RU2803082C2 - Antibodies against hepatitis b virus and their use - Google Patents

Abstract

FIELD: biotechnology and medicine.

SUBSTANCE: antibody capable of specifically binding to HBsAg, an isolated nucleic acid molecule that encodes an antibody, an expression vector, a host cell for producing an antibody are proposed. A method of producing an antibody, a pharmaceutical composition for the treatment of an HBV infection or a disease associated with an HBV infection, and a pharmaceutical composition for the prevention of an HBV infection or a disease associated with an HBV infection in a subject, the use of an antibody or a pharmaceutical composition in the manufacture of a drug for the treatment of HBV infection or a disease associated with an HBV infection in a subject, as well as in the manufacture of a medicinal product for the prevention of an HBV infection or a disease associated with an HBV infection in a subject are proposed. A method of treating an HBV infection or a disease associated with an HBV infection in a subject, a method of preventing an HBV infection or a disease associated with an HBV infection in a subject are provided. Anti-HBsAg antibodies have a higher binding affinity for HBsAg at neutral pH than at acidic pH, thereby significantly increasing the efficiency of virus clearance and prolonging the time of virus inhibition.

EFFECT: invention allows to neutralize HBV virulence in the body of a subject, such as a human, to reduce the serum level of HBV DNA and/or HBsAg in the subject's body, or to activate the humoral immune response of a subject, for example, a person infected with chronic HBV infection, or a patient who has chronic hepatitis B.

37 cl, 7 tbl, 22 dwg, 5 ex

Description

Technical field

The present invention relates to the field of molecular virology and immunology, in particular to the field of treatment of infection caused by the hepatitis B virus (HBV). Specifically, the present invention relates to an antibody against hepatitis B virus surface antigen (HBsAg) and a nucleic acid encoding the antibody and their use. The anti-HBsAg antibody of the present invention has a higher binding affinity to HBsAg at neutral pH than at acidic pH. The novel antibody may be used to prevent and/or treat HBV infection or a disease associated with HBV infection (eg, hepatitis B), to neutralize the virulence of HBV in a subject (eg, human), or to reduce the serum level of HBV DNA and/or HBsAg in a subject . Therefore, the present invention further relates to the use of an antibody and a variant thereof in the manufacture of a pharmaceutical composition for the prevention and/or treatment of HBV infection or a disease associated with HBV infection (e.g., hepatitis B) to neutralize the virulence of HBV in a subject (e.g., a human), to reduce serum levels of HBV DNA and/or HBsAg in a subject or to activate a humoral immune response to HBV in a subject (eg, a person with chronic HBV infection or a patient with chronic hepatitis B).

State of the art

Hepatitis B virus infection, especially chronic HBV infection, is one of the most serious health problems in the world (Dienstag J.L., Hepatitis B virus influenza, N. Engl. J. Med., 2008 Oct 2; 359 (14): 1486- 1500). Chronic HBV infection can lead to a number of liver diseases, such as chronic hepatitis B (CHB), liver cirrhosis (LC), and primary hepatocellular carcinoma (HCC) (Liaw Y.F., Chu C.M., Liaw Y.F., Chu C.M., Hepatitis B virus infection, Lancet , 2009 Feb 14;373(9663):582-592). It is estimated that there are currently approximately 2 billion people worldwide infected with HBV, and there are also currently approximately 350 million people with chronic hepatitis B virus infections, and the risk that these infected people will eventually die from the disease liver disease associated with HBV infection can range from 15% to 25%, and more than one million people die annually from these diseases worldwide (Dienstag J.L., ibid.; and Liaw Y.F. et al., ibid.).

Current drugs for the treatment of chronic HBV infection can be divided into interferons (IFN) and nucleoside or nucleotide analogues (NA) (Dienstag J.L., ibid.; Kwon H., Lok A.S., Hepatitis B therapy, Nat. Rev. Gastroenterol. Hepatol., 2011 May;8(5): 275-284; and Liaw Y.F. et al., ibid.). For patients infected with HBV (eg, patients with chronic hepatitis B), the above drugs alone or in combination can effectively suppress viral replication in the body and significantly reduce HBV DNA levels; in particular, after 52 weeks or more of such courses of treatment, the response rate when the level of HBV DNA in the body is below the lower limit of detection (virological response) can reach 40-80% (Kwon H. et al., ibid.). However, treatment with the above drugs alone or in combination cannot completely eliminate the HBV virus from infected people, and the response rate of HBsAg-negative conversion or HBsAg seroconversion (complete clearance of the HBV virus in an infected person) caused by this is usually less than 5% (Kwon H. et al., ibid.).

The development of new drugs for the treatment of chronic HBV infection based on immunological agents is one of the important areas of research in this area. Immunotherapy for chronic HBV infection is usually carried out in two ways: active immunotherapy (appropriate dosage forms, including vaccines, etc.) and passive immunotherapy (appropriate dosage forms, including antibodies, etc.). Active immunotherapy refers to the administration of a therapeutic vaccine (including protein vaccine, peptide vaccine, nucleic acid vaccine, etc.) to stimulate the body of a person with chronic HBV infection to actively generate a cellular immune response (CTL effect, etc. .) and/or a humoral immune response against HBV (antibodies, etc.) to achieve the goal of inhibiting or eliminating HBV. Currently, there is no clearly significant and effective active immunotherapy drug/vaccine that could be used to treat chronic HBV infection. Passive immunotherapy (as exemplified by antibodies) refers to the administration of an antibody with therapeutic properties to an HBV-infected person, and the therapeutic effect can be achieved by antibody-mediated neutralization of the virus, blocking HBV from infecting the hepatocytes of newborns, or antibody-mediated immune clearance, removing viruses and infected liver cells from body. Currently, polyclonal anti-HBs antibodies isolated from the serum/plasma of subjects who have responded to hepatitis B vaccine or those who have recovered from HBV infection, namely hepatitis B immunoglobulin (HBIG), are widely used to block vertical transmission of HBV from mother to child, prevention of reinfection with HBV after liver transplantation in patients with chronic HBV infection, and prevention of infection in people accidentally exposed to HBV. However, the direct use of HBIG to treat patients infected with HBV (eg, patients with chronic hepatitis B) has no obvious effect and has many limitations, such as a small number of sources of highly active plasma, high cost, unstable nature, and potential safety concerns.

Therefore, it is urgent and necessary to develop innovative treatments and drugs for HBV-infected people that can more effectively eliminate the HBV virus, in particular HBsAg.

The essence of the invention

The present inventors have previously developed a humanized anti-HBsAg antibody with excellent properties that can neutralize HBV virulence in vivo and reduce serum levels of HBV DNA and/or HBsAg. Based on the results of previous studies, the inventors of the present invention have invested a lot of creative work in conducting in-depth research and designing a humanized antibody, thereby developing an anti-HBsAg antibody with a pH-dependent antigen binding ability. The anti-HBsAg antibody of the present invention has a higher binding affinity to HBsAg at neutral pH than at acidic pH, thereby resulting in reusability of the antibody, the half-life of the antibody is significantly increased, and the clearance efficiency of HBV is improved. In addition, the present inventors prepared a scavenger antibody and further prolonged the half-life of the antibody by introducing a mutation in the Fc region of the above antibody to increase its affinity for hFcRn or mFcγRII under neutral conditions.

The antibody of the present invention is extremely advantageous because it not only retains activity in reducing serum levels of HBV DNA and/or HBsAg, but also has a longer antigen suppression time, thereby significantly reducing the injection dose and frequency of administration in treatment, and has a great clinical benefit. meaning.

Antibody of the invention

Therefore, in one aspect, the present invention provides 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.

In some embodiments, the neutral pH is between pH 6.7 and pH 7.5, such as pH 7.4.

In some embodiments, the acidic pH is between pH 4.0 and pH 6.5, such as pH 6.0.

In some embodiments, the ratio of the _KD of binding to HBsAg at an acidic pH (e.g., pH 6.0) to the _KD of binding to HBsAg at a neutral pH (e.g., pH 7.4) (i.e., the _KD value (acidic pH) /K _D (neutral pH)) of the antibody or its antigen-binding fragment is higher than 1, for example not lower than 1.5, not lower than 2, not lower than 3, not lower than 4, not lower than 5, not lower than 6, not lower than 7, not lower 8, not lower than 9, not lower than 10, not lower than 15, not lower than 20, not lower than 30, not lower than 40, not lower than 50, not lower than 60, not lower than 70, not lower than 80, not lower than 90, not lower than 100, not less than 300, not less than 500, not less than 800, not less than 1000, not less than 2000, not less than 5000, or not less than 10000. In some embodiments, the _KD (acidic pH)/ _KD (neutral pH) value is greater than 1 and not above 10000, for example, not above 5000, not above 2000, not above 1000, not more than 900, not above 800, not above 700, not above 600, not above 500, not above 400, not above 300, not above 200, not more than 100, not more than 90, not more than 80, not more than 70, not more than 60, not more than 50, not more than 40, not more than 30, not more than 20 or not more than 10. K _D can be measured using a method known in the art technical fields, for example, by the SPR method (for example, Biacore).

In some embodiments, the ratio of the HBsAg binding _KD at pH 6.0 to the HBsAg binding _KD at pH 7.4 of the antibody or antigen binding fragment thereof is greater than 1, such as not less than 1.5, not less than 2. In some embodiments, the K _D value of the antibody of the invention at neutral pH may be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or lower. In some embodiments, the K _D value of an antibody of the present invention at an acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or more.

In some embodiments, the ratio of the EC ₅₀ of HBsAg binding at an acidic pH (e.g., pH 6.0) to the EC ₅₀ of HBsAg binding at a neutral pH (e.g., pH 7.4) (i.e., the EC ₅₀ value (acidic) The pH/EC ₅₀ (neutral pH)) of an antibody or antigen-binding fragment thereof is greater than 1, for example not less than 1.5, not less than 2, not less than 3, not less than 4, not less than 5, not less than 6, not less than 7, not lower than 8, not lower than 9, not lower than 10, not lower than 15, not lower than 20, not lower than 30, not lower than 40, not lower than 50, not lower than 60, not lower than 70, not lower than 80, not lower than 90, not lower 100, not less than 300, not less than 500, not less than 800, not less than 1000, not less than 2000, not less than 5000, or not less than 10000. In some embodiments, the EC ₅₀ (acidic pH)/EC ₅₀ (neutral pH) value is greater 1 and not higher than 10000, for example, not higher than 5000, not higher than 2000, not higher than 1000, not higher than 900 and not higher than 800, not higher than 700, not higher than 600, not higher than 500, not higher than 400, not higher than 300, not greater than 200, not greater than 100, not greater than 90, not greater than 80, not greater than 70, not greater than 60, not greater than 50, not greater than 40, not greater than 30, not greater than 20, or not greater than 10. In some embodiments, EC ₅₀ is measured ELISA, for example, is calculated using regression analysis of the dose-response curve generated using the ELISA method.

In some embodiments, the ratio of the HBsAg binding EC ₅₀ at pH 6.0 to the HBsAg binding EC ₅₀ at pH 7.4 of the antibody or antigen binding fragment thereof is greater than 1, e.g., not less than 1.5, or not less than 2.

In some embodiments, the antibody or antigen binding fragment thereof of the present invention is derived from a humanized anti-HBV 162 antibody (which is described in detail in Chinese Patent Application 201610879693.5).

In some embodiments, an antibody or antigen binding fragment thereof of the present invention binds to amino acids 121-124 of HBsAg with higher affinity at neutral pH than at acidic pH.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, which has one or more of the following characteristics:

(i) HCDR1 has at least one amino acid (eg, 1, 2, 3, 4 or 5 amino acids) replaced by histidine from the sequence shown in SEQ ID NO: 11;

(ii) HCDR2 has at least one amino acid (eg, 1, 2, 3, 4 or 5 amino acids) replaced by histidine from the sequence shown in SEQ ID NO: 12; and/or

(iii) HCDR3 has at least one amino acid (e.g., 1, 2, 3, 4, or 5 amino acids) replaced by histidine from the sequence shown in SEQ ID NO: 13.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, which has one or more of the following characteristics:

(i) LCDR1 has at least one amino acid (eg, 1, 2, 3, 4 or 5 amino acids) replaced by histidine compared to the sequence shown in SEQ ID NO: 14;

(ii) LCDR2 has at least one amino acid (eg, 1, 2, 3, 4 or 5 amino acids) replaced by histidine compared to the sequence shown in SEQ ID NO: 15; and/or

(iii) LCDR3 has at least one amino acid (e.g., 1, 2, 3, 4, or 5 amino acids) replaced by histidine from the sequence shown in SEQ ID NO: 16.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region (VH) containing the following 3 CDRs:

(i) HCDR1 with the sequence X ₁ X ₂ YHX ₃ N (SEQ ID NO: 26), where X ₁ is selected from Y or H, X ₂ is selected from G or R, X ₃ is selected from W or Y;

(ii) HCDR2 with the sequence YIX ₄ X ₅ DGSVX ₆ YNPSLEN (SEQ ID NO: 27), where X ₄ is selected from S, N or H, X ₅ is selected from Y or H, X ₆ is selected from L, H or Q; And

(iii) HCDR3 with the sequence GFDH (SEQ ID NO: 13);

and/or

(b) a light chain variable region (VL) containing the following 3 CDRs:

(iv) LCDR1 with the sequence RSSQSLVHSYGDX ₇ YLH (SEQ ID NO: 28), where X ₇ is selected from T or N;

(v) LCDR2 with the sequence KVSNRFS (SEQ ID NO: 15); And

(vi) LCDR3 with the sequence SQNTHX ₈ PYT (SEQ ID NO: 29), where X ₈ is selected from V, L or H.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region (VH) containing the following 3 CDRs:

(i) HCDR1, which consists of a sequence selected from the following sequences: SEQ ID NO: 17, 21, 24;

(ii) HCDR2, which consists of a sequence selected from: SEQ ID NO: 18, 20, 22, 12; And

(iii) HCDR3, which consists of the sequence shown in SEQ ID NO: 13;

and/or

(b) a light chain variable region (VL) containing the following 3 CDRs:

(iv) LCDR1, which consists of a sequence selected from the following sequences: SEQ ID NO: 14, 25;

(v) LCDR2, which consists of the sequence shown in SEQ ID NO: 15; And

(vi) LCDR3, which consists of a sequence selected from the following sequences: SEQ ID NO: 19, 16, 23.

In some embodiments, X ₁ is selected from H, X ₂ is selected from G, X ₃ is selected from W or Y, X ₄ is selected from S or H, X ₅ is selected from Y, X ₆ is selected from L or H, X ₇ is selected from T or N, X ₈ selected from V, L or H.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region (VH) containing the following 3 CDRs:

(i) HCDR1, which consists of a sequence selected from the following sequences: SEQ ID NO: 17, 24;

(ii) HCDR2, which consists of a sequence selected from: SEQ ID NO: 18, 12; And

(iii) HCDR3, which consists of the sequence shown in SEQ ID NO: 13;

and/or

(b) a light chain variable region (VL) containing the following 3 CDRs:

(iv) LCDR1, which consists of a sequence selected from the following sequences: SEQ ID NO: 14, 25;

(v) LCDR2, which consists of the sequence shown in SEQ ID NO: 15; And

(vi) LCDR3, which consists of a sequence selected from the following sequences: SEQ ID NO: 19, 16, 23.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(1) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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.

In some embodiments, the antibody or antigen binding fragment thereof further comprises a human immunoglobulin framework region (e.g., 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 (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10) back mutations from human remains to mouse remains.

In some embodiments, the antibody or antigen binding fragment thereof comprises: a heavy chain framework region contained in an amino acid sequence encoded by a human germline heavy chain gene, and/or a light chain framework region contained in an amino acid sequence encoded by a human germline light chain gene.

In some embodiments, the antibody or antigen binding fragment thereof comprises: a heavy chain framework region contained in the amino acid sequence encoded by the human germline heavy chain gene 4-28-02 (SEQ ID NO: 38), and a light chain framework region contained in the amino acid sequence encoded by the human germline light chain 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human remains to mouse remains.

In some embodiments, the VH of an 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.

In some embodiments, the VL of an 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.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region (VH) which contains an amino acid sequence selected from the following sequences:

(i) the sequence shown in any of SEQ ID NO: 3, 5, 6, 8;

(ii) sequences with a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any of SEQ ID NO: 3, 5, 6, 8; or

(iii) sequences with 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% with the sequence shown in any of SEQ ID NO: 3, 5, 6, 8;

And

(b) a light chain variable region (VL) which contains an amino acid sequence selected from the following sequences:

(iv) the sequence shown in any of SEQ ID NO: 4, 2, 7, 9, 10;

(v) a sequence with a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any of SEQ ID NO: 4, 2, 7, 9, 10; or

(vi) sequences with 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% with the sequence shown in any of SEQ ID NO: 4, 2, 7 , 9, 10.

Preferably, the substitution described in (ii) or (v) is a conservative substitution.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(1) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 4;

(2) VH with the sequence shown in SEQ ID NO: 5, and VL with the sequence shown in SEQ ID NO: 2;

(3) VH with the sequence shown in SEQ ID NO: 6, and VL with the sequence shown in SEQ ID NO: 7;

(4) VH with the sequence shown in SEQ ID NO: 8, and VL with the sequence shown in SEQ ID NO: 9;

(5) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 10; or

(6) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 9.

In some embodiments, the antibody or antigen binding fragment thereof further comprises a constant region derived from human immunoglobulin.

In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment comprises a light chain constant region derived from a human immunoglobulin (for example, κ or λ).

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) a heavy chain constant region (CH) of a human immunoglobulin or variant thereof, wherein the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (for example, a substitution, deletion or addition of at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) compared to the wild type sequence from which it is derived; and/or

(b) a light chain constant region (CL) of a human immunoglobulin or variant thereof, wherein the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (for example, a substitution, deletion or addition of a maximum of 20, a maximum of 15, a maximum of 10, or a maximum of 5 amino acids or any combination thereof; e.g. substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) compared to the wild type sequence from which it is derived.

In some embodiments, the antibody or antigen binding fragment thereof comprises a human IgG1 or IgG4 heavy chain constant region. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 40.

In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a human immunoglobulin heavy chain constant region (CH) variant, wherein the variant has increased affinity for hFcRn or mFcγRII at neutral pH (e.g., pH 7.4) compared to the wild-type sequence the type from which it is derived. In such embodiments, the variant typically has at least one amino acid substitution relative to the wild type sequence from which it is derived.

In some embodiments, the antibody or antigen binding fragment thereof comprises a human IgG1 heavy chain constant region variant, wherein the variant has the following substitutions relative to the wild type sequence from which it is derived: (i) M252Y, N286E, N434Y; or (ii) K326D, L328Y; wherein the above amino acid positions are those according to the Kabat numbering system. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 42 or 43.

In some embodiments, the light chain constant region is a κ light chain constant region. In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 41.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(1) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41;

(2) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41;

(3) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41;

(4) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41;

(5) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41;

(6) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41;

(7) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41;

(8) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41;

(9) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41;

(10) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41;

(11) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41;

(12) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41;

(13) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41;

(14) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41;

(15) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41;

(16) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41;

(17) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; or

(18) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41.

Antibody production

The antibody of the present invention can be produced by various methods known in the art, for example, can be produced using genetic engineering or recombinant DNA technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specified conditions and the antibody of the present invention is expressed.

The antigen binding fragment of the present invention can be obtained by hydrolysis of the entire antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods, 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985) ). In addition, these antigen binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol., 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically linked to form F(ab') ₂ fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab') ₂ fragments can also be directly isolated from the culture medium of recombinant host cells. Those skilled in the art are fully aware of other methods for obtaining these antigen binding fragments.

Therefore, in yet another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen binding fragment thereof of the present invention, or a heavy chain variable region and/or a light chain variable region thereof. In some preferred embodiments, the isolated nucleic acid molecule encodes an antibody or antigen binding fragment thereof of the present invention or a heavy chain variable region and/or a light chain variable region thereof.

In yet another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) containing an isolated nucleic acid molecule of the present invention. In some preferred embodiments, the vector of the present invention is, for example, a plasmid, cosmid, bacteriophage, or the like.

In another aspect, the present invention relates to a host cell containing an isolated nucleic acid molecule of the present invention or a vector of the present invention. Such a host cell includes, but is not limited to, a prokaryotic cell such as an E. coli cell, and a eukaryotic cell such as a yeast cell, an insect cell, a plant cell, and an animal cell (e.g., a mammalian cell such as a mouse cell, a human cell etc.). In some preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (eg, CHO-K1, CHO-S, CHO DG44).

In yet another aspect, the present invention provides a method for producing an antibody or antigen binding fragment thereof of the present invention, which comprises culturing a host cell of the present invention under conditions that allow expression of the antibody or antigen binding fragment thereof, and isolating the antibody or antigen binding fragment thereof from the cultured cells. host cells.

Antibody derivative

An antibody or antigen binding fragment thereof of the present invention can be derivatized, for example, linked to another molecule (eg, another polypeptide or protein). In general, derivatization (eg, labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to HBsAg. Therefore, the antibody or antigen binding fragment thereof of the present invention also includes such derivatized forms. For example, an antibody or antigen-binding fragment of the present invention may be operably linked (by chemical linkage, gene fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (eg, to form a bispecific antibody), an agent for detection, pharmaceutical agent and/or protein or polypeptide capable of mediating the binding of an antibody or antigen binding fragment to another molecule (eg, an avidin or polyhistidine tag).

Therefore, in some embodiments, the antibody or antigen binding fragment thereof of the present invention is labeled. In some embodiments, an antibody or antigen binding fragment thereof of the present invention carries a detectable label, such as an enzyme, radionuclide, fluorescent dye, luminescent substance (eg, chemiluminescent substance), or biotin. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclide (e.g., ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), fluorescent dye (e.g. fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g. Cy7, Alexa 750)), luminescent substance (e.g. chemiluminescent substance such as acridine ester derivative), magnetic particles (e.g. Dynabeads®), calorimetric marker such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex etc.), and biotin used for ligation of avidin (eg streptavidin) modified with the above marker. In some embodiments, such a label may be suitable for an immunoassay (eg, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, a detectable label, as described above, can be ligated to an antibody or antigen binding fragment thereof of the present invention through a linker of varying length to reduce potential steric hindrance.

Pharmaceutical composition and therapeutic use

An antibody or antigen binding fragment thereof of the present invention can be used to prevent or treat HBV infection in a subject (e.g., a human) or a disease associated with HBV infection (e.g., hepatitis B), to neutralize the virulence of HBV in vitro or in a subject (e.g., a human) ), to reduce serum levels of HBV DNA and/or HBsAg in a subject (eg, a human) and to activate a humoral immune response to HBV in a subject (eg, a patient with chronic HBV infection or chronic hepatitis B).

Therefore, in yet another aspect, the present invention provides a pharmaceutical composition that includes an antibody or antigen binding fragment thereof of the present invention and a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition of the present invention may also include an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug used to prevent or treat HBV infection or a disease associated with HBV infection (eg, hepatitis B), such as an interferon drug such as interferon or pegylated interferon.

In yet another aspect, the present invention relates to the use of an antibody or antigen binding fragment thereof of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for the prevention and/or treatment of HBV infection (eg, in humans) or a disease associated with HBV infection (eg , hepatitis B) in a subject, to neutralize the virulence of HBV in vitro or in a subject (eg, a human), to reduce serum levels of HBV DNA and/or HBsAg in a subject (eg, a human), and/or to activate a humoral immune response to HBV in a subject (eg, a patient with chronic HBV infection or chronic hepatitis B).

In yet another aspect, the present invention provides a method for preventing or treating HBV infection or a disease associated with HBV infection (eg, hepatitis B) in a subject (eg, a human), to neutralize the virulence of HBV in vivo or in a subject (eg, a human). ), to reduce the serum level of HBV DNA and/or HBsAg in a subject (eg, a human) and/or to activate a humoral immune response to HBV in a subject (eg, a patient with chronic HBV infection or chronic hepatitis B), where the method includes administering an effective amount of an antibody or antigen binding fragment thereof of the present invention or a pharmaceutical composition of the present invention to a subject in need thereof.

The drugs and pharmaceutical compositions provided by the present invention can be used alone or in combination, and can also be used in combination with other pharmaceutically active agents (eg, other antiviral agents, such as interferon-based drugs, such as interferon or pegylated interferon).

The antibody or antigen binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be administered by conventional routes of administration, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops) or nasal route. The antibody or antigen binding fragment thereof of the present invention can be administered by various methods known in the art. However, for many therapeutic applications, the preferred route/mode of administration is parenteral (eg, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). One skilled in the art will appreciate that the route and/or method of administration will vary depending on the intended purpose. In a preferred embodiment, the antibody or antigen binding fragment thereof of the present invention is administered by intravenous infusion or injection.

The antibody or antigen binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be formulated in various dosage forms, such as liquid, semi-solid and solid forms, for example, solution (for example, injection solution), dispersion or suspension, tablet, powder, granule, emulsion, pill, syrup, powder, liposome, capsule and suppository. The preferred dosage form depends on the intended route of administration and therapeutic use.

For example, one preferred dosage form is an injection solution. Such an injection solution may be a sterile injection solution. For example, a sterile injection solution can be prepared in the following manner: the required dose of an antibody or antigen binding fragment thereof of the invention is included in a suitable diluent and, optionally, other contemplated ingredients (including, but not limited to, pH adjuster, surfactant, adjuvant , ionic strength enhancer, isotonic agent, preservative, diluent or any combination thereof) are included simultaneously, and then filter sterilization is carried out. Additionally, the sterile injection solution may be prepared as a sterile powder (eg, vacuum-dried or freeze-dried) for ease of storage and use. Such sterile powder may be dispersed in a suitable vehicle such as sterile pyrogen-free water before use.

Another preferred dosage form is a dispersion. The dispersion can be prepared in the following manner: the antibody or antigen-binding fragment thereof of the invention is included in a sterile carrier containing a basic dispersion medium and optionally other intended ingredients (including, but not limited to, pH adjuster, surfactant, adjuvant, ionic enhancer strength, isotonic agent, preservative, diluent or any combination thereof). In addition, an absorption delaying agent such as a monostearate salt and gelatin may also be included in the dispersion to obtain the expected pharmacokinetic properties.

Another preferred dosage form is an oral solid dosage form including capsule, tablet, powder, granules and the like. Such solid dosage form typically contains at least one of the following: (a) an inert excipient (or carrier) for the drug, such as sodium citrate and calcium phosphate; (b) a filler such as starch, lactose, sucrose, mannose and silicic acid; (c) a binder such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum acacia; (d) a humectant such as glycerin; (e) a leavening agent such as agar, calcium carbonate, potato or tapioca starch; (f) a absorption retarder such as an olefin; (g) an absorption accelerator such as a quaternary ammonium compound; (h) a wetting agent such as cetyl alcohol and glycerol monostearate; (i) an adsorbent such as kaolin and bentonite; (j) a lubricant such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or any combination thereof. In the case of tablet and capsule dosage forms, a buffer may also be included.

In addition, a release rate modifier (ie, an agent capable of changing the rate of release of a drug) can also be added to a solid oral dosage form to form a modified release or pulsed release dosage form. Such release rate modifier includes, but is not limited to, carboxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, xanthan gum, isoacrylic acid copolymer, hydrogenated flavor oil, carnauba wax, paraffin, cellulose acetate, carboxypropyl methylcellulose phthalate, methacrylic copolymer acids or any of them combination. A modified release or pulsed release dosage form may contain one or a group of release rate modifiers.

Another preferred dosage form is an oral liquid dosage form including an emulsion, solution, suspension, syrup and the like. In addition to the active ingredients, such a liquid oral dosage form may further contain inert solvents commonly used in the art, such as water or other solvents such as ethyl alcohol, isopropanol, propylene glycol, 1,3-butylene glycol, oil (e.g. such as cottonseed oil, peanut oil, corn oil, olive oil, flavor oil and sesame oil), glycerin, polyethylene glycol and sorbitan fatty acid ester and any combination thereof. In addition to these inert solvents, such an oral liquid dosage form may further contain a humectant, an emulsifying agent, a suspending agent, a sweetening agent, a flavoring agent, a flavoring agent, and the like.

In addition, the antibody or antigen binding fragment thereof of the invention may be in unit dosage form in a pharmaceutical composition for ease of administration. The pharmaceutical composition according to the invention must be sterile and stable under the conditions of production and storage.

The drug and pharmaceutical composition provided by the invention may be used alone or in combination, or may be used in combination with an additional pharmaceutically active agent (eg, other antiviral agents, eg interferon-type agents such as interferon or pegylated interferon). In some preferred embodiments, an antibody or antigen binding fragment thereof of the invention is used in combination with other antiviral agent(s) for the prevention and/or treatment of disease associated with HBV infection. The antibody or antigen binding fragment thereof of the invention and such antiviral agent(s) can be administered simultaneously, separately or sequentially. Such antiviral agents include, but are not limited to, agents such as interferon, ribavirin, adamantane, hydroxyurea, IL-2, L-12 and pentacarboxycytosolic acid, etc.

The pharmaceutical composition of the invention may contain a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen binding fragment thereof of the invention. “Prophylactically effective amount” refers to an amount that is sufficient to prevent, inhibit, or delay the progression of a disease (such as HBV infection or disease associated with HBV infection). "Therapeutically effective amount" refers to an amount that is sufficient to treat or at least partially suppress a disease and its complications in a patient with the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention may vary depending on the following factors: the severity of the disease being treated, the general condition of the patient's immune system, the patient's general characteristics such as age, weight and gender, methods of drug administration, additional methods treatments used simultaneously, etc.

The dosage regimen may be adjusted to provide the optimal desired effect (eg, therapeutic or prophylactic effect). For example, a single dose may be administered, or multiple doses may be administered over a period of time, or the dose may be proportionally reduced or increased depending on the requirements of the therapeutic situation.

For an antibody or antigen binding fragment thereof of the invention, an exemplary and non-limiting range of a therapeutically or prophylactically effective amount is from 0.025 to 50 mg/kg, more preferably from 0.1 to 50 mg/kg, more preferably from 0.1 to 25 mg/kg, from 0.1 to 10 mg/kg. It should be noted that the dose may vary depending on the type and severity of the disease being treated. In addition, one skilled in the art will appreciate that for any given patient, a particular dosage regimen must be adjusted over time depending on the needs of the patient and the professional judgment made by the physician; the dosage range presented here is for purposes of illustration only and not as a definition of the use or scope of the pharmaceutical composition of the invention.

Recruitment and detection for application

The antibody or antigen binding fragment thereof of the present invention can specifically bind to HBsAg such that it can be used to detect the presence or level of HBsAg in a sample.

Therefore, in yet another aspect, the present invention provides a kit containing an antibody or antigen binding fragment thereof of the present invention. In some embodiments, an antibody or antigen binding fragment thereof of the present invention carries a detectable label. In yet other embodiments, the kit further includes a second antibody that specifically recognizes an antibody or antigen binding fragment thereof of the present invention. Preferably, the second antibody further contains a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, and an enzyme (eg, horseradish peroxidase), and the like.

In yet another aspect, the present invention provides a method for detecting the presence or level of HBsAg protein in a sample, which comprises: using an antibody or antigen binding fragment thereof of the present invention. In some embodiments, the antibody or antigen binding fragment thereof of the present invention further comprises a detectable label. In yet other embodiments, the method further includes using a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof of the present invention. The method may be used for diagnostic purposes or for non-diagnostic purposes (eg, the sample is a sample of cells and not a sample from a patient).

In some embodiments, the method includes: (1) contacting a sample with an antibody or antigen-binding fragment thereof of the present invention; (2) detecting the formation of a complex between the antibody or an antigen-binding fragment thereof and the HBsAg protein or determining the amount of the complex. The formation of a complex indicates the presence of HBsAg and/or HBV protein.

In yet another aspect, the present invention provides a method for diagnosing whether a subject is infected with HBV, which comprises: using an antibody or antigen binding fragment thereof of the present invention to detect the presence of HBsAg protein in a sample from the subject. In some embodiments, the antibody or antigen binding fragment thereof of the present invention further comprises a detectable label. In yet other embodiments, the method further includes using a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof of the present invention.

In yet another aspect, the present invention relates to the use of an antibody or antigen binding fragment thereof of the present invention in the manufacture of a kit for detecting the presence or level of HBsAg protein in a sample or for diagnosing whether a subject is infected with HBV.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the steps of cell culture, biochemistry, nucleic acid chemistry, immunological laboratory tests and other operating steps used in this description are common steps widely used in their respective fields. At the same time, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term “antibody” refers to an immunoglobulin molecule typically consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be divided into κ (kappa) and λ (lambda) light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of the CL domain. The constant domain is not directly involved in antibody-antigen binding, but exhibits various effector functions, such as mediating the binding of immunoglobulin to host tissue or factor, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with relatively more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively. The assignment of amino acids to each region or domain can be made according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol., 196: 901-917; Chothia et al. (1989) Nature, 342: 878-883.

As used herein, the term “complementarity determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy chain and light chain variable regions each contain three CDRs, named CDR1, CDR2 and CDR3. The exact boundaries of these CDRs can be determined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol., 27: 55-77, 2003). For any given antibody, CDRs are readily identified by those skilled in the art using definitions according to each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol., 27: 55-77, 2003).

In the present invention, the CDRs contained in an antibody or an antigen-binding fragment thereof of the present invention can be defined in accordance with various numbering systems known in the art. In some embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the present invention are preferably defined using the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the present invention are preferably defined using the Kabat numbering system.

As used herein, the term “framework region” or “FR” residues refers to those amino acid residues in the variable region of an antibody that are not CDR residues as defined herein above.

The term "antibody" is not limited to any particular method of producing an antibody. For example, the term includes recombinant antibody, monoclonal antibody, and polyclonal antibody. The antibody may be of a different isotype, such as an IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen that also called "antigen binding site". See generally the monograph Fundamental Immunology, Ch. 7 (Paul W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen binding fragment of an antibody can be produced using recombinant DNA technology or enzymatic or chemical digestion of an intact antibody Non-limiting examples of antigen binding fragment include Fab, Fab', F(ab') ₂ , Fd, Fv, complementarity determining region (CDR), scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (Domantis technology platform), Probody and those polypeptides that contain at least a portion of an antibody sufficient to impart to the polypeptides a specific antigen-binding ability.Engineered antibody variants are discussed in Holliger et al., 2005; Nat. Biotechnol., 23: 1126-1136.

As used herein, the term “full-length antibody” refers to an antibody consisting of two “full-length heavy chains” and two “full-length light chains.” The term “full-length heavy chain” refers to a polypeptide consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N -end to C-end; and, when the full-length antibody is of the IgE isotype, it optionally also contains a CH4 domain of the heavy chain constant region. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N-terminus to C-terminus. A “full-length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from the N-terminus to the C-terminus. The two pairs of full-length antibody chains are linked by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human; it may also be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention contains two antigen binding sites formed by pairs VH and VL, respectively, and the two antigen binding sites specifically recognize/bind the same antigen.

As used herein, the term "Fd" refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of a VH domain (Ward et al., Nature, 341: 544,546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" refers to an antibody fragment consisting of two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab'fragment" refers to the fragment obtained by reduction of the disulfide bond connecting two heavy chain fragments in the F(ab') ₂ fragment, and consists of an intact light chain and a Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of one arm of the antibody. The Fv fragment is generally considered the smallest antibody fragment that can form a complete antigen-binding site. The six CDRs are generally believed to confer antigen-binding specificity to an antibody. However, even a single variable region (eg, an Fd fragment that contains only three antigen-specific CDRs) can recognize and bind to an antigen, although its affinity may be lower than the full binding site.

As used herein, the term "Fc" refers to an antibody fragment that is formed by linking a second, third constant region of a first heavy chain of an antibody and a second, third constant region of a second heavy chain via a disulfide bond. The Fc fragment of an antibody has many different functions but is not involved in antigen binding.

As used herein, the term “scFv” refers to a single polypeptide chain containing the VL and VH regions, where VL and VH are linked by a linker (see, for example, Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated amino acid sequences GGGGS or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA, 90: 6444-6448). Other linkers that can be used in the present invention are described in the works of Alfthan et al. (1995), Protein Eng., 8:725-731, Choi et al. (2001), Eur. J. Immunol., 31: 94-106, Hu et al. (1996), Cancer Res., 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol., 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, scFv may also have disulfide bonds between VH and VL.

As used herein, the term "diabody" refers to an antibody fragment in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing of two domains of the same chain, thereby forcing one domain to pair with a complementary domain of the other chain and form two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) and Poljak R. J. et al. ,Structure, 2: 1121-1123 (1994)).

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or to compete with the full-length antibody for specific antigen binding.

Conventional methods known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation) can be used to obtain antigen binding antibody fragments (e.g., the above antibody fragments) from a particular antibody (e.g., the antibody of the present invention) and tested for specificity in the same way as intact antibodies.

Here, unless the context clearly dictates otherwise, when the term “antibody” is mentioned, it includes not only the intact antibody, but also antigen-binding fragments of the antibody.

As used herein, the terms "monoclonal antibody", "McAb" and "mAb" have the same meaning and can be used interchangeably. The term refers to an antibody or antibody fragment from a population of highly homologous antibody molecules, i.e., a population of completely identical antibody molecules, except for a natural mutation that may occur spontaneously. Monoclonal antibodies are highly specific for one epitope of an antigen. A polyclonal antibody, compared to a monoclonal antibody, typically comprises at least two or more different antibodies that typically recognize different epitopes on an antigen. In addition, the designation “monoclonal” simply refers to the nature of the antibody as being derived from a highly homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

As used herein, the term "chimeric antibody" refers to an antibody whose light chain and/or heavy chain part is derived from one antibody (which may be from a particular species or belong to a particular class or subclass of antibodies) and another part of its light chain and/or the heavy chain is derived from another antibody (which may be from the same or different species or from the same or a different class or subclass of antibodies), but in either case still retains binding activity to the target antigen (U.S. Pat. No. 4816567, Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)). For example, the term "chimeric antibody" may include an antibody (eg, a human-mouse chimeric antibody) in which the variable regions of the heavy and light chains of the antibody are derived from the first antibody (eg, a mouse antibody), whereas the constant regions of the heavy chain and light chain antibodies are derived from a second antibody (eg, a human antibody). To produce a chimeric antibody, methods known in the art can be used to link immunoglobulin variable regions from an immunized animal to human immunoglobulin constant regions (see, for example, US Pat. No. 4,816,567 to Cabilly et al.). For example, DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to produce a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat E. A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 ), and DNA fragments containing these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to produce a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, for example, Kabat E. A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 ), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a κ or λ constant region, but is generally preferred to be a κ constant region.

As used herein, the term “humanized antibody” refers to an engineered antibody of non-human origin, the amino acid sequence of which has been modified to increase homology to the sequence of the human antibody. In essence, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (eg, FR variable region and/or constant region) are derived from a human immunoglobulin (recipient antibody). In some embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and/or constant regions) are derived from a human immunoglobulin (recipient antibody). The humanized antibody typically retains the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. The donor antibody may be a mouse, rat, rabbit, or non-human primate antibody (e.g., cynomolgus monkey) with the desired properties (for example, antigen specificity, affinity, reactivity, etc.). To obtain a humanized antibody, methods known in the art can be used to insert CDR regions of an immunized animal into human framework sequences (see US Pat. No. 5,225,539, Winter; US Pat. No. 5,530,101, Queen et al.; US Pat. Nos. 5,585,089; 5,693,762 and 6180370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

As used herein, the term “germline antibody gene” or “germline antibody gene segment” refers to a sequence present in the genome of an immunoglobulin-encoding organism that has not undergone a maturation process that may lead to genetic rearrangements and mutations for the expression of a particular immunoglobulin . As used herein, the expression “germline heavy chain gene” refers to a germline antibody gene or gene fragment encoding an immunoglobulin heavy chain, which includes a V (variable) gene, a D (diversity) gene, a J (junction) gene, and a C (constant); similarly, the expression “germline light chain gene” refers to a germline antibody gene or gene fragment encoding an immunoglobulin light chain, which includes the V gene (variable), the J gene (junction) and the C gene (constant). In the context of the present invention, the amino acid sequence encoded by a germline antibody gene or a fragment of a germline antibody gene is also referred to as a “germline sequence.” A germline antibody gene or germline antibody gene fragment and corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from professional databases (eg, IMGT, unswag, NCBI or VBASE2).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant ( _KD ) of the interaction. In the present invention, the term “K _D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The lower the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between antibody and antigen. Specific binding parameters between two molecules can be measured using methods known in the art, for example, using surface plasmon resonance (SPR) on the BIACORE instrument.

As used herein, the expression "binding at neutral pH with an affinity greater than that at acidic pH" or the equivalent expression "pH-dependent binding" refers to the antibody of the present invention having a K _D value or EC ₅₀ value for binding HBsAg at an acidic pH value that is higher than the K _D value or EC ₅₀ value for HBsAg binding at neutral pH. K _D can be measured using a method known in the art, for example, using the SPR method (eg, Biacore). In the present invention, the term "EC ₅₀ " refers to the antibody-antigen half-maximal effect concentration, i.e., the antibody concentration required to achieve 50% of the maximum binding effect between a particular antibody and an antigen, and is used to describe the binding ability between an antibody and antigen. The lower the EC ₅₀ value, the higher the binding capacity between antibody and antigen. The half-maximal antibody-antigen binding concentration (EC ₅₀ ) can be determined using methods known in the art, for example, using an enzyme-linked immunosorbent assay (ELISA), in which the antigen is bound to a solid-phase carrier and the antibody specifically binds to the antigen. .

As used herein, the term “neutralizing antibody” refers to an antibody or antigen-binding fragment thereof that can significantly reduce or completely inhibit the virulence (eg, the ability to infect cells) of a target virus. In essence, neutralizing antibodies can recognize and bind to a target virus and prevent the target virus from entering/infecting the cells of a subject. The antibody of the present invention is a neutralizing antibody.

However, it should be understood that, as used herein, the ability of an antibody to neutralize a virus is not equivalent to the ability of an antibody to clear a virus. As used herein, the term “virus neutralization” means that the virulence of the target virus is neutralized (ie, the virulence of the target virus is significantly reduced or completely inhibited) by inhibiting entry of the target virus/infection of the target virus into a cell of a subject. As used herein, the term “virus clearance” means that the target virus (whether it infects a cell or not) is cleared from the body, and therefore the body returns to the state before infection with the virus (for example, the result of a serological test of the virus becomes negative). Therefore, as a rule, neutralizing antibodies do not necessarily have the ability to “clear” the virus. However, within the scope of the present application, the inventors unexpectedly discovered that the antibodies of the invention can not only neutralize HBV, but also eliminate the virus (i.e., can “clear” HBV DNA and/or HBsAg in vivo, “clear” HBV and HBV-infected cells in vivo), and therefore have important clinical significance.

For purposes of the present invention, the term “isolated” refers to a state obtained from a natural state using artificial means. If a certain "isolated" substance or component is present in nature, it is possible because its natural environment is altered, or the substance is isolated from its natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide occurs naturally in the body of a certain living animal, and the same polynucleotide or polypeptide with high purity isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude any mixed artificial or synthesized substance, nor other impure substances that do not affect the activity of the isolated substance.

As used herein, the term “vector” refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector provides expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, such that elements of genetic material carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or phage P1 DNA artificial chromosomes (PAC); bacteriophages such as phage λ or phage M13, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40) . The vector may contain various elements that control expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may contain an origin of replication.

As used herein, the term “host cell” refers to a cell into which the vector can be introduced, which includes, but is not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis , a fungal cell such as a yeast cell or Aspergillus , an insect cell such as a Drosophila S2 or Sf9 cell, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell or human cell.

As used herein, the term “identity” refers to the degree of correspondence between two polypeptides or between two nucleic acids. When two sequences to be compared contain the same monomeric subunit, base or amino acid, at a specific position (for example, two DNA molecules each have an adenine at a specific position, or two polypeptides each have a lysine at a specific site), then the two molecules identical in this position. The percentage identity between two sequences is a function of the number of identical positions shared by the two sequences times the total number of positions compared multiplied by 100. For example, if 6 out of 10 positions of two sequences match, the two sequences have 60% identity. For example, the DNA sequences: CTGACT and CAGGTT are 50% identical (3 out of 6 positions are the same). Typically, the comparison of two sequences is carried out in such a way as to ensure maximum identity. Such alignment can be performed using a computer program such as the Align program (DNAstar, Inc.), which is based on the method of Needleman, et al. (J. Mol. Biol., 48: 443-453, 1970). The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which was included in the ALIGN program (version 2.0), using PAM120 residue weight tables, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol., 48: 444 -453 (1970)), which was included in the GAP program in the GCG software package (available at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and the gap weights are 16, 14, 12 , 10, 8, 6, or 4, and the sequence length weight is 1, 2, 3, 4, 5, or 6.

The twenty common amino acids involved here are expressed in a normal manner. See, for example, Immunology-A Synthesis ( ^2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Also herein, amino acids are generally represented by one-letter and three-letter symbols known in the art. For example, alanine can be represented by the symbols A or Ala. Additionally, as used herein, the terms “monoclonal antibody” and “McAb” have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "PcAb" have the same meaning and can be used interchangeably.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active substance, which is well known in the art (see, for example, Remington's Pharmaceutical monograph Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjuster, surfactant, adjuvant, ionic strength enhancer, diluent, osmotic pressure adjusting agent, retardant absorption, and preservative. For example, a pH regulator includes, but is not limited to, a phosphate buffer. The surfactant includes, but is not limited to, a cationic, anionic, or nonionic surfactant, such as Tween-80. The ionic strength enhancer includes, but is not limited to, sodium chloride. The preservative includes, but is not limited to, various antibacterial agents and antifungal agents such as paraben, chlorobutanol, phenol and sorbic acid. The osmotic pressure adjusting agent includes, but is not limited to, sugar, NaCl, and analogs thereof. The absorption retarding agent includes, but is not limited to, monostearate and gelatin.

As used herein, the term “prevention” refers to a method that is performed to suppress or delay the progression of a disease, disorder, or symptom (such as HBV infection or a disease associated with HBV infection) in a subject. In the context of the present invention, the term “treating/treating” refers to a method that is carried out to obtain a positive or desired clinical result. For purposes of the invention, a beneficial or desired clinical outcome includes, but is not limited to, symptomatic relief, narrowing of the extent of disease, stabilization (i.e., not exacerbation) of disease severity, delay or slowing of disease progression, and relief of symptoms (partially or completely), whether whether they are detectable or not. In addition, "treatment" also refers to longer survival than expected survival (if no treatment is given). In the present application, the antibody of the invention has the ability to neutralize HBV and, therefore, can be used to prevent/protect a healthy subject or its cells from HBV infection. In addition, the antibody of the invention has the ability to “clear” HBV (i.e., is able to “clear” HBV DNA and/or HBsAg in vivo, “clear” HBV and HBV-infected cells in vivo) and therefore can used for the treatment of HBV infection or disease associated with HBV infection in an infected subject.

As used herein, the term “subject” refers to a mammal, such as a mammalian primate, such as a human.

As used herein, the term "effective amount" refers to an amount that is sufficient to achieve, or at least partially achieve, the intended effect. For example, an amount effective to prevent a disease (such as HBV infection or a disease associated with HBV infection) refers to an amount effective to prevent, suppress or delay the progression of a disease (such as an HBV infection or a disease associated with an HBV infection). An effective amount for treating a disease refers to an amount effective to treat or at least partially block a disease and its complications in a patient suffering from the disease. Determination of such effective amount is within the competence of one skilled in the art. For example, the amount effective for therapeutic use depends on the severity of the disease being treated, the general condition of the patient's immune system, general characteristics of the patient such as age, weight and sex, routes of drug administration, additional treatments used concurrently, and things like that.

Advantageous Effects of the Present Invention

The antibody of the present invention not only can specifically recognize/bind HBsAg, but can neutralize the virulence of HBV, can reduce the serum level of HBV DNA and/or HBsAg in a subject, and can effectively eliminate HBV and HBV-infected cells from the body, but also has a significantly increased antigen clearance effect and antigen suppression time. What is particularly surprising is that it is known in the art that patients with chronic hepatitis B tend to deplete the immune system (tolerize) to HBV due to high levels of HBsAg in the body, thereby prolonging the course of the infection, but the antibody of the present invention can provide activation in a subject (eg, patients with chronic HBV infection or patients with chronic hepatitis B) with restoration of the humoral immune response against HBV, thereby increasing the rate of clinical cure. Therefore, the antibody of the present invention is particularly suitable for the prevention and treatment of HBV infection and diseases associated with HBV infection (eg, hepatitis B). In addition, the antibody of the present invention has pH-dependent antigen-binding properties, and one antibody molecule can bind to multiple antigen molecules, and as a result, it can also reduce the frequency and dosage of administration, and has great clinical significance.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying figures and examples. However, it will be understood by those skilled in the art that the following figures and examples are used only to illustrate the present invention and not to limit the scope of the present invention. Referring to the accompanying drawings and the following detailed description of preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art.

Brief description of the figures

In fig. Figure 1 shows a schematic representation of the principle of operation of an antibody with pH-dependent antigen-binding activity. Human blood plasma is neutral with a pH of approximately 7.4, while the intracellular environment is acidic with a pH of approximately 6.0. An antibody with pH-dependent antigen-binding activity can bind to an antigen in the plasma, after which the antigen-antibody complex is 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 circulate outside the cell. In an extracellular neutral environment, FcRn releases the antibody, and the antibody, returning to the plasma, can again bind to another antigen, thereby realizing the cycle of antibody use.

In fig. Figure 2 shows the results of docking the Fab crystal structure according to the structural analysis of antibody 162 with a short antigen-mimicking peptide, where the blue structure represents the short antigen peptide and the red structure is part of the binding region of antibody 162.

In fig. 3 is a schematic representation of a recombinant vector (pCGMT-scFv) encoding a scFv antibody, wherein the scFv antibody has the structure: NH ₂ -VH-linker-VL-COOH.

In fig. 4A-4D show ELISA results of a phage library displaying a pH-dependent scFv antibody derived from antibody 162 and HBsAg antigen. In fig. 4A: HBsAg binding detection results at pH 7.4 and pH 6.0 for a phage library obtained from antibody 162 after the third round of screening, the x-axis represents the number of phage antibodies, and the y-axis represents the OD value. The results show that all of these single clones have high antigen-binding activity and have a significant decrease in binding activity at pH 6.0. In fig. 4B: pH-dependent HBsAg binding detection results for 13 individual clones with high OD _(450/630) at pH 7.4 in the third round and showing the largest difference between OD _(450/630) at pH 7.4 and pH 6.0, with 8 gradients and 3-fold dilution, where the x-axis represents the dilution factor and the y-axis represents the OD value. The results show that the pH-dependent antigen binding effect is better after gradient dilution, in which C32, C27, C26 and C19 are more effective, and the C27 molecule gives the best effect (the other 9 molecules are not shown). In fig. 4C: HBsAg binding detection results at pH 7.4 and pH 6.0 for the phage library obtained from antibody 162 after the fourth round of screening, the x-axis represents the number of phage antibodies, and the y-axis represents the OD value. In fig. 4D: pH-dependent HBsAg binding detection results for 8 individual clones with high OD value _(450/630) at pH 7.4 in the fourth round and showing the largest difference between OD values _(450/630) at pH 7.4 and pH 6.0, with 8 gradients and 3-fold dilution, where the x-axis represents the dilution factor and the y-axis represents the OD value. The results show that the pH-dependent antigen-binding effect is better after gradient dilution, in which D3, D4 and D5 are more effective, and the D5 molecule gives the best effect (the remaining 5 molecules are not shown).

In fig. Figure 5 presents summarized data on mutation sites C26, C27, C32, D3, D4 and D5.

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

In fig. Figure 7 shows the principle of operation of the “scavenger” antibody. In cells, pH-dependent antigen-binding activity plays a role. Thus, if this first limiting factor of cell entry is not disrupted, then the pH-dependent antigen-binding properties will not be applied subsequently and the benefit of the modification will be greatly reduced. A scavenger antibody generated by additional mutation of amino acids in the Fc region can enhance binding to the hFcRn receptor at neutral pH or enhance binding to the FcγRs receptor. The “scavenger” antibody is located outside the cell and functions as a “transport assistant”, respectively, for the transport of antigens into the cell, therefore, the half-life of the antibody can be greatly prolonged, and the antibody can again bind to the antigen, thereby leading to an increase in the efficiency of penetration of antigens into the cell. cell, and significantly improving clearance efficiency.

In fig. 8A-8B show the results of protein separation in gel electrophoresis for pH-dependent antibodies and antibodies with DY modification. In fig. 8A: Image of proteins in gel electrophoresis of pH-dependent antibodies, where antibody 162 is a positive control, and the results show that the expressed antibodies C26, D3, D4 and D5 are single-component. In fig. 8B: Gel electrophoresis image of antibodies with DY modification, where antibody 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.

In fig. 9A-9D show the results of detecting the binding of pH-dependent antibodies and antibodies with DY modification to HBsAg at pH 7.4 and pH 6.0 in example 4, where the abscissa represents the antibody concentration (Log ₁₀ ng/ml), and the ordinate represents OD value. The results show that D26, D3, D4 and D5 retain antigen-binding activity equivalent to that of the parent antibody (162) at neutral pH and have a significant decrease in antigen-binding activity at pH 6.0, and the corresponding DY-modified antibodies also retain antigen-binding activity at neutral pH and pH-dependent antigen-binding activity comparable to that of the parent antibody.

In fig. 10A shows the results of an immunofluorescence assay experiment on primary murine macrophages for pH-dependent and DY-modified antibodies in Example 4, where green fluorescence represents hFcRn, blue fluorescence represents the nucleus, and red fluorescence represents HBsAg. The results indicate that the DY modification enhances the phagocytic activity of murine macrophages through antigen-antibody complexes. In fig. 10B shows the results of an experiment evaluating phagocytosis on phagocytic human THP-1 cells for pH-dependent antibodies and antibodies with DY modification in Example 4. The results show that the DY modification enhances the phagocytic activity of phagocytic human THP-1 cells through antigen-antibody complexes.

In fig. 11A-11B show the therapeutic effects of scavenger antibody DY C26 and antibody 162 in HBV transgenic mice following a single tail vein injection at a dose of 5 mg/kg in Example 4. FIG. 11C shows the therapeutic effects of D3 DY, D3 DY, D4 DY and D5 DY in HBV transgenic mice after a single tail vein injection at a dose of 5 mg/kg in Example 4. FIG. 11A: The abscissa represents the number of days (d) after antibody injection, and the ordinate represents the level of HBsAg in mouse serum after clearance (log ₁₀ IU/ml). In fig. 11B shows changes in antibody concentration in mouse serum, where the abscissa represents the number of days (d) after injection of the antibody, and the ordinate represents the antibody concentration (ng/ml). In fig. 11C: The abscissa represents the number of days (d) after antibody injection, and the ordinate represents the level of HBsAg in mouse serum after clearance (log ₁₀ IU/ml). The results show that scavenger antibodies with the modification DY C26 DY, D3 DY, D4 DY and D5 DY have a higher ability to clear antigen by more than one order of magnitude than antibody 162. This indicates that the antibodies are Scavengers with DY modification C26 DY, D3 DY, D4 DY and D5 DY can perform the function of cyclic antigen binding and enhance the effect of antigen clearance when injected at a low dose of 5 mg/kg.

Sequence information

Information on partial sequences included in the present invention is presented in Table 1 below.

Table 1
Description of sequences SEQ ID NO Description Sequence information 1 162 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYGYHWNWIRQFPGNKLEWIGYISYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS 2 C27 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIK 3 C26 D4 D5 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS 4 C26 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHHPYTFGGGTKLEIK 5 C27 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS 6 C32 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLEWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS 7 C32 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHLPYTFGGGTKLEIK 8 D3 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS 9 D3 D5 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIK 10 D4 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHLPYTFGGGTKLEIK eleven 162
HCDR1 YGYHWN 12 162 D3 D4 D5
HCDR2 YISYDGSVLYNPSLEN 13 162 C26 C27 C32 D3 D4 D5
HCDR3 GFDH 14 162 C26 C27 C32
LCDR1 RSSQSLVHSYGDTYLH 15 162 C26 C27 C32 D3 D4 D5
LCDR2 KVSNRFS 16 162 C27 D3 D5
LCDR3 SQNTHVPYT 17 C26 C27 D4 D5
HCDR1 HGYHWN 18 C26
HCDR2 YIHYDGSVLYNPSLEN 19 C26
LCDR3 SQNTHHPYT 20 C27
HCDR2 YINHDGSVQYNPSLEN 21 C32
HCDR1 YRYHWN 22 C32
HCDR2 YINYDGSVHYNPSLEN 23 C32 D4
LCDR3 SQNTHLPYT 24 D3
HCDR1 HGYHYN 25 D3 D4 D5
LCDR1 RSSQSLVHSYGDNYLH 26 General formula of HCDR1 X ₁ X ₂ YHX ₃ N 27 General formula HCDR2 YIX ₄ X ₅ DGSVX ₆ YNPSLEN 28 General formula LCDR1 RSSQSLVHSYGDX ₇ YLH 29 General formula LCDR3 SQNTHX ₈ PYT thirty C26 C27 C32 D3 D4 D5
HFR1 EVQLQESGPGLVKPSQTLSLTCAVSGSSIT 31 C26 C27 C32 D3 D4 D5
HFR2 WIRQFPGNKLEWIG 32 C26 C27 C32 D3 D4 D5
HFR3 RVTITRDTSKNQFFLKLSSVTAEDTAKYYCAS 33 C26 C27 C32 D3 D4 D5
HFR4 WGQGTTLTVSS 34 C26 C27 C32 D3 D4 D5
LFR1 DVVMTQSPLSLPVTLGEPASISC 35 C26 C27 C32 D3 D4 D5
LFR2 WYLQKPGQSPKLLIY 36 C26 C27 C32 D3 D4 D5
LFR3 GVPDRFSGSGSGTDFTLKISRVETEDLGVYYC 37 C26 C27 C32 D3 D4 D5
LFR4 FGGGTKLEIK 38 4-28-02 QVQLQESGPGLVKPSQTLSLTCAVSGYSISSSNWWGWIRQPPGKGLEWIGYIYYSGSIYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCAR 39 2D-28-01 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 40 Human IgG1 heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 Human κ light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDSALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 42 Human IgG1 heavy chain constant region with V4 mutation ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHEAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK 43 Human IgG1 heavy chain constant region with DY mutation ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNDAYPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44 Primer 5'>GTTATTACTCGTGGCCCAGCCGGCCATGGCAGAGGTGCAGCTGCAGGAGTC <3' 45 Primer 5'>CTCCAGCTTGTTCCCTGGGAACTGCCGGATCCAGTTSYRGTGGTRGYSGTRGGTGATGGAGCTACCAGA <3' 46 Primer 5'>GTTCCCAGGGAACAAGCTGGAGTGGATTGGGYACMWCMRCYACSACGGCAGCSWYCWSYACAATCCATCTCTCG <3' 47 Primer 5'>GACTGTGAGAGTTGTGCCTTGGCCCCAGTGGTSGWRACCACTCGCACAGTA <3' 48 Primer 5'>CCAGATCCGCCACCTCCACTCCCGCCTCCACCTGAGGAGACTGTGAGAGTTGTGCCTT <3' 49 Primer 5'>GTGGAGGTGGCGGATCTGGAGGGGGTGGTAGCGATGTTGTGATGACCCAATC <3' 50 Primer 5'>CTTTGGAGACTGGCCTGGCTTCTGCAGGTACCAATGSWGGTRGKKGTCTCCATAGYKGTGRWS <3' 51 Primer 5'>AGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTG <3' 52 Primer 5'>TTTCCAGCTTGGTCCCCCCTCCGAAGKKGTRGKGRWSATGGKKGTKSTGAGAGCAGTAATAAAC <3' 53 Primer 5'>TAGTCGACCAGGCCCCCGAGGCCTTTTATTTCCAGCTTGGTCCCCCCT <3' 54 Signal peptide MGWSCIILFLVATATGVHS 55 Primer 5'-
AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTC 56 Primer 5'-
GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGACGGTGG 57 Primer 5'-
AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTCTC 58 Primer 5'-
ATGGTGCAGCCACCGTACG TTTGATTTCCACCTTGGTCC

Examples

The present invention will now be described with reference to the following examples, which are intended to illustrate the present invention and not to limit the present invention.

Unless otherwise noted, the experimental molecular biology and immunoassay methods used in the present invention are substantially those described in 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; restriction enzymes were used according to the conditions recommended by the product manufacturer. It will be understood by those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of protection to be protected by the present invention.

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

1.1 Determination of mutation sites for pH-dependent modification of antibodies

Humanized anti-HBV antibody 162 (detailed in Chinese patent application 201610879693.5) developed in the laboratory was used as the parent antibody, and its variable region was modified for pH-dependent antigen binding. As shown in FIG. 1, the modified antibody 162 retained antigen-binding activity under neutral conditions, but its antigen-binding activity under acidic conditions was significantly reduced. The dissociated modified 162 could bind to intracellular FcRn and thus return to the plasma and bind again to the antigen, so that one molecule of modified 162 antibody with pH-dependent antigen-binding ability could repeatedly bind and neutralize multiple antigen molecules. Histidine was protonated under acidic conditions and was the key amino acid for pH-dependent antigen-binding properties. Fab 162 has an established crystal structure, the established crystal structure was docked through modeling with a short antigen peptide, and part of the results are shown in FIG. 2, where the blue structure represents the short antigen peptide, and the red structure represents part of the binding region of antibody 162. According to the docking results, a total of 14 key amino acids were identified that are important for antigen-antibody binding. Considering that the docking simulation results had a higher reference value, amino acids at the interface and amino acids on both sides were selected for mutation, and 26 sites were identified.

1.2 Construction of a phage library of pH-dependent scFv antibodies derived from antibody 162

Using the light and heavy chain variable regions of antibody 162 as a template, established sites in the CDR of the antibody variable region were mutated to provide pH-dependent modification, and the target fragments were amplified using the primers shown in Table 2 to produce gene fragments encoding pH- dependent scFv antibodies derived from antibody 162. PCR conditions were as follows: 95°C, 5 min; 95°C, 30 s; 57°C, 30 s; 72°C, 30 s; 72°C, 10 min; 25 amplification cycles; SOE-PCR reaction conditions: 95°C, 5 min; 95°C, 30 s; 57°C, 30 s; 72°C, 30 s; 72°C, 10 min; 5 amplification cycles. The amplified products were analyzed by agarose gel electrophoresis, and the amplification products were isolated/purified using a DNA purification and extraction kit (TianGen, DP214-03), thereby obtaining HK gene fragments encoding humanized scFv antibodies derived from antibody 162. Structure The scFv antibody was as follows: NH ₂ -VH-linker-VL-COOH, and the linker sequence could be (G ₄ S) ₃ . Each of the HK gene fragments was digested with SfiI, and then ligated into the pCGMT vector (Scripps Research Institute, Making Chemistry Selectable by Linking to Infectivity) at a molar ratio of 10:1 (gene fragment:vector). The ligation products were transformed into competent Escherichia coli ER2738 using electroporation (electroporation conditions: 25 μF, 2.5 kV, 200 Ω). Transformed Escherichia coli were isolated in SOC medium for 45 min, and then 200 μl of bacterial solution was plated on LB plates (containing 100 g/l ampicillin+tetracycline+2 g/ml glucose) and incubated at 37°C overnight. All plated colonies were libraries where the mutation sites identified in the variable regions were randomly mutated to histidine, which were used for subsequent screening. Monoclonal colonies were picked from the plates and sequenced to ensure the correct sequences of the recombinant vectors encoding the scFv antibodies. A schematic representation of the recombinant vector (pCGMT-scFv) encoding the scFv antibody is shown in FIG. 3.

table 2
Primers for mutation of pH-dependent scFv antibodies derived from antibody 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

The library obtained in the previous step was screened in several rounds, and the positive monoclonal colonies obtained from the screening were cultured in 2xYT medium containing ampicillin (100 g/l) and glucose (2 g/ml) to achieve OD of 0.6, and then M13KO7 helper phage was added for superinfection. After 2 h, 100 g/L kanamycin was added and superinfection was carried out 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 medium containing ampicillin and kanamycin (100 g/L) and cultured with shaking at 30°C overnight. The culture was then centrifuged at 12,000 rpm for 10 min, and the cells and supernatant were collected and stored at 4°C for testing.

An ELISA plate coated with HBsAg antigen (200 ng/ml) was used and 100 μl of test supernatant was added to each well and incubated at 37°C for 1 hour (two wells per supernatant). The ELISA plate was then washed once with PBST, and then 120 μl of PBS pH 7.4 and pH 6.0 were added to two wells of each supernatant, respectively, and incubated at 37°C for 30 min. After washing the wells with PBST 5 times at the appropriate pH, 100 μl of anti-M13-HRP diluted 1:5000 was added and incubated at 37°C for 30 min. The ELISA plate was then washed 5 times with PBST and TMB substrate solution was added. After 15 minutes of color development, the color reaction was stopped by adding H ₂ SO ₄ and the reading was taken at OD450/630. The detection results of the third round of ELISA are shown in FIG. 4A-4D. The results showed that all phages displaying these scFv antibodies were reactive in ELISA detection and weakly bound to antigens at pH 6.0; Initially, six strains of pH-dependent phage antibodies with good effects were 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 a recombinant vector for a eukaryotic expression system

In the present invention, it was necessary to carry out large recombination of antibodies, therefore, it was necessary to construct a series of light and heavy chain vectors that can efficiently produce recombinant antibodies. In the present invention, an existing laboratory eukaryotic expression vector pTT5 was specifically modified to construct a series of recombinant light and heavy chain vectors for dual plasmid cotransfection. The sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 54) was used as the signal peptide for the light and heavy chains. Sequences encoding the human antibody light and heavy chain constant regions were individually ligated to the downstream signal peptide to construct a series of eukaryotic expression vectors pTT5-CH, pTT5-Cκ and pTT5-Cλ, which produced recombinant antibodies.

Six scFv antibodies prepared as described in section 1.3 were used to amplify light and heavy chain variable region fragments using the primers shown in Table 3. Specific amplification reaction conditions were as follows: 95°C, 5 min; 95°C, 30 s; 57°C, 30 s; 72°C, 30 s; 72°C, 10 min; 25 amplification cycles. And the amplification products were extracted from the gel.

A laboratory-prepared Gibson assembly reaction solution was used to ligate the constructed eukaryotic expression vector described above with the reconstituted antibody variable region gene PCR product (a primer carrying sequence homologous to the vector) to produce recombinant vectors VH+pTT5-CH (containing CH shown in SEQ ID NO: 40) and VH+pTT5-Cκ (containing the CL shown in SEQ ID NO: 41). The recombinant vector was transformed into E. coli strain DH5α, plated on an LB plate, and cultured overnight in an incubator at 37°C. Monoclonal colonies were picked on a plate and sequenced, and the sequencing results were subjected to sequence comparison using MEGA to confirm the correctness of its genes and eliminate genes with incorrect information.

Table 3
Primers for the 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-scale and large-scale expression of antibody genes

The constructed recombinant vectors VH+pTT5-CH and VH+ pTT5-Cκ were cotransfected into HEK293 cells, and dual small-scale expression plasmids were cotransfected into a 24-well plate at 500 μL per well; if the cell supernatant in small-scale expression had antigenic activity, then the volume of the transfection system was increased to 100 ml (determined by the amount of antibody used) of FreeStyle ^TM 293F cell suspension (cell density was approximately 2 x 10 ⁶ cells/ml). Transfected cells were cultured in a shake flask in an incubator at 32°C, 5% CO ₂ , and the supernatant was collected after 7 days of expression.

2.3 Antibody purification

The supernatant of the expressing cells was collected and purified on a protein A column according to the manufacturer's instructions. The specific steps were as follows: the collected cell culture supernatant was centrifuged at 8000 rpm for 10 min, the supernatant was stored, the pH was adjusted to 8.4 using dry Na ₂ HPO ₄ powder, and then filtered through a 0.0 pore diameter filter membrane. 22 microns. 10 ml of Sepharose 4B coupled to protein A was loaded onto a column which was connected to the AKTA Explorer100 system, pump A was connected to 0.2 M sodium hydrogen phosphate solution, and pump B was connected to 0.2 M citric acid solution. The detection wavelength was UV 280 nm, the flow rate was 5 mL/min, and the sample injection ratio from the A/B pumps was adjusted. The column was first washed with 100% B (pH 2.3) to remove protein impurities, the pH was equilibrated with 10% B (pH 8.0), the signal at the detection wavelength returned to zero after it had stabilized, then the sample was loaded. After peak flow, 10% B was used to equilibrate until the signal at the detection wavelength dropped to zero and stabilized, elution was performed using 70% B (pH 4.0), and the elution peak was collected. The elution peak sample was dialyzed against PBS buffer and subjected to concentration analysis and SDS-PAGE and HPLC analysis to determine the purity of the IgG antibody.

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

Using the method described in Example 1, six strains of pH-dependent phage antibodies that bound to HBsAg were obtained by preliminary screening and were named C26, C27, C32, D3, D4 and D5, respectively. In addition, small-scale eukaryotic expression and purification of the 6 phage antibody strains was carried out using the method described in Example 2. The amino acid sequences of the VH and VL 6 antibodies are shown in Table 4 below. In addition, the CDR sequences of the 6 antibodies were determined, and the amino acid sequences of the CDR heavy chain variable regions and light chain variable regions are given in Table 5. Mutation sites that give antibodies C26, C27, C32, D3, D4 and D5 pH-dependent binding properties to antigen with HBsAg are shown in Fig. 5.

Table 4
Amino acid sequences of the light and heavy chain variable regions C26/C27/C32/D3/D4/D5 Sequence name SEQ ID NO Amino acid sequence C26 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS C26 VK 4 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHHPYTFGGGTKLEIK C27 VH 5 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS C27 VK 2 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIK C32 VH 6 EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLEWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS C32 VK 7 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHLPYTFGGGTKLEIK D3 VH 8 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS D3 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIK D4 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS D4 VK 10 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHLPYTFGGGTKLEIK D5 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS D5 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIK

Table 5
Light and heavy chain CDR sequences C26/C27/C32/D3/D4/D5 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

We first performed small-scale transfection for six monoclonal antibodies; After quantification of the supernatant obtained after transfection, the pH-dependent antigen-binding capacity with HBsAg was determined by ELISA, and the antibody concentration was uniformly diluted to 1111.11 ng/ml. 20% NBS was then used to dilute the antibody in a 3-fold concentration gradient, for a total of 8 concentration gradients. The diluted antibody was then added to a commercial HBsAg plate (purchased from Beijing Wantai) and incubated at 37°C for 1 h (two wells per supernatant). The ELISA plate was then washed once with PBST and dried in a spin dryer. Then, 120 μL of PBS with pH 7.4 and pH 6.0, respectively, was added to two wells of each supernatant and incubated at 37°C for 30 min. The wells at the appropriate pH were then washed 5 times with PBST and dried in a spin dryer. GAH-HRP-labeled secondary antibody was then added, incubated for 30 min, and the plate was washed 5 times with PBST and spin-dried. TMB substrate solution was added. After 15 minutes of color development, the color reaction was stopped by adding H ₂ SO ₄ and readings were taken at OD450/630.

The results are shown in Fig. 6A-6B. From the data obtained, it follows that all candidate molecules retained antigen binding activity comparable to that of the parent antibody 162 at neutral pH, and antigen binding activity was significantly reduced at pH 6.0. The results of determining EC ₅₀ are presented in Table 6.

Table 6
EC ₅₀ values for detecting pH-dependent activity for C26, C27, C32, D3, D4 and D5 Antibody EC ₅₀ at pH 6.0
(ng/ml) EC ₅₀ at pH 7.4
(ng/ml) EC ₅₀ (pH 6.0)/EC ₅₀ (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 a Scavenger Antibody

A pH-dependent antibody needs to enter the cell to exhibit its pH-dependent antigen-binding activity. Therefore, if the first limiting factor of cell entry is not disrupted, then subsequent pH-dependent antigen-binding properties will not have any chance to "play". Therefore, in this example, the scavenger antibody was generated by additional amino acid mutation in the Fc region, which can enhance binding to the hFcRn receptor at neutral pH or enhance binding to the FcγRs receptor. As shown in FIG. 7, the “scavenger” antibody is located outside the cell and plays the role of a “transport assistant”, which accordingly transports antigens into the cell, thereby significantly increasing the half-life of the antibody, and it can bind to the antigen again, thereby increasing the efficiency of antigen penetration into cells and significantly increasing clearance efficiency.

C26, D3, D4 and D5 were selected as antibodies for subsequent evaluation and subjected to Fc DY mutations (K326D, L328Y) to increase binding affinity to mFcγRII under neutral conditions (C26 Fc modification was carried out by General Biologicals, order number G122413) to obtain scavenger antibodies C26 DY, D3 DY, D4 DY and D5 DY, which bound to mFcγRII. The above antibodies were subjected to large-scale eukaryotic expression and purification, and the specific steps were the same as described in Examples 1.2 and 1.3.

In fig. 8A-8B show the results of protein separation in gel electrophoresis of the original antibody and modified antibodies. In fig. 8A: Gel electrophoresis image of proteins from parent antibody, where antibody 162 was a positive control. The results showed that the parent antibody expressed was single-component. In fig. 8B: Image of proteins in gel electrophoresis of antibodies with DY modification, where antibody 162 was a positive control. The results showed that the expressed antibodies with DY modification are single-component.

4.1 Assessment of pH-dependent antigen-binding activity of scavenger antibodies binding to mFcγRII

For the parent and DY-modified antibodies, after expression and purification, we used ELISA to determine their pH-dependent antigen-binding ability with HBsAg. First, a BCA protein quantitation kit was used to determine the concentrations of purified antibodies, and the antibodies were diluted uniformly to a concentration of 1111.11 ng/mL. Then, 20% NBS was used to carry out dilutions in a 3-fold concentration gradient, for a total of 8 concentration gradients. The diluted antibody was then added to a commercial HBsAg plate (purchased from Beijing Wantai) and incubated at 37°C for 1 h (two wells per supernatant). The ELISA plate was then washed once with PBST and dried in a spin dryer. Then, 120 μl of PBS pH 7.4 and PBS pH 6.0 were added to two wells with each supernatant, respectively, incubated at 37°C for 30 min, wells with the corresponding pH were washed 5 times with PBST and dried in a centrifugal dryer. GAH-HRP-labeled secondary antibody was then added and incubated for 30 min, the plate was washed 5 times with PBST and spin-dried. And TMB substrate solution was added. After 15 minutes of color development, the color reaction was stopped by adding H ₂ SO ₄ and the reading was measured at OD450/630.

The results are shown in Fig. 9A-9D, wherein the C26, D3, D4, D5 and their DY modified antibodies all have antigen binding activity equivalent to that of antibody 162, but exhibit weak antigen binding at pH 6.0, thereby demonstrating good pH-dependent antigen binding activity.

4.2 Testing the cellular function of mFcγRII-binding scavenger antibodies

4.2.1 Fluorescent labeling of HBsAg 488

Labeling of 1 mg of HBsAg is given as an example; the entire process was carried out in a place protected from light.

(1) 1 ml of 1 mg/ml HBsAg was dialyzed against borate buffer (pH 8.5, 500 ml), 4°C, 4 h;

(2) the molar ratio of HBsAg to 488 tag was 1:5, and after calculation, 0.1988 mg of fluorescent 488 tag was required;

(3) prepare a solution of fluorescent label 488 with a concentration of 10 mg/ml in DMF and mix well;

(4) 19.88 μL of fluorescent tag 488 was added to 1 mL of dialyzed HBsAg, mixed well, and incubated at room temperature for 1 h;

(5) the incubation mixture was dialyzed against PBS at 4°C overnight.

4.2.2 Immunofluorescence assay experiment on primary murine macrophages

(1) 4 days before the experiment, 1.5 ml of a 3% sodium thioglycolate solution was injected into the abdominal cavity of each mouse without entering the intestine;

(2) two mice were euthanized and the carcass was soaked in 75% alcohol for 3 min;

(3) the mouse was fixed horizontally on a foam board to open the abdominal cavity; the abdominal skin was incised with soft tissue scissors, the peritoneum was disinfected and dissected to expose the abdominal cavity, the abdominal incision skin was stretched with surgical two-prong clamps held in the left hand and fixed, culture medium 1640 was added with a Pasteur pipette held in the right hand for peritoneal lavage from calculation 4 ml/time, only two times. The pipette was used to gently and completely mix the abdominal contents to make the lavage more complete and thorough. After complete mixing for approximately 2 min and allowing for approximately 5 min to allow complete release of macrophages, the lavage solution was pipetted and transferred to a centrifuge tube;

(4) 4°C, 1100 g, 5 min;

(5) the supernatant was carefully discarded, 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 medium;

(6) After cell counting, the cell density was adjusted to 10 ⁶ cells/ml, the cells were cultured in a 24-well glass bottom plate for cell imaging, 250 μl/well, the medium was replaced after 2 h and washing was done once using RPM1640, after After non-adherent cells were removed, the cells were incubated overnight in a CO ₂ incubator at 37°C;

(7) the antibody and antigen, labeled with the appropriate fluorescent label, were diluted with serum-free medium to: 800 ng/ml for antigen and 20 μg/ml for antibody;

(8) 125 μl of antigen and 125 μl of antibody were mixed evenly, and then left for 1 hour in a CO ₂ incubator at 37°C;

(9) the cell supernatant in the culture plate for cell imaging was removed, the antigen-antibody complex was added, shaken evenly and left for 2 hours in a CO ₂ incubator at 37°C;

(10) the supernatant was removed, and 1 ml of sterile PBS, pre-incubated at 37°C, was used to “wash” the cell surface 3-5 times, and then completely removed with a pipette;

(11) Dio diluted 1:2000 was added in an amount sufficient to immerse the cells and kept at room temperature for 20 min;

(12) the supernatant was removed, and 1 ml of sterile PBS, pre-incubated at 37°C, was used to “wash” the cell surface 3-5 times, and then completely removed with a pipette;

(13) added live cell nuclear dye (2 drops added to 1 mL volume), kept at room temperature for 20 min, and placed in a high-resolution imager for imaging.

The results of the experiment are shown in Fig. 10A. From the data obtained, it follows that the DY modification enhances the phagocytic activity of mouse macrophages due to antigen-antibody complexes, which leads to greater degradation of the antigen.

4.2.3 Confirmation by chemiluminescent method based on phagocytic human THP-1 cells

(1) Adherent THP-1 cells were coated on a plate at a rate of 2×10 ⁵ cells/well, medium 1640 containing 10% serum was added, placed in a CO ₂ incubator, and cultured at 37°C for 24 hours;

(2) HBsAg was diluted with serum-free 1640 medium to a concentration of 800 ng/ml, and the test antibody with a concentration of 20 μg/ml as the starting concentration was subjected to a 2-fold gradient dilution, for a total of 11 gradients. 300 μl of diluted HBsAg and 300 μl of test antibody were mixed in a 1:1 ratio and left for 1 hour at 37°C;

(3) the supernatant of THP-1 cells was removed, 250 μl of HBsAg-antibody mixture was added to THP-1 cells, placed in a CO ₂ incubator and cultured at 37°C for 1 hour;

(4) the supernatant of THP-1 cells was removed and washed 3 times with sterile PBS;

(5) 120 μL of DDM cell lysis solution was added to each well of THP-1 cells, kept and reacted at 4°C for 1 h;

(6) The concentration of HBsAg in the lysate supernatant was determined using a hepatitis B virus surface antigen quantitation kit (Beijing Wantai).

The results of the experiment are shown in Fig. 10B. From the data obtained it follows that the DY modification enhances the phagocytic activity of human phagocytic THP-1 cells due to antigen-antibody complexes.

4.3 Determination of the therapeutic effect of mFcγRII-binding scavenger antibodies in animal models

HBV transgenic mice were chosen as animal models. Scavenger antibodies C26 DY, D3 DY, D4 DY and D5 DY and antibody 162 were administered as a single dose of 5 mg/kg into the tail vein (4 mice in each group) to HBV transgenic mice aged 6-8 weeks. Antigen clearance rates and in vivo half-lives of scavenger antibodies were analyzed by determining serum concentrations of HBsAg, antibodies, and HBV DNA.

Quantitative determination of HBsAg

(1) Reaction plate preparation: mouse monoclonal antibody HBs-45E9 was diluted with 20 mM PB buffer (Na ₂ HPO ₄ / NaH ₂ PO ₄ buffer, pH 7.4) to 2 μg/ml, and into each well of the chemiluminescence assay plate 100 µl of coating solution was added and coating was carried out at 2-8°C for 16-24 hours, then another 2 hours at 37°C, the plate was washed once with PBST wash solution and dried in a spin dryer. After washing, 200 μl of blocking solution was added to each well, and blocking was performed at 37°C for 2 hours. The blocking solution was then removed, the plate was placed in an oven to dry, and stored at 2-8°C for further use.

(2) Sample dilution: The collected mouse serum was diluted with PBS solution containing 20% NBS (fetal bovine serum) in two gradients of 1:30 and 1:150 for subsequent quantification.

(3) Sample denaturation: 15 μL of serum sample diluted as above was thoroughly 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.

(4) Sample reaction: 100 μL of the above denatured serum sample was added to the reaction plate and reacted at 37°C for 1 hour. The reaction plate was then washed 5 times with PBST and dried in a spin dryer.

(5) Enzyme label reaction: The HBs-A6A7-HRP reaction solution was added at 100 μL/well to the chemiluminescence assay plate and reacted at 37°C for 1 hour. The plate was then washed with PBST 5 times and dried in a spin dryer.

(6) Luminescence reaction and measurement: Luminescence solution (100 μL/well) was added to the chemiluminescence assay plate and the light intensity was measured.

(7) Calculation of HBsAg concentration in mouse serum sample: Conduct parallel experiments using standards, and construct a standard curve based on the measurement results of the standards. The light intensity value for the mouse serum sample was then plugged into the standard curve and the concentration of HBsAg in the test serum sample was calculated.

The results of detection of HBsAg in serum are shown in Fig. 11A and FIG. 11C. From the data in Fig. 11A shows that the DY-modified C26 DY scavenger antibody had a clearance capacity greater than one order of magnitude greater than that of antibody 162, which is consistent with the serum half-life of the antibody (FIG. 11B). When comparing antibody concentrations in serum, the half-life of C26 DY was longer than that of antibody 162 by almost 12 days, indicating that the scavenger antibody C26 DY functions as a circular and analog binding antigen, thereby increasing the duration of antigen clearance . The experimental results shown in Fig. 11C showed that the antigen clearance capacity of D3 DY, D4 DY and D5 DY was equivalent to that of C26 DY, and the functioning time was longer than that of C26 DY, indicating that when administered at a low dose of 5 mg/kg antibody - "scavengers" with DY modification D3 DY, D4 DY and D5 DY had better circular and analogous antigen binding function, thereby providing better antigen clearance.

Example 5: Determination of the affinity of antibodies 162 and C26

HBsAg was dissolved in sodium acetate (pH 4.5) at a concentration of 5 μg/ml, and the chip coating program was run on a Biacore 3000 device to coat HBsAg on the CM5 chip. The HBsAg coating volume was 2400RU. 2-fold serial dilutions of the analyte were prepared starting from 100 nM, preparing samples with 7 concentrations. The affinity program was run on a Biacore 3000, flow rate was set to 50 μl/min, binding time was set to 90 s, dissociation time was set to 600 s, sample chamber temperature was set to 10 °C, regenerating 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 are shown in Table 7.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes may be made to the details in accordance with all published teachings of the invention, and these changes are within the scope of protection of the present invention . The present invention is fully defined by the appended claims and any equivalents thereof.

Claims (79)

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 wherein the antibody or antigen-binding fragment thereof contains: (1) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region and the framework region optionally contains from 1 to 10 back mutations from human residues to mouse 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 germline heavy chain gene, and/or a light chain framework region contained in an amino acid sequence encoded by a human germline light chain gene. 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region contained in the amino acid sequence encoded by the human germline heavy chain gene SEQ ID NO: 38, and a light chain framework region containing in the amino acid sequence encoded by the human germline light chain gene SEQ ID NO: 39, and the heavy chain framework region and/or the light chain framework region optionally contains from 1 to 10 back mutations from human residues to mouse residues. 4. The antibody or antigen binding fragment thereof according to claim 1, wherein 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; and/or The VL of an 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 as shown in SEQ ID NO: 37. 5. An antibody or an antigen-binding fragment thereof according to any one of claims 1-4, where said antibody or an antigen-binding fragment thereof contains: (a) a heavy chain variable region (VH) which contains an amino acid sequence selected from the following sequences: (i) the sequence shown in any of SEQ ID NO: 3, 5, 6, 8; (ii) sequences with a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any of SEQ ID NO: 3, 5, 6, 8; or (iii) sequences with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% sequence identity, according to at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the sequence shown in any of SEQ ID NO: 3, 5, 6, 8; And (b) a light chain variable region (VL) which contains an amino acid sequence selected from the following sequences: (iv) the sequence shown in any of SEQ ID NO: 2, 4, 7, 9, 10; (v) a sequence with a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any of SEQ ID NO: 2, 4, 7, 9, 10; or (vi) sequences with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% sequence identity, according to at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the sequence shown in any of SEQ ID NO: 2, 4, 7, 9, 10; preferably the substitution described in (ii) or (v) is a conservative substitution. 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1-5, where said antibody or an antigen-binding fragment thereof contains: (1) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 4; (2) VH with the sequence shown in SEQ ID NO: 5, and VL with the sequence shown in SEQ ID NO: 2; (3) VH with the sequence shown in SEQ ID NO: 6, and VL with the sequence shown in SEQ ID NO: 7; (4) VH with the sequence shown in SEQ ID NO: 8, and VL with the sequence shown in SEQ ID NO: 9; (5) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 10; or (6) VH with the sequence shown in SEQ ID NO: 3, and VL with the sequence shown in SEQ ID NO: 9. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof further comprises a constant region derived from human immunoglobulin. 8. The antibody or antigen binding fragment thereof according to claim 7, wherein the heavy chain of the antibody or antigen binding fragment comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), and the light chain of the antibody or antigen binding fragment comprises a light chain constant region derived from human immunoglobulin (eg κ or λ). 9. The antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 41. 10. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises a human IgG1 heavy chain constant region variant, wherein the variant has the following substitution relative to the wild type sequence from which it is derived: (i) M252Y, N286E , N434Y; or (ii) K326D, L328Y; wherein the above amino acid positions are those according to the Kabat numbering system. 11. The antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 42 or 43. 12. The antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 40. 13. An antibody or an antigen-binding fragment thereof according to any one of claims 1-4, where said antibody or an antigen-binding fragment thereof contains: (1) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41; (2) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41; (3) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 4 and CL shown in SEQ ID NO: 41; (4) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41; (5) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41; (6) a heavy chain containing VH shown in SEQ ID NO: 5 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 41; (7) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41; (8) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41; (9) a heavy chain containing VH shown in SEQ ID NO: 6 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 7 and CL shown in SEQ ID NO: 41; (10) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; (11) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; (12) a heavy chain containing VH shown in SEQ ID NO: 8 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; (13) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41; (14) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41; (15) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 10 and CL shown in SEQ ID NO: 41; (16) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 40, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; (17) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 47, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41; or (18) a heavy chain containing VH shown in SEQ ID NO: 3 and CH shown in SEQ ID NO: 48, and a light chain containing VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 41. 14. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or an antigen-binding fragment thereof is selected from the group consisting of a scFv fragment, Fab, Fab', (Fab') ₂ , Fv, Probody, chimeric antibody or humanized antibodies; preferably the antibody is a chimeric antibody or a humanized antibody. 15. The antibody or antigen binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen binding fragment thereof is capable of specifically binding to HBsAg, neutralizing HBV virulence and/or reducing serum levels of HBV DNA and/or HBsAg in a subject. 16. An isolated nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15. 17. An expression vector containing the nucleic acid molecule according to claim 16. 18. A host cell for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15, which contains a nucleic acid molecule according to claim 16 or a vector according to claim 17. 19. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15, which includes culturing a host cell according to claim 18 under conditions that ensure expression of the antibody or an antigen-binding fragment thereof and isolating the antibody or an antigen-binding fragment thereof from the culture of cultured cells -owners. 20. A pharmaceutical composition for the treatment of HBV infection or a disease associated with HBV infection, which contains an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier and/or excipient. 21. A pharmaceutical composition for the prevention of HBV infection or a disease associated with HBV infection in a subject, which contains an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier and/or excipient. 22. The pharmaceutical composition according to claim 20 or 21, wherein the subject's serum level of HBV DNA and/or HBsAg is reduced. 23. The pharmaceutical composition according to claim 20 or 21, wherein a humoral immune response against HBV is activated in the subject. 24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 in the manufacture of a medicament for the treatment of HBV infection or a disease associated with HBV infection in a subject. 25. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 in the manufacture of a medicament for the prevention of HBV infection or a disease associated with HBV infection in a subject. 26. Use according to claim 24 or 25, wherein the subject's serum level of HBV DNA and/or HBsAg is reduced. 27. Use according to claim 24 or 25, wherein a humoral immune response against HBV is activated in the subject. 28. Use according to claim 24 or 25, where the subject is a human. 29. Use according to claim 24 or 25, wherein the disease associated with HBV infection is hepatitis B. 30. Use according to claim 24, wherein the subject is a person with chronic HBV infection or a patient with chronic hepatitis B. 31. A method that is used for treating HBV infection or a disease associated with HBV infection in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 to a subject in need thereof . 32. A method that is used for the prevention of HBV infection or a disease associated with HBV infection in a subject, comprising administering an effective amount of an antibody or antigen binding fragment thereof according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 to a subject in need thereof . 33. The method of claim 31 or 32, wherein the subject's serum level of HBV DNA and/or HBsAg is reduced. 34. The method of claim 31 or 32, wherein a humoral immune response against HBV is activated in the subject. 35. The method according to claim 31 or 32, where the subject is a person. 36. The method according to claim 31 or 32, wherein the disease associated with HBV infection is hepatitis B. 37. The method of claim 31, wherein the subject is a person with chronic HBV infection or a patient with chronic hepatitis B.