3 Determination of Therapeutic Effect of Scavenger Antibody Capable of Binding to mFcγRII in Animal Model

[0294] 4.2.3.1 Evaluation of Therapeutic Effect of Scavenger Antibody in Transgenic Mice

[0295] HBV transgenic mice aged 6-8 weeks were selected, and injected with A31-73 DY scavenger antibody and M1D via tail vein at a single dose of 5 mg/kg (4 mice per group). By detecting the concentrations of HBsAg, antibody and HBV DNA in serum, the in vivo antigen clearance rate and antibody half-life of scavenger antibody were analyzed.

[0296] 4.2.3.1.1 Quantitative Detection of HBsAg

[0297] The specific steps were shown in Example 3.2.1, and the detection results of A31-73 DY were shown in FIG. 17A.

[0298] 4.2.3.1.2 Quantitative Detection of HBV DNA

[0299] The quantitative detection of HBV DNA was carried out in accordance with the instructions of the HBV DNA real-time fluorescence quantitative detection kit (the kit was purchased from Beijing Jinmaige Biotechnology Co., Ltd.).

[0300] In this example, the virus clearance abilities of A31-73 DY and reference antibody M1D in animals were determined. The experimental results were shown in FIG. 17B.

[0301] 4.2.3.1.3 Quantitative Detection of Antibody in Serum

[0302] The specific steps were shown in Example 3.2.2, and the detection results of A31-73 DY were shown in FIG. 17C.

[0303] The results of FIGS. 17A to 17C showed that the scavenger antibody A31-73 DY with DY modification was more than an order of magnitude stronger than M1D in antigen clearance ability. The A31-73 DY could not only quickly clear HBsAg in HBV transgenic mice (FIG. 17A), but also effectively reduce the HBV DNA titer in mice (FIG. 17B), which was consistent with the detection result of antibody half-life in serum (FIG. 17C). Comparing the antibody concentrations in the serum, the half-life of A31-73 DY was longer than that of M1D by nearly 12 days, which showed that the scavenger antibody A31-73 DY had the function of reciprocally binding antigen, thereby increasing the time of antigen clearance.

[0304] 4.2.3.2 Evaluation of Therapeutic Effect of Scavenger Antibody in HBV Mouse Model Transfected with Adeno-Associated Virus

[0305] As shown in FIG. 18A, 4-6 weeks old C57 mice were injected with 1×10.sup.11 rAAV8-1.3HBV adr to establish an HBV mouse model generated by transfection by adeno-associated virus (Day −28), and the mouse virus titer was continuously monitored. After the virus titer became stable, the baseline HBsAg level in mice was detected and the mice were divided into groups (Day −1). scavenger antibody A31-73 DY (73 DY for short) and the negative control 16G12 were injected at a dose of 5 mg/kg, for 5 times; all mice were sacrificed 2 weeks after the 5.sup.th treatment, the mouse serum was collected, and the serum HBsAg and Anti-HBs levels were analyzed.

[0306] 4.2.3.2.1 Quantitative Detection of HBsAg

[0307] The specific steps were shown in Example 3.2.1, and the detection results of A31-73DY were shown in FIG. 18B.

[0308] 4.2.3.2.2 Qualitative Detection of Anti-HBs

[0309] (1) Sample dilution: the serum sample was diluted with 20% NBS into 5 gradients: 100 times, 500 times, 2500 times, 12500 times and 62500 times;

[0310] (3) Sample addition: 100 μL/well of the sample diluted in the previous step and the corresponding antibody diluent were added to a commercial HBsAb plate (purchased from Beijing Wantai), and incubated at 37° C. for 1 h.

[0311] (4) Washing: the plate was washed 5 times with 300 μL/well of PBST by a plate washer and spin-dried;

[0312] (5) Adding enzyme-labeled secondary antibody: 100 μL/well of GAM-HRP-labeled secondary antibody diluent was added, and incubated at 37° C. for 30 min;

[0313] (6) Washing: the plate was washed 5 times with 300 μL/well of PBST by a plate washer, and spin-dried;

[0314] (7) Color development: 100 μL/well of color development solution, 37° C., 10-15 min;

[0315] (8) Stop and read: 50 μL/well stop solution was added, the reading value was measured by a microplate reader at OD450/630 within 10 minutes.

[0316] (9) Calculation of Anti-HBs concentration in mouse serum sample: the reading value between 0.1 and 1 was multiplied by the dilution factor, and its logarithmic value (log.sub.10) was taken.

[0317] The detection results of Anti-HBs level in mouse serum after antibody treatment were shown in FIG. 18C.

[0318] The results of FIG. 18B showed that the scavenger antibody A31-73 DY could control the HBsAg level in the HBV mice model generated by adeno-associated virus below 100 IU/ml for a long time with a dose of 5 mg/kg and a dosing frequency of 15 days/injection. The results of FIG. 18C showed the Anti-HBs levels in mice on days 0, 7, 37, 52, 67 and 82, and it could be seen that the A31-73 DY antibody-treated mice had higher Anti-HBs levels, 3 out of 4 mice produced obvious Anti-HBs (only the mice continuously treated to the end were counted), indicating that the production of Anti-HBs could be related to the continuous inhibition of HBsAg. This showed that the scavenger antibody A31-73 DY broke through the limitations of traditional antibodies and achieved low-dose, low-frequency treatment; and more importantly, after long-term treatment, the humoral immune response in mice was activated that allowed the production of Anti-HBs.

Example 5: Affinity Determination of M1D, A31-73 and A41-8

[0319] 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 2400RU. 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 M1D, A31-73 and A41-8 KD(pH 6.0)/ KD(M) in pH 7.4 KD(M) in pH 6.0 KD(pH 7.4) M1D 5.34E−10 A31-73  7.3E−10 3.62E−09 4.95 A41-8 1.68E−09 1.93E−09 1.15

[0320] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that according to all the teachings that have been published, various modifications and changes can be made to the details, 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.