Hepatitis B virus surface antibody and application thereof

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof directed against hepatitis B virus surface antigen (HBsAg), and methods of making and using the neutralizing antibodies and antigen-binding fragments thereof. The antibody provided by the invention is derived from a hepatitis B vaccinator and a hepatitis B infection convalescent person, shows an excellent effect in the aspect of identifying different hepatitis B virus surface antigens, and has more advantages in the aspects of neutralizing hepatitis B virus mutants and reducing hepatitis B mutant antigens.

Description

Technical Field

[0001] The present invention relates to an antibody against hepatitis B virus surface antigen (HBsAg) and an antigen-binding fragment thereof, as well as a method for preparing and using the neutralizing antibody and the antigen-binding fragment thereof. Background Art

[0002] Acute and chronic infections caused by hepatitis B virus (HBV) are global public health issues that seriously endanger human health. With the vaccination of hepatitis B vaccine, the spread of the virus has been greatly controlled, but my country is still the country with the largest number of hepatitis B patients in the world. The drugs currently used in clinical treatment of chronic hepatitis B, including interferon and nucleoside / nucleotide analogs, can effectively inhibit HBV replication, but cannot completely eliminate the virus and thus cure chronic hepatitis B virus infection, and drug resistance may also occur.

[0003] HBV is an enveloped DNA virus belonging to the Hepadnaviridae family. There are currently nine confirmed HBV genotypes AI and one undetermined genotype J, which are further subdivided into many subgenotypes with specific geographical distributions. After HBV infection, the natural history of the patient's course of disease varies depending on the HBV genotype they are infected with, including the mode of transmission, the time of HBeAg seroconversion, the speed and severity of liver disease progression, such as progression to hepatocellular carcinoma, and the possibility of HBsAg clearance. HBsAg particles have three forms: large, medium, and small. Among them, small HBsAg is encoded by 226 amino acids and is the main component that causes the body to produce protective antibodies. The HBsAg protein has four transmembrane regions, including an extracellular domain of about 70 amino acids (amino acids 99-169). This extracellular domain is exposed on the surface of the virus particle, which is particularly important for the virus to infect host cells. It is also an important site for the body's immune system (such as antibodies) to recognize. In this extracellular region, there is an immunodominant region called the "a" antigenic determinant cluster, located between amino acids 124-147, between transmembrane regions 2 and 3, and is an important epitope recognized by anti-HBs, the surface antibody of hepatitis B. Due to the natural selection, time and space pressure of survival of the fittest during the chronic infection of HBV, a series of mutant strains will appear, and the above-mentioned "a" antigenic determinant cluster is the region where HBsAg mutation occurs with a high frequency.

[0004] Hepatitis B surface antigen (HBsAg) can be derived from HBV cccDNA and / or integrated HBV DNA fragments. It not only participates in the journey of complete virus particles as an envelope protein of HBV, but also forms non-infectious subviral particles with host lipids, namely HBsAg particles, which exist freely in the blood of infected people to form surface antigenemia. More and more studies have shown that the large amount of HBsAg in patients with chronic hepatitis B may be the cause of abnormal or exhausted immune response of the body, and may affect the pathobiology of liver disease and the occurrence of cancer. Therefore, reducing or eliminating HBsAg can not only prevent HBV infection, but also help achieve "functional cure", thereby reducing the incidence of complications (cirrhosis and hepatocellular carcinoma). The use of monoclonal antibodies (anti-HBs) against HBsAg surface protein to reduce or eliminate HBsAg protein in the human body is a clinical strategy of "functional cure of HBsAg" that is worth exploring and developing.

[0005] At present, the most economical and effective way to prevent and treat hepatitis B is to get vaccinated with hepatitis B vaccine. The hepatitis B surface antibody anti-HBs produced after vaccination is a protective antibody against hepatitis B. Since the implementation of the policy of vaccinating newborns with hepatitis B vaccine in 1992, the positive rate of hepatitis B virus antigen in the population has decreased year by year. As of 2020, the positive rate of hepatitis B surface antigen in the Chinese population is about 6%, but due to the large population base, there are still 86 million people infected with HBV in my country, with about 1 million new cases each year.

[0006] Antibodies are secreted by plasma cells differentiated from B lymphocytes. Each B lymphocyte can only produce a proprietary antibody against a specific antibody determinant cluster, and antibodies obtained from a single cell or a single cell line are called monoclonal antibodies. With the development of biotechnology, monoclonal antibody screening technology has also been continuously improved. There have been many studies that have obtained anti-HBs monoclonal antibodies using B cells from the peripheral blood of vaccine immunized persons through different technologies, such as the construction of phage display Fab library technology, Epstein-Barr virus-mediated B cell immortalization technology, and B cell activation culture technology, to obtain monoclonal antibodies that can bind to HBsAg and have certain neutralizing activity, and the antigen recognition epitopes are all in the "a" determinant cluster of S-HBsAg. The monoclonal antibodies obtained in the above studies have a common disadvantage, which is the loss of the naturalness of humanized antibodies. Existing research data show that volunteers with high serum neutralization efficacy against hepatitis B virus can produce anti-HBs monoclonal antibodies with high in vitro neutralization efficacy and can effectively inhibit the proliferation of hepatitis B virus in vivo (1,2). Recombinantly expressed multiple natural humanized anti-HBs, most of which had neutralizing efficacy in in vitro experiments; it is worth mentioning that in the in vivo infection experiment of human liver chimeric mice, after a few weeks of single antibody treatment, the virus in the mice showed obvious virus escape; and the use of monoclonal antibodies that recognize different epitopes and have complementarity can more effectively inhibit the viremia of mice and prevent virus escape. It can be seen that the anti-HBs isolated from hepatitis B vaccine recipients have the effect of blocking HBV infection and inhibiting viral replication in both in vivo and in vitro experimental models.

[0007] Although the hepatitis B virus is a DNA virus, the replication process involves an intermediate process through RNA reverse transcriptase, and DNA polymerase does not have the function of self-correction. Compared with other DNA viruses, it is more likely to produce genomic nucleotide mutations, generating HBV mutants in this process. Most of these mutated new sequences cannot effectively compete with wild-type viruses or cannot survive, but when there is selection pressure, these new sequences provide a starting point for the emergence of mutants. When talking about viral mutants, we usually mention RNA viruses such as the new coronavirus or influenza virus, which can produce large and rapid mutations. The difference is that the circular DNA genome of HBV has 4 partially overlapping open reading frames (ORFs), which can encode 7 proteins. Because gene overlap limits the variability of the virus, mutants or variant forms have been identified for 4 genes. HBV has a certain tendency to mutate. The "a" determinant cluster on HBsAg is a region with a higher probability of mutation. The human immune response to HBsAg is also mainly directed against the disulfide bond conformational epitope of the "a" determinant cluster. When the mutation causes changes in the amino acid sites of the corresponding protein and changes in the spatial structure and characteristics of the protein (conformational epitope), the antibody will be unable to neutralize the virus infection and may also affect diagnostic testing. Q129R (3), M133T (4), D144A, D144E, G145R (5) and G145K (6) have been reported in the literature. Among them, the G145R mutant is the most important HBsAg mutant mentioned in the literature. This mutant was first reported in children born to HBV-positive mothers who still had breakthrough infection despite vaccination. Although the children had anti-HBs protective antibodies against wild-type virus, the DNA and HBsAg of this G145R mutant persisted (5,7). This suggests that the antibodies produced by the hepatitis B vaccine are protective against wild-type HBV, but their protective effect against HBV mutant strains is limited.

[0008] The existing human hepatitis B surface antibody products mainly include hepatitis B immunoglobulin (HBIg), which is a concentrated passive immunization preparation for preventing the invasion and replication of hepatitis B virus. It is produced by screening plasma containing high titers of anti-HBs from healthy blood donors, and then using a bioconcentration process to make high-titer hepatitis B immunoglobulin. However, there are some problems with blood-derived HBIg. For example, the raw material required for HBIg is high-titer anti-HBs plasma, and the production and use scale of the product are relatively limited. At the same time, given the differences in individual plasma supply between batches, there are also differences in quality and effectiveness between different product batches. In particular, in terms of effectiveness, only some antibodies in the concentrated HBIg may have the effect of neutralizing HBV; although the country has strict control over blood products, the various indicators for testing "healthy blood donors" are still very limited. From a biosafety perspective, the safety of HBIg is poor, and the dose of HBIg used clinically is also limited to 200 IU; finally, patients who receive HBIg injections may have allergic reactions, such as IgG4 allergies. For example, patients in immunosuppressive states such as liver transplantation may also experience adverse symptoms such as nausea, urticaria, and joint pain. The use of HBIg in clinically blocking HBV transmission shows that anti-HBs is feasible in blocking HBV infection, but its existing problems also suggest that it is urgent to develop anti-HBs that can be used for engineering mass production, is efficient and safe, and can preferably block mutant strains at the same time.

[0009] In recent years, many laboratories at home and abroad have used biotechnology to separate peripheral blood cells from hepatitis B vaccine recipients and then clone human antibodies from B cells, but there are problems such as a single source and the inability to cover different genotypes or clinical mutants. Therefore, there has always been a need in this field for hepatitis B surface antibodies with improved performance. Summary of the invention

[0010] In the present invention, the inventor screened patients who had recovered from hepatitis B infection and recipients of hepatitis B vaccine with high serum neutralizing activity, and obtained a series of monoclonal antibodies against HBsAg by isolating single B cells in their bodies. Both in vivo and in vitro experiments have shown that these monoclonal antibodies have excellent antigen binding ability, in vitro neutralization efficacy, and the ability to recognize a broad spectrum of various mutant strains. In addition, by comparing with antibodies in recipients of hepatitis B vaccine, the inventor found that antibodies from patients who had recovered from hepatitis B infection had more diverse characteristics of recognizing and neutralizing mutant strains. In vivo experiments in mice also further confirmed that this series of antibodies had excellent in vivo antiviral efficacy, thereby completing the present invention.

[0011] The present invention provides an isolated antibody or an antigen-binding fragment thereof, wherein the isolated antibody or the antigen-binding fragment thereof specifically binds to hepatitis B surface antigen (HBsAg), characterized in that the isolated antibody or the antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises complementary determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region VL comprises complementary determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences:

[0012] a) CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, CDRH3 of SEQ ID NO:4, CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8;

[0013] b) CDRH1 of SEQ ID NO: 10, CDRH2 of SEQ ID NO: 11, CDRH3 of SEQ ID NO: 12, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16;

[0014] c) CDRH1 of SEQ ID NO: 18, CDRH2 of SEQ ID NO: 19, CDRH3 of SEQ ID NO: 20, CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24;

[0015] d) CDRH1 of SEQ ID NO:26, CDRH2 of SEQ ID NO:27, CDRH3 of SEQ ID NO:28, CDRL1 of SEQ ID NO:30, CDRL2 of SEQ ID NO:31, and CDRL3 of SEQ ID NO:32;

[0016] e) CDRH1 of SEQ ID NO:34, CDRH2 of SEQ ID NO:35, CDRH3 of SEQ ID NO:36, CDRL1 of SEQ ID NO:38, CDRL2 of SEQ ID NO:39, and CDRL3 of SEQ ID NO:40;

[0017] f) CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:46, CDRL2 of SEQ ID NO:47, and CDRL3 of SEQ ID NO:48;

[0018] g) CDRH1 of SEQ ID NO:50, CDRH2 of SEQ ID NO:51, CDRH3 of SEQ ID NO:52, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56;

[0019] h) CDRH1 of SEQ ID NO:58, CDRH2 of SEQ ID NO:59, CDRH3 of SEQ ID NO:60, CDRL1 of SEQ ID NO:62, CDRL2 of SEQ ID NO:63, and CDRL3 of SEQ ID NO:64;

[0020] i) CDRH1 of SEQ ID NO:66, CDRH2 of SEQ ID NO:67, CDRH3 of SEQ ID NO:68, CDRL1 of SEQ ID NO:70, CDRL2 of SEQ ID NO:71, and CDRL3 of SEQ ID NO:72;

[0021] j) CDRH1 of SEQ ID NO:74, CDRH2 of SEQ ID NO:75, CDRH3 of SEQ ID NO:76, CDRL1 of SEQ ID NO:78, CDRL2 of SEQ ID NO:79, and CDRL3 of SEQ ID NO:80;

[0022] k) CDRH1 of SEQ ID NO:82, CDRH2 of SEQ ID NO:83, CDRH3 of SEQ ID NO:84, CDRL1 of SEQ ID NO:86, CDRL2 of SEQ ID NO:87, and CDRL3 of SEQ ID NO:88;

[0023] 1) CDRH1 of SEQ ID NO:90, CDRH2 of SEQ ID NO:91, CDRH3 of SEQ ID NO:92, CDRL1 of SEQ ID NO:94, CDRL2 of SEQ ID NO:95, and CDRL3 of SEQ ID NO:96;

[0024] m) CDRH1 of SEQ ID NO:98, CDRH2 of SEQ ID NO:99, CDRH3 of SEQ ID NO:100, CDRL1 of SEQ ID NO:102, CDRL2 of SEQ ID NO:103, and CDRL3 of SEQ ID NO:104;

[0025] n) CDRH1 of SEQ ID NO: 106, CDRH2 of SEQ ID NO: 107, CDRH3 of SEQ ID NO: 108, CDRL1 of SEQ ID NO: 110, CDRL2 of SEQ ID NO: 111, and CDRL3 of SEQ ID NO: 112;

[0026] o) CDRH1 of SEQ ID NO: 114, CDRH2 of SEQ ID NO: 115, CDRH3 of SEQ ID NO: 116, CDRL1 of SEQ ID NO: 118, CDRL2 of SEQ ID NO: 119, and CDRL3 of SEQ ID NO: 120;

[0027] p) CDRH1 of SEQ ID NO: 122, CDRH2 of SEQ ID NO: 123, CDRH3 of SEQ ID NO: 124, CDRL1 of SEQ ID NO: 126, CDRL2 of SEQ ID NO: 127, and CDRL3 of SEQ ID NO: 128;

[0028] q) CDRH1 of SEQ ID NO: 130, CDRH2 of SEQ ID NO: 131, CDRH3 of SEQ ID NO: 132, CDRL1 of SEQ ID NO: 134, CDRL2 of SEQ ID NO: 135, and CDRL3 of SEQ ID NO: 136;

[0029] r) CDRH1 of SEQ ID NO: 138, CDRH2 of SEQ ID NO: 139, CDRH3 of SEQ ID NO: 140, CDRL1 of SEQ ID NO: 142, CDRL2 of SEQ ID NO: 143, and CDRL3 of SEQ ID NO: 144;

[0030] s) CDRH1 of SEQ ID NO: 146, CDRH2 of SEQ ID NO: 147, CDRH3 of SEQ ID NO: 148, CDRL1 of SEQ ID NO: 150, CDRL2 of SEQ ID NO: 151, and CDRL3 of SEQ ID NO: 152;

[0031] t) CDRH1 of SEQ ID NO: 154, CDRH2 of SEQ ID NO: 155, CDRH3 of SEQ ID NO: 156, CDRL1 of SEQ ID NO: 158, CDRL2 of SEQ ID NO: 159, and CDRL3 of SEQ ID NO: 160;

[0032] u) CDRH1 of SEQ ID NO: 162, CDRH2 of SEQ ID NO: 163, CDRH3 of SEQ ID NO: 164, CDRL1 of SEQ ID NO: 166, CDRL2 of SEQ ID NO: 167, and CDRL3 of SEQ ID NO: 168;

[0033] v) CDRH1 of SEQ ID NO: 170, CDRH2 of SEQ ID NO: 171, CDRH3 of SEQ ID NO: 172, CDRL1 of SEQ ID NO: 174, CDRL2 of SEQ ID NO: 175, and CDRL3 of SEQ ID NO: 176;

[0034] w) CDRH1 of SEQ ID NO: 178, CDRH2 of SEQ ID NO: 179, CDRH3 of SEQ ID NO: 180, CDRL1 of SEQ ID NO: 182, CDRL2 of SEQ ID NO: 183, and CDRL3 of SEQ ID NO: 184;

[0035] x) CDRH1 of SEQ ID NO: 186, CDRH2 of SEQ ID NO: 187, CDRH3 of SEQ ID NO: 188, CDRL1 of SEQ ID NO: 190, CDRL2 of SEQ ID NO: 191, and CDRL3 of SEQ ID NO: 192;

[0036] y) CDRH1 of SEQ ID NO: 194, CDRH2 of SEQ ID NO: 195, CDRH3 of SEQ ID NO: 196, CDRL1 of SEQ ID NO: 198, CDRL2 of SEQ ID NO: 199, and CDRL3 of SEQ ID NO: 200;

[0037] z) CDRH1 of SEQ ID NO:202, CDRH2 of SEQ ID NO:203, CDRH3 of SEQ ID NO:204, CDRL1 of SEQ ID NO:206, CDRL2 of SEQ ID NO:207, and CDRL3 of SEQ ID NO:208;

[0038] aa) CDRH1 of SEQ ID NO:210, CDRH2 of SEQ ID NO:211, CDRH3 of SEQ ID NO:212, CDRL1 of SEQ ID NO:214, CDRL2 of SEQ ID NO:215, and CDRL3 of SEQ ID NO:216;

[0039] bb) CDRH1 of SEQ ID NO:218, CDRH2 of SEQ ID NO:219, CDRH3 of SEQ ID NO:220, CDRL1 of SEQ ID NO:222, CDRL2 of SEQ ID NO:223, and CDRL3 of SEQ ID NO:224;

[0040] cc) CDRH1 of SEQ ID NO:226, CDRH2 of SEQ ID NO:227, CDRH3 of SEQ ID NO:228, CDRL1 of SEQ ID NO:230, CDRL2 of SEQ ID NO:231, and CDRL3 of SEQ ID NO:232;

[0041] dd) CDRH1 of SEQ ID NO:234, CDRH2 of SEQ ID NO:235, CDRH3 of SEQ ID NO:236, CDRL1 of SEQ ID NO:238, CDRL2 of SEQ ID NO:239, and CDRL3 of SEQ ID NO:240;

[0042] ee) CDRH1 of SEQ ID NO:242, CDRH2 of SEQ ID NO:243, CDRH3 of SEQ ID NO:244, CDRL1 of SEQ ID NO:246, CDRL2 of SEQ ID NO:247, and CDRL3 of SEQ ID NO:248;

[0043] ff) CDRH1 of SEQ ID NO:250, CDRH2 of SEQ ID NO:251, CDRH3 of SEQ ID NO:252, CDRL1 of SEQ ID NO:254, CDRL2 of SEQ ID NO:255, and CDRL3 of SEQ ID NO:256;

[0044] gg) CDRH1 of SEQ ID NO:258, CDRH2 of SEQ ID NO:259, CDRH3 of SEQ ID NO:260, CDRL1 of SEQ ID NO:262, CDRL2 of SEQ ID NO:263, and CDRL3 of SEQ ID NO:264;

[0045] hh) CDRH1 of SEQ ID NO:266, CDRH2 of SEQ ID NO:267, CDRH3 of SEQ ID NO:268, CDRL1 of SEQ ID NO:270, CDRL2 of SEQ ID NO:271, and CDRL3 of SEQ ID NO:272;

[0046] ii) CDRH1 of SEQ ID NO:274, CDRH2 of SEQ ID NO:275, CDRH3 of SEQ ID NO:276, CDRL1 of SEQ ID NO:278, CDRL2 of SEQ ID NO:279, and CDRL3 of SEQ ID NO:280;

[0047] jj) CDRH1 of SEQ ID NO:282, CDRH2 of SEQ ID NO:283, CDRH3 of SEQ ID NO:284, CDRL1 of SEQ ID NO:286, CDRL2 of SEQ ID NO:287, and CDRL3 of SEQ ID NO:288;

[0048] kk) CDRH1 of SEQ ID NO:290, CDRH2 of SEQ ID NO:291, CDRH3 of SEQ ID NO:292, CDRL1 of SEQ ID NO:294, CDRL2 of SEQ ID NO:295, and CDRL3 of SEQ ID NO:296;

[0049] 11) CDRH1 of SEQ ID NO:298, CDRH2 of SEQ ID NO:299, CDRH3 of SEQ ID NO:300, CDRL1 of SEQ ID NO:302, CDRL2 of SEQ ID NO:303, and CDRL3 of SEQ ID NO:304;

[0050] mm) CDRH1 of SEQ ID NO:306, CDRH2 of SEQ ID NO:307, CDRH3 of SEQ ID NO:308, CDRL1 of SEQ ID NO:310, CDRL2 of SEQ ID NO:311, and CDRL3 of SEQ ID NO:312;

[0051] nn) CDRH1 of SEQ ID NO:314, CDRH2 of SEQ ID NO:315, CDRH3 of SEQ ID NO:316, CDRL1 of SEQ ID NO:318, CDRL2 of SEQ ID NO:319, and CDRL3 of SEQ ID NO:320;

[0052] oo) CDRH1 of SEQ ID NO:322, CDRH2 of SEQ ID NO:323, CDRH3 of SEQ ID NO:324, CDRL1 of SEQ ID NO:326, CDRL2 of SEQ ID NO:327, and CDRL3 of SEQ ID NO:328;

[0053] pp) CDRH1 of SEQ ID NO:330, CDRH2 of SEQ ID NO:331, CDRH3 of SEQ ID NO:332, CDRL1 of SEQ ID NO:334, CDRL2 of SEQ ID NO:335, and CDRL3 of SEQ ID NO:336;

[0054] qq) CDRH1 of SEQ ID NO:338, CDRH2 of SEQ ID NO:339, CDRH3 of SEQ ID NO:340, CDRL1 of SEQ ID NO:342, CDRL2 of SEQ ID NO:343, and CDRL3 of SEQ ID NO:344;

[0055] rr) CDRH1 of SEQ ID NO:346, CDRH2 of SEQ ID NO:347, CDRH3 of SEQ ID NO:348, CDRL1 of SEQ ID NO:350, CDRL2 of SEQ ID NO:351, and CDRL3 of SEQ ID NO:352;

[0056] ss) CDRH1 of SEQ ID NO:354, CDRH2 of SEQ ID NO:355, CDRH3 of SEQ ID NO:356, CDRL1 of SEQ ID NO:358, CDRL2 of SEQ ID NO:359, and CDRL3 of SEQ ID NO:360;

[0057] tt) CDRH1 of SEQ ID NO:362, CDRH2 of SEQ ID NO:363, CDRH3 of SEQ ID NO:364, CDRL1 of SEQ ID NO:366, CDRL2 of SEQ ID NO:367, and CDRL3 of SEQ ID NO:368;

[0058] uu) CDRH1 of SEQ ID NO:370, CDRH2 of SEQ ID NO:371, CDRH3 of SEQ ID NO:372, CDRL1 of SEQ ID NO:374, CDRL2 of SEQ ID NO:375, and CDRL3 of SEQ ID NO:376;

[0059] vv) CDRH1 of SEQ ID NO:378, CDRH2 of SEQ ID NO:379, CDRH3 of SEQ ID NO:380, CDRL1 of SEQ ID NO:382, CDRL2 of SEQ ID NO:383, and CDRL3 of SEQ ID NO:384;

[0060] ww) CDRH1 of SEQ ID NO:386, CDRH2 of SEQ ID NO:387, CDRH3 of SEQ ID NO:388, CDRL1 of SEQ ID NO:390, CDRL2 of SEQ ID NO:391, and CDRL3 of SEQ ID NO:392;

[0061] xx) CDRH1 of SEQ ID NO:394, CDRH2 of SEQ ID NO:395, CDRH3 of SEQ ID NO:396, CDRL1 of SEQ ID NO:398, CDRL2 of SEQ ID NO:399, and CDRL3 of SEQ ID NO:400;

[0062] yy) CDRH1 of SEQ ID NO:402, CDRH2 of SEQ ID NO:403, CDRH3 of SEQ ID NO:404, CDRL1 of SEQ ID NO:406, CDRL2 of SEQ ID NO:407, and CDRL3 of SEQ ID NO:408;

[0063] zz) CDRH1 of SEQ ID NO:410, CDRH2 of SEQ ID NO:411, CDRH3 of SEQ ID NO:412, CDRL1 of SEQ ID NO:414, CDRL2 of SEQ ID NO:415, and CDRL3 of SEQ ID NO:416;

[0064] aaa) CDRH1 of SEQ ID NO:418, CDRH2 of SEQ ID NO:419, CDRH3 of SEQ ID NO:420, CDRL1 of SEQ ID NO:422, CDRL2 of SEQ ID NO:423, and CDRL3 of SEQ ID NO:424;

[0065] bbb) CDRH1 of SEQ ID NO:426, CDRH2 of SEQ ID NO:427, CDRH3 of SEQ ID NO:428, CDRL1 of SEQ ID NO:430, CDRL2 of SEQ ID NO:431, and CDRL3 of SEQ ID NO:432;

[0066] ccc) CDRH1 of SEQ ID NO:434, CDRH2 of SEQ ID NO:435, CDRH3 of SEQ ID NO:436, CDRL1 of SEQ ID NO:438, CDRL2 of SEQ ID NO:439, and CDRL3 of SEQ ID NO:440;

[0067] ddd) CDRH1 of SEQ ID NO:442, CDRH2 of SEQ ID NO:443, CDRH3 of SEQ ID NO:444, CDRL1 of SEQ ID NO:446, CDRL2 of SEQ ID NO:447, and CDRL3 of SEQ ID NO:448;

[0068] eee) CDRH1 of SEQ ID NO:450, CDRH2 of SEQ ID NO:451, CDRH3 of SEQ ID NO:452, CDRL1 of SEQ ID NO:454, CDRL2 of SEQ ID NO:455, and CDRL3 of SEQ ID NO:456;

[0069] fff) CDRH1 of SEQ ID NO:458, CDRH2 of SEQ ID NO:459, CDRH3 of SEQ ID NO:460, CDRL1 of SEQ ID NO:462, CDRL2 of SEQ ID NO:463, and CDRL3 of SEQ ID NO:464;

[0070] ggg) CDRH1 of SEQ ID NO:466, CDRH2 of SEQ ID NO:467, CDRH3 of SEQ ID NO:468, CDRL1 of SEQ ID NO:470, CDRL2 of SEQ ID NO:471, and CDRL3 of SEQ ID NO:472;

[0071] hhh) CDRH1 of SEQ ID NO:474, CDRH2 of SEQ ID NO:475, CDRH3 of SEQ ID NO:476, CDRL1 of SEQ ID NO:478, CDRL2 of SEQ ID NO:479, and CDRL3 of SEQ ID NO:480;

[0072] iii) CDRH1 of SEQ ID NO:482, CDRH2 of SEQ ID NO:483, CDRH3 of SEQ ID NO:484, CDRL1 of SEQ ID NO:486, CDRL2 of SEQ ID NO:487, and CDRL3 of SEQ ID NO:488; and

[0073] jjj) CDRH1 of SEQ ID NO:490, CDRH2 of SEQ ID NO:491, CDRH3 of SEQ ID NO:492, CDRL1 of SEQ ID NO:494, CDRL2 of SEQ ID NO:495, and CDRL3 of SEQ ID NO:496.

[0074] In one embodiment, the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences:

[0075] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0076] b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 13;

[0077] c) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 21;

[0078] d) VH of SEQ ID NO:25 and VL of SEQ ID NO:29;

[0079] e) VH of SEQ ID NO:33 and VL of SEQ ID NO:37;

[0080] f) VH of SEQ ID NO:41 and VL of SEQ ID NO:45;

[0081] g) VH of SEQ ID NO:49 and VL of SEQ ID NO:53;

[0082] h) VH of SEQ ID NO:57 and VL of SEQ ID NO:61;

[0083] i) VH of SEQ ID NO:65 and VL of SEQ ID NO:69;

[0084] j) VH of SEQ ID NO:73 and VL of SEQ ID NO:77;

[0085] k) VH of SEQ ID NO:81 and VL of SEQ ID NO:85;

[0086] 1) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0087] m) VH of SEQ ID NO:97 and VL of SEQ ID NO:101;

[0088] n) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0089] o) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0090] p) VH of SEQ ID NO: 121 and VL of SEQ ID NO: 125;

[0091] q) VH of SEQ ID NO: 129 and VL of SEQ ID NO: 133;

[0092] r) VH of SEQ ID NO: 137 and VL of SEQ ID NO: 141;

[0093] s) VH of SEQ ID NO: 145 and VL of SEQ ID NO: 149;

[0094] t) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0095] u) VH of SEQ ID NO: 161 and VL of SEQ ID NO: 165;

[0096] v) VH of SEQ ID NO: 169 and VL of SEQ ID NO: 173;

[0097] w) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0098] x) VH of SEQ ID NO: 185 and VL of SEQ ID NO: 189;

[0099] y) VH of SEQ ID NO: 193 and VL of SEQ ID NO: 197;

[0100] z) VH of SEQ ID NO: 201 and VL of SEQ ID NO: 205;

[0101] aa) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0102] bb) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0103] cc) VH of SEQ ID NO: 225 and VL of SEQ ID NO: 229;

[0104] dd) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0105] ee) VH of SEQ ID NO: 241 and VL of SEQ ID NO: 245;

[0106] ff) VH of SEQ ID NO: 249 and VL of SEQ ID NO: 253;

[0107] gg) VH of SEQ ID NO: 257 and VL of SEQ ID NO: 261;

[0108] hh) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0109] ii) VH of SEQ ID NO: 273 and VL of SEQ ID NO: 277; jj) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285;

[0110] kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293;

[0111] 11) VH of SEQ ID NO:297 and VL of SEQ ID NO:301;

[0112] mm) VH of SEQ ID NO: 305 and VL of SEQ ID NO: 309;

[0113] nn) VH of SEQ ID NO:313 and VL of SEQ ID NO:317;

[0114] oo) VH of SEQ ID NO:321 and VL of SEQ ID NO:325;

[0115] pp) VH of SEQ ID NO: 329 and VL of SEQ ID NO: 333;

[0116] qq) VH of SEQ ID NO: 337 and VL of SEQ ID NO: 341;

[0117] rr) VH of SEQ ID NO:345 and VL of SEQ ID NO:349;

[0118] ss) VH of SEQ ID NO:353 and VL of SEQ ID NO:357;

[0119] tt) VH of SEQ ID NO:361 and VL of SEQ ID NO:365;

[0120] uu) VH of SEQ ID NO: 369 and VL of SEQ ID NO: 373;

[0121] vv) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0122] ww) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389;

[0123] xx) VH of SEQ ID NO: 393 and VL of SEQ ID NO: 397;

[0124] yy) VH of SEQ ID NO:401 and VL of SEQ ID NO:405;

[0125] zz) VH of SEQ ID NO:409 and VL of SEQ ID NO:413;

[0126] aaa) VH of SEQ ID NO:417 and VL of SEQ ID NO:421;

[0127] bbb) VH of SEQ ID NO:425 and VL of SEQ ID NO:429;

[0128] ccc) VH of SEQ ID NO:433 and VL of SEQ ID NO:437;

[0129] ddd) VH of SEQ ID NO:441 and VL of SEQ ID NO:445;

[0130] eee) VH of SEQ ID NO:449 and VL of SEQ ID NO:453;

[0131] fff) VH of SEQ ID NO:457 and VL of SEQ ID NO:461;

[0132] ggg) VH of SEQ ID NO: 465 and VL of SEQ ID NO: 469;

[0133] hhh) VH of SEQ ID NO:473 and VL of SEQ ID NO:477;

[0134] iii) VH of SEQ ID NO: 481 and VL of SEQ ID NO: 485; and

[0135] jjj) VH of SEQ ID NO:489 and VL of SEQ ID NO:493.

[0136] In one embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to an epitope in the S region of the extracellular domain of HBsAg.

[0137] In one embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to HBV and its mutants. In one embodiment, the HBV mutant is a naturally occurring HBV mutant. In one embodiment, the HBV mutant is selected from G145R, K122R-G145R and F134L.

[0138] In one embodiment, the isolated antibodies or antigen-binding fragments thereof described herein have a 50% inhibitory concentration IC of less than 0.5 μg / ml. 50 The value is to neutralize HBV and its mutants in vitro, as determined by the method based on the present disclosure. In one embodiment, the HBV mutant is a naturally occurring HBV mutant. In one embodiment, the HBV mutant is selected from G145R, K122R-G145R and F134L.

[0139] In one embodiment, the isolated antibody or antigen-binding fragment thereof has neutralizing activity against different genotypes of HBV. In one embodiment, the genotype is selected from genotype A, genotype B, genotype C and genotype D.

[0140] In one embodiment, the isolated antibody or antigen-binding fragment thereof described herein is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof.

[0141] In one embodiment, the antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule.

[0142] In one embodiment, the isolated antibody or antigen-binding fragment thereof described herein is of the IgG1, IgG2, IgG3 or IgG4 type.

[0143] The present invention also provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof described herein.

[0144] The present invention also provides an expression vector comprising the polynucleotide described herein.

[0145] The present invention also provides a host cell, wherein the host cell comprises the expression vector described herein.

[0146] The present invention also provides a method for producing the isolated antibody or antigen-binding fragment thereof described herein, characterized in that the method comprises culturing the host cell described herein under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof produced by the host cell.

[0147] The present invention also provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0148] The present invention also provides the use of the isolated antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing HBV infection in a subject in need thereof.

[0149] The present invention also provides the use of the isolated antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing HBV and its mutant strain infection in a subject in need thereof.

[0150] In certain aspects, the present invention provides methods for treating or preventing HBV infection in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one isolated antibody or antigen-binding fragment thereof disclosed herein.

[0151] In certain aspects, the present invention provides a method for treating or preventing HBV and its mutants infection in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one isolated antibody or antigen-binding fragment thereof disclosed herein.

[0152] In certain aspects, the present invention provides an isolated antibody or antigen-binding fragment thereof disclosed herein for use in treating or preventing HBV and its mutants infection in a subject in need thereof.

[0153] In certain aspects, the present invention provides a hepatitis B virus surface antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope in the S region of the extracellular domain of HBsAg. In one embodiment, the epitope is located in amino acid residues 99-169 of the S region of HBsAg. The epitope is located in amino acid residues 124-147 of HBsAg. In one embodiment, the HBsAg has an amino acid sequence as shown in SEQ ID NO: 497. In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in an amino acid sequence having at least 90% identity with SEQ ID NO: 497 or a subsequence thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] The present invention will be better understood with reference to the accompanying drawings.

[0155] Figure 1 A schematic diagram of the structure of the extracellular domain of HBsAg is shown, illustrating the configuration of amino acid residues 99-169.

[0156] Figure 2 The antibody responses of recovered hepatitis B virus (HBV) infected individuals and recipients of HBV vaccine are shown. The neutralization of HBV was evaluated in the HBV cell infection model in the presence of different serum dilutions.

[0157] Figure 3 The gating strategy for cell sorting is shown. Double positive (bait protein-APC+ and bait protein-PE+) cells were single cell sorted for antibody cloning.

[0158] Figure 4 The results of flow cytometry of B cell sorting with different donor specificities are shown, showing the frequency of B lymphocytes recognizing HBsAg in a control donor of a HBV vaccine-immunized person (low anti-HBs level) and selected recovered HBV-infected persons and HBV vaccine-immunized persons. Representative flow cytometry results are shown for binding to HBsAg labeled with phycocyanin (APC) and phycoerythrin (PE) after biotinylation for dual fluorescence-dye sorting strategy.

[0159] Figure 5 The area under the curve (AUC) values ​​of antibody binding to HBsAg protein ELISA are shown.

[0160] Figure 6 The neutralizing activity of the antibodies evaluated in the HBV cell infection model is shown. In the cell model based on HBV infection in vitro in HepG2-NTCP cells, the levels of HBV antigens - HBsAg and HBeAg in the cell supernatant represent the neutralizing potency of the human monoclonal antibodies.

[0161] Figure 7 Competitive ELISA and 3 major HBsAg surface antibody groupings are shown. Competitive ELISA to validate 3 non-competing epitopes on HBsAg. The first antibody is an unlabeled HBsAg-binding antibody and is used to block epitopes on coated HBsAg. The second antibody is a biotinylated HBsAg antibody for streptavidin-HRP dependent detection. Among them, H019 is the group I control antibody, H017 is the group III control antibody, and H007 is the group II control antibody. PBS buffer was used as a normalization reference instead of the first blocking antibody.

[0162] Figure 8The results of antibody amino acid scanning are shown. Consistent with the competitive ELISA results, the four grouped antibodies were compared to the HBsAg binding levels at specific mutated amino acid sites. The level of each antibody binding to wild-type HBsAg was used as a normalization reference for its binding to other HBsAg mutants, and PBS was used as a negative control.

[0163] Fig. 9 The activity of antibodies in in vitro models against different genotypes (genotype A, genotype B and genotype C) of the virus is shown. The level of HBV antigen-HBsAg in the cell supernatant represents the neutralizing efficacy of human monoclonal antibodies. The antibodies shown here can neutralize the HBV virus.

[0164] Fig.10 The results of antibody binding to different HBV mutants are shown. The binding effect of different HBsAg mutants produced by transfected cells with antibodies was detected by ELISA. The level of each antibody binding to wild-type HBsAg was used as a standardized reference for its binding to other HBsAg mutants, and PBS was used as a negative control.

[0165] Fig.11 Neutralizing activity against HBV mutant strains (HBV-G145R, HBV-K122R G145R and F134L) viruses was shown. DETAILED DESCRIPTION

[0166] Disclosed herein are antibodies or antigen binding fragments thereof that specifically bind to HBsAg. In certain embodiments, the antibody or antigen binding fragment thereof binds to an epitope in the extracellular domain of HBsAg. In certain embodiments, the antibody or antigen binding fragment thereof has excellent antigen binding ability, in vitro neutralization potency, and the ability to recognize a broad spectrum of various mutant strains. In certain embodiments, the antibody is a human monoclonal antibody.

[0167] For convenience, the following section summarizes the various meanings of the terms used herein, followed by a discussion of various aspects of the antibodies or antigen-binding fragments thereof, followed by specific examples demonstrating various embodiments of the antibodies or antigen-binding fragments thereof.

[0168] I. Definition

[0169] It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory and are not intended to limit the invention as claimed. In this application, unless otherwise expressly stated, the use of the singular includes the plural. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" as well as other forms such as "comprising" is not limiting and encompasses the term "consisting of".

[0170] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. For any purpose, all documents or portions of documents cited in this application (including but not limited to patents, patent applications, articles, books and papers) are hereby incorporated by reference in their entirety.

[0171] The term "hepatitis B surface antigen (HBsAg)" refers to the surface antigen contained in the outer shell of the hepatitis B virus, which is particularly important for the virus to infect host cells and is also an important site for the body's immune system (such as antibodies) to recognize. The HBsAg protein contains four transmembrane regions, among which the S region contains an extracellular domain of about 70 amino acids, and its structure is as follows: Figure 1 The full-length amino acid sequence of HBsAg protein is shown in SEQ ID NO: 497, wherein the extracellular domain of amino acid residues 99-169 is underlined.

[0172] MESTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLL DYQGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSD GNCTCIPIPSSWAFARFLWEWASVR FSWLSLLLVPFVQWFAGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI (SEQ ID NO: 497).

[0173] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or any type of nucleotides in modified form. The modifications include: base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2', 3'-dideoxyribose; and internucleotide linkage modifications, such as phosphorothioates, phosphorodithioates, selenophosphates, diselenose phosphorothioates, and phosphoramidates. The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotides. In certain embodiments, the oligonucleotide is 10-60 bases long. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. The oligonucleotide may be single-stranded or double-stranded, such as for constructing a mutant gene. The oligonucleotide may be a sense or antisense oligonucleotide. Oligonucleotides may include labels for detection assays, including radioactive labels, fluorescent labels, hapten or antigenic labels. Oligonucleotides may be used, for example, as PCR primers, cloning primers or hybridization probes.

[0174] The term "vector" is intended to refer to any molecule or entity (eg, nucleic acid, plasmid, phage or virus) used to transfer protein coding information into a host cell.

[0175] The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or regulates the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or regulate transcription, translation, and, if introns are present, sequences that affect RNA splicing of the coding region operably linked thereto.

[0176] As used herein, "operably linked" means that the components to which the term is used are in a relationship that allows the components to perform their inherent functions under appropriate conditions. For example, a regulatory sequence in a vector that is "operably linked" to a protein coding sequence is linked to the protein coding sequence so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the regulatory sequence.

[0177] The term "host cell" means a cell that has been or can be transformed by a nucleic acid sequence and thereby expresses a target gene. Regardless of whether the offspring is identical to the original mother cell in morphology or genetic makeup, as long as the offspring has the target gene, the term includes the offspring of the mother cell.

[0178] The term "transfection" means the uptake of foreign or exogenous DNA by a cell. A cell is "transfected" when the exogenous DNA is introduced into the cell membrane. A variety of transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0179] The term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed when the native state of a cell is genetically modified by the introduction of new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the transformed DNA may be recombined with the cell's DNA by physically integrating into the cell's chromosomes, or may be retained temporarily as an extrachromosomal element that is not replicated, or may replicate independently as a plasmid. When the transformed DNA replicates as the cell divides, the cell is considered to have been "stably transformed."

[0180] The term "amino acid" includes its general meaning in the art. Conventional one-letter amino acid codes and three-letter amino acid codes are used herein, as shown in Table 1.

[0181] Table 1

[0182]

[0183] Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the antibodies of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxyl terminal direction, consistent with standard usage and convention.

[0184] A "variant" of a protein (eg, an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted relative to another protein sequence. Variants include fusion proteins.

[0185] The term "identity" refers to the relationship between two or more polypeptide molecules or two or more nucleic acid molecule sequences determined by alignment and comparison of sequences. "Identity percentage" means the percentage of identical residues between amino acids or nucleotides within the compared molecules, calculated based on the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, ed.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., ed.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0186] When calculating the percent identity, the sequences to be compared are usually aligned in a manner that results in the maximum match between the sequences. An example of a computer program that can be used to determine the percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides to be determined for the percent identity of the sequence. The sequences are aligned according to the best match of their respective amino acids or nucleotides (the "match span" is determined by the algorithm). In conjunction with the method, a gap open penalty (calculated as 3x average diagonal, wherein the "average diagonal" is the average value of the diagonal of the comparison matrix used; the "diagonal" is the score or value assigned to each perfect amino acid match by a specific comparison matrix) and a gap extension penalty (which is generally 1 / 10 times the gap open penalty) and a comparison matrix such as PAM 250 or BLOSUM 62 are used. In certain embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0187] Examples of parameters that can be used in determining the percent identity of polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparison matrix: from Henikoff et al., 1992, BLOSUM 62 mentioned above; Gap penalty: 12 (but no penalty for terminal gaps); Gap length penalty: 4; Similarity threshold: 0.

[0188] Certain alignment designs for aligning two amino acid sequences may result in only short regions of the two sequences matching, even if there is no significant relationship between the two full-length sequences, such small alignment regions may have very high sequence identity. Therefore, if it is necessary to generate an alignment spanning at least 50 or other numbers of contiguous amino acids of the target polypeptide, the selected alignment method (GAP program) may be adjusted.

[0189] The term "derivative" refers to a molecule that includes chemical modifications other than insertion, deletion or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives include covalent modifications, including but not limited to chemical bonding to polymers, lipids or other organic or inorganic parts. In certain embodiments, chemically modified antigen-binding proteins may have a longer circulation half-life than antigen-binding proteins that are not chemically modified. In certain embodiments, chemically modified antigen-binding proteins can improve the ability to target cells, tissues and / or organs as desired. In certain embodiments, the derived antigen-binding proteins are covalently modified to include one or more water-soluble polymeric links, including but not limited to polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. In certain embodiments, the derived antigen-binding proteins include one or more polymers, including but not limited to monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., ethylene glycol) and polyvinyl alcohol and mixtures of the polymers.

[0190] In certain embodiments, the derivative is covalently modified with a polyethylene glycol (PEG) subunit. In certain embodiments, one or more water-soluble polymers are bonded at one or more specific positions (e.g., at the amino terminus of the derivative). In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve the therapeutic ability of an antibody or its antigen-binding fragment. In certain embodiments, PEG is used to improve the therapeutic ability of a humanized antibody. Some of the methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is incorporated herein by reference for any purpose.

[0191] The term "antibody" refers to a complete immunoglobulin of any isotype or a fragment thereof that can compete with a complete antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, human whole antibodies, and bispecific antibodies. "Antibodies" are a class of antigen-binding proteins. Complete antibodies will generally contain at least two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, such as antibodies naturally occurring in camels that may contain only heavy chains. Antibodies may be derived from only a single source, or may be "chimeric", i.e., different parts of the antibody may be derived from two different antibodies. Antigen-binding proteins, antibodies, or binding fragments may be produced in hybridomas; by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Unless otherwise indicated, the term "antibody" includes derivatives, variants, and fragments thereof in addition to antibodies comprising two full-length heavy chains and two full-length light chains. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates") and fragments thereof, respectively.

[0192] Naturally occurring antibody structural units typically comprise tetramers. Each tetramer is typically composed of two identical polypeptide chain pairs, each pair having a full-length "light chain" (in certain embodiments, about 25 kDa) and a full-length "heavy chain" (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically comprises a variable region of about 100-110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain is typically defined as a constant region that may be responsible for effector function. Human light chains are typically divided into kappa and lambda light chains. Heavy chains are typically divided into μ, δ, γ, α or ε chains, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is also subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)) (which is incorporated herein by reference in its entirety for all purposes). The variable regions of each light chain / heavy chain pair typically form an antigen binding site.

[0193] In certain embodiments, different antibody variable regions vary extensively in amino acid sequence, even between antibodies of the same species. The antibody variable region generally determines the specificity of a particular antibody for its target.

[0194] The variable region usually exhibits the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, which are also called complementarity determining regions or CDRs. The CDRs from each pair of two chains are usually positioned by the framework regions, and the CDRs allow binding to specific epitopes. The two light chain and heavy chain variable regions usually contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. The amino acid distribution to each domain is usually consistent with the following definitions: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989). The International Immunogenetics (IMGT) database (http: / / www.imgt.org) provides standardized numbering and definitions of antigen binding sites. The correspondence between CDRs and IMGT divisions is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. Unless otherwise indicated, CDR sequences referred to in the present disclosure are numbered and defined according to IMGT.

[0195] By convention, the CDR regions in the heavy chain are usually referred to as H1, H2 and H3, and are numbered sequentially from the amino terminus to the carboxyl terminus. The CDR regions in the light chain are usually referred to as L1, L2 and L3, and are numbered sequentially from the amino terminus to the carboxyl terminus.

[0196] The term "light chain" includes full-length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length light chain comprises variable region domains V L and constant region domain C L The variable region domain of the light chain is located at the amino terminus of the polypeptide. The light chain includes kappa chain and lambda chain.

[0197] The term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length heavy chain comprises variable region domains V H and three constant region domains C H 1. C H 2 and CH 3. V H The domain is located at the amino terminus of the polypeptide, while the C H The domain is located at the carboxyl terminus, and the C H 3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

[0198] The term "antigen-binding fragment" includes, but is not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, and single-chain antibody molecules.

[0199] The "Fc" region contains two H 1 and C H The two heavy chain fragments are connected by two or more disulfide bonds and C H The 3 domains are held together by hydrophobic interactions.

[0200] The "Fab fragment" contains one light chain and one heavy chain C H 1 and variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0201] The "Fab' fragment" contains a light chain and a VH domain and a C H 1 domain also has C H 1 and C H The region between the two Fab' domains is part of one heavy chain, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0202] The "F(ab')2 fragment" contains two light chains and a C H 1 and C H The two heavy chains contain portions of the constant region between the two heavy chains, thereby forming an interchain disulfide bond between the two heavy chains. The F(ab')2 fragment is therefore composed of two Fab' fragments held together by the disulfide bonds between the two heavy chains.

[0203] An "Fv fragment" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0204] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions are linked together by a flexible linker to form a single polypeptide chain, thereby forming an antigen binding region. Single-chain antibodies are described in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the contents of which are incorporated herein by reference.

[0205] "Monoclonal antibody" refers to a homogeneous population of antibodies with a single molecular composition. Monoclonal antibodies can be non-specific or polyspecific.

[0206] "Domain antibodies" are immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or a light chain. H The two V domains of the bivalent domain antibody are covalently linked to a peptide linker to form a bivalent domain antibody. H The regions may target the same or different antigens.

[0207] "Multispecific antibodies" are antibodies that target more than one antigen or epitope. "Bispecific," "dual specific," or "bifunctional" antibodies are hybrid antibodies with two different antigen binding sites. The two binding sites of a bispecific antibody will bind to two different epitopes, which may be located on the same or different protein targets.

[0208] An antibody is "selective" when it binds one target more tightly than it binds a second target.

[0209] A "recombinant antibody" is an antibody produced using recombinant technology. Methods and techniques for producing recombinant antibodies are well known in the art.

[0210] Bispecific or bifunctional antibodies are generally artificial hybrid antibodies that have two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including but not limited to fusing hybridomas or connecting Fab' fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148: 1547-1553 (1992).

[0211] Each individual immunoglobulin chain is usually composed of several "immunoglobulin domains", each of which is composed of approximately 90-110 amino acids and has a unique folding pattern. These domains are the basic units that make up antibody polypeptides. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region usually contains one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. For example, the IgG heavy chain contains three called C H 1. C H 2 and C H The C region domain of 3. The provided antibodies may be of any of these isotypes and subtypes.

[0212] The term "neutralization" refers to binding to a ligand and preventing or reducing the biological effect of the ligand. This can be achieved, for example, by directly blocking the binding site on the ligand or by binding to the ligand and changing the binding ability of the ligand by indirect means (e.g., structural or energetic changes of the ligand). In assessing the binding and / or specificity of an antibody or its antigen-binding fragment, an antibody or fragment can substantially inhibit the binding of a ligand to its binding partner when an excess of antibody reduces the amount of binding to the ligand's binding partner by at least about 1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured by in vitro competitive binding assays). In certain embodiments, the neutralization ability is characterized and / or described via competitive assays. In certain embodiments, the neutralization ability is measured at a 50% inhibitory concentration (IC 50 ) value to describe.

[0213] The term "target" refers to a molecule or portion of a molecule that can be bound by an antibody or its antigen binding fragment. In certain embodiments, a target may have one or more epitopes. In certain embodiments, a target is an antigen. The use of "antigen" in the phrase "antigen binding fragment" simply means a protein sequence that contains an antigen that can be bound by an antibody. In this case, the protein is not necessarily exogenous or capable of inducing an immune response.

[0214] When the term "competition" is used in the context of neutralizing antibodies that compete for the same epitope, it means competition between antibodies, which is determined by the following assay: in the assay, the antibody or antigen-binding fragment thereof to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antibody to a common antigen (e.g., S protein or its domain). Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9 :242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibodies, laboratory manual), Cold Spring Harbor Press); solid phase direct labeling RIA with I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25 :7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176 :546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32 :77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled detection antibody and a labeled reference antibody. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the antibody being tested. Typically the antibody being tested is present in excess. Typically when the competing antibody is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antibody to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0215] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antibody or an immunologically functional fragment thereof. In certain embodiments, an antigen can be administered to an animal to produce antibodies that can bind to the antigen. An antigen may possess one or more epitopes that can interact with different antibodies.

[0216] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen. When the antigen is a protein, it contains specific amino acids that come into direct contact with the antibody. In most cases, epitopes are located on proteins, but in some cases, they can be located on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface clusters of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0217] The term "therapeutically effective amount" refers to the amount of the antibody or antigen-binding fragment thereof of the present invention determined to produce a therapeutic response in a mammal. The therapeutically effective amount can be easily determined by a person skilled in the art.

[0218] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with a formally diagnosed disease, subjects with an unformally diagnosed disease, subjects receiving medical treatment, subjects at risk of developing a disease, etc.

[0219] The term "treatment" includes therapeutic treatment, prophylactic treatment, and use in reducing a subject's risk of developing a disease or other risk factors. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or underlying risk factors are mitigated.

[0220] The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring in the presence of the antibody or method is reduced.

[0221] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be implemented according to the manufacturer's instructions, or can be completed or carried out as described herein according to methods commonly used in the art. Conventionally, the aforementioned techniques and methods can be implemented according to conventional methods well known in the art, and various general and more specific references cited and discussed in this specification sheet are described. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which are cited herein as references for any purpose. Unless clearly defined, the terms and experimental techniques and techniques used related to analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry described herein are well known in the art and are often used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery and treatment of patients.

[0222] 2. Antibodies of the present invention and methods for preparing the same

[0223] The present disclosure provides effective antibodies or antigen-binding fragments thereof (hereinafter collectively referred to as "antibodies") that can bind to HBV and neutralize its activity. The inventors have also found that antibodies cloned from donors of hepatitis B infection survivors show better effects in recognizing different hepatitis B virus surface antigens, and have more advantages in neutralizing hepatitis B virus mutants and reducing hepatitis B mutant antigens compared to donors of hepatitis B vaccine recipients.

[0224] In some embodiments, the antibodies described herein are expressed at a 50% inhibitory concentration (IC) of less than 0.5 μg / ml. 50) value in vitro neutralizes HBV and its mutants. In some embodiments, the antibodies described herein have a 50% inhibitory concentration (IC) of less than 0.25 μg / ml. 50 ) value in vitro neutralizes HBV and its mutants. In some embodiments, the antibodies described herein have a 50% inhibitory concentration (IC) of less than 0.1 μg / ml. 50 ) values ​​for in vitro neutralization of HBV and its mutants. The IC values ​​of the antibodies described herein were measured as described in the cell-based in vitro neutralization assay in the Examples. 50 value.

[0225] The present invention relates to an isolated antibody or antigen binding fragment thereof for HBV and its mutants. In one embodiment, the present invention relates to an isolated antibody or antigen binding fragment thereof that binds to the HBsAg protein. In another embodiment, the present invention relates to an isolated antibody or antigen binding fragment thereof that binds to an epitope in the extracellular domain of HBsAg. In another embodiment, the present invention relates to an isolated antibody or antigen binding fragment thereof that binds to an epitope in amino acid residues 99-169 of HBsAg. In another embodiment, the present invention relates to an isolated antibody or antigen binding fragment thereof that binds to an epitope in amino acid residues 124-147 of HBsAg.

[0226] In one embodiment, the HBsAg protein comprises a single or double site mutation. In one embodiment, the mutation is selected from Q101R, Q101K, M103I, P105L, L109I, L109R, I110L, S113N, T114R, T115N, T116N, T118K, P120S, P120T, P120K-T123D, K122I, K122R-G145R, T123N, T123N-T143S, C124R, T125M, T126S, A12 In one embodiment, the mutation is G145R. In one embodiment, the mutation is K122R-G145R. In one embodiment, the mutation is F134L.

[0227] In one aspect, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, wherein the isolated antibody or the antigen-binding fragment thereof specifically binds to HBsAg, characterized in that the isolated antibody or the antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises complementary determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region VL comprises complementary determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences:

[0228] a) CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, CDRH3 of SEQ ID NO:4, CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8;

[0229] b) CDRH1 of SEQ ID NO: 10, CDRH2 of SEQ ID NO: 11, CDRH3 of SEQ ID NO: 12, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16;

[0230] c) CDRH1 of SEQ ID NO: 18, CDRH2 of SEQ ID NO: 19, CDRH3 of SEQ ID NO: 20, CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24;

[0231] d) CDRH1 of SEQ ID NO:26, CDRH2 of SEQ ID NO:27, CDRH3 of SEQ ID NO:28, CDRL1 of SEQ ID NO:30, CDRL2 of SEQ ID NO:31, and CDRL3 of SEQ ID NO:32;

[0232] e) CDRH1 of SEQ ID NO:34, CDRH2 of SEQ ID NO:35, CDRH3 of SEQ ID NO:36, CDRL1 of SEQ ID NO:38, CDRL2 of SEQ ID NO:39, and CDRL3 of SEQ ID NO:40;

[0233] f) CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:46, CDRL2 of SEQ ID NO:47, and CDRL3 of SEQ ID NO:48;

[0234] g) CDRH1 of SEQ ID NO:50, CDRH2 of SEQ ID NO:51, CDRH3 of SEQ ID NO:52, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56;

[0235] h) CDRH1 of SEQ ID NO:58, CDRH2 of SEQ ID NO:59, CDRH3 of SEQ ID NO:60, CDRL1 of SEQ ID NO:62, CDRL2 of SEQ ID NO:63, and CDRL3 of SEQ ID NO:64;

[0236] i) CDRH1 of SEQ ID NO:66, CDRH2 of SEQ ID NO:67, CDRH3 of SEQ ID NO:68, CDRL1 of SEQ ID NO:70, CDRL2 of SEQ ID NO:71, and CDRL3 of SEQ ID NO:72;

[0237] j) CDRH1 of SEQ ID NO:74, CDRH2 of SEQ ID NO:75, CDRH3 of SEQ ID NO:76, CDRL1 of SEQ ID NO:78, CDRL2 of SEQ ID NO:79, and CDRL3 of SEQ ID NO:80;

[0238] k) CDRH1 of SEQ ID NO:82, CDRH2 of SEQ ID NO:83, CDRH3 of SEQ ID NO:84, CDRL1 of SEQ ID NO:86, CDRL2 of SEQ ID NO:87, and CDRL3 of SEQ ID NO:88;

[0239] 1) CDRH1 of SEQ ID NO:90, CDRH2 of SEQ ID NO:91, CDRH3 of SEQ ID NO:92, CDRL1 of SEQ ID NO:94, CDRL2 of SEQ ID NO:95, and CDRL3 of SEQ ID NO:96;

[0240] m) CDRH1 of SEQ ID NO:98, CDRH2 of SEQ ID NO:99, CDRH3 of SEQ ID NO:100, CDRL1 of SEQ ID NO:102, CDRL2 of SEQ ID NO:103, and CDRL3 of SEQ ID NO:104;

[0241] n) CDRH1 of SEQ ID NO: 106, CDRH2 of SEQ ID NO: 107, CDRH3 of SEQ ID NO: 108, CDRL1 of SEQ ID NO: 110, CDRL2 of SEQ ID NO: 111, and CDRL3 of SEQ ID NO: 112;

[0242] o) CDRH1 of SEQ ID NO: 114, CDRH2 of SEQ ID NO: 115, CDRH3 of SEQ ID NO: 116, CDRL1 of SEQ ID NO: 118, CDRL2 of SEQ ID NO: 119, and CDRL3 of SEQ ID NO: 120;

[0243] p) CDRH1 of SEQ ID NO: 122, CDRH2 of SEQ ID NO: 123, CDRH3 of SEQ ID NO: 124, CDRL1 of SEQ ID NO: 126, CDRL2 of SEQ ID NO: 127, and CDRL3 of SEQ ID NO: 128;

[0244] q) CDRH1 of SEQ ID NO: 130, CDRH2 of SEQ ID NO: 131, CDRH3 of SEQ ID NO: 132, CDRL1 of SEQ ID NO: 134, CDRL2 of SEQ ID NO: 135, and CDRL3 of SEQ ID NO: 136;

[0245] r) CDRH1 of SEQ ID NO: 138, CDRH2 of SEQ ID NO: 139, CDRH3 of SEQ ID NO: 140, CDRL1 of SEQ ID NO: 142, CDRL2 of SEQ ID NO: 143, and CDRL3 of SEQ ID NO: 144;

[0246] s) CDRH1 of SEQ ID NO: 146, CDRH2 of SEQ ID NO: 147, CDRH3 of SEQ ID NO: 148, CDRL1 of SEQ ID NO: 150, CDRL2 of SEQ ID NO: 151, and CDRL3 of SEQ ID NO: 152;

[0247] t) CDRH1 of SEQ ID NO: 154, CDRH2 of SEQ ID NO: 155, CDRH3 of SEQ ID NO: 156, CDRL1 of SEQ ID NO: 158, CDRL2 of SEQ ID NO: 159, and CDRL3 of SEQ ID NO: 160;

[0248] u) CDRH1 of SEQ ID NO: 162, CDRH2 of SEQ ID NO: 163, CDRH3 of SEQ ID NO: 164, CDRL1 of SEQ ID NO: 166, CDRL2 of SEQ ID NO: 167, and CDRL3 of SEQ ID NO: 168;

[0249] v) CDRH1 of SEQ ID NO: 170, CDRH2 of SEQ ID NO: 171, CDRH3 of SEQ ID NO: 172, CDRL1 of SEQ ID NO: 174, CDRL2 of SEQ ID NO: 175, and CDRL3 of SEQ ID NO: 176;

[0250] w) CDRH1 of SEQ ID NO: 178, CDRH2 of SEQ ID NO: 179, CDRH3 of SEQ ID NO: 180, CDRL1 of SEQ ID NO: 182, CDRL2 of SEQ ID NO: 183, and CDRL3 of SEQ ID NO: 184;

[0251] x) CDRH1 of SEQ ID NO: 186, CDRH2 of SEQ ID NO: 187, CDRH3 of SEQ ID NO: 188, CDRL1 of SEQ ID NO: 190, CDRL2 of SEQ ID NO: 191, and CDRL3 of SEQ ID NO: 192;

[0252] y) CDRH1 of SEQ ID NO: 194, CDRH2 of SEQ ID NO: 195, CDRH3 of SEQ ID NO: 196, CDRL1 of SEQ ID NO: 198, CDRL2 of SEQ ID NO: 199, and CDRL3 of SEQ ID NO: 200;

[0253] z) CDRH1 of SEQ ID NO:202, CDRH2 of SEQ ID NO:203, CDRH3 of SEQ ID NO:204, CDRL1 of SEQ ID NO:206, CDRL2 of SEQ ID NO:207, and CDRL3 of SEQ ID NO:208;

[0254] aa) CDRH1 of SEQ ID NO:210, CDRH2 of SEQ ID NO:211, CDRH3 of SEQ ID NO:212, CDRL1 of SEQ ID NO:214, CDRL2 of SEQ ID NO:215, and CDRL3 of SEQ ID NO:216;

[0255] bb) CDRH1 of SEQ ID NO:218, CDRH2 of SEQ ID NO:219, CDRH3 of SEQ ID NO:220, CDRL1 of SEQ ID NO:222, CDRL2 of SEQ ID NO:223, and CDRL3 of SEQ ID NO:224;

[0256] cc) CDRH1 of SEQ ID NO:226, CDRH2 of SEQ ID NO:227, CDRH3 of SEQ ID NO:228, CDRL1 of SEQ ID NO:230, CDRL2 of SEQ ID NO:231, and CDRL3 of SEQ ID NO:232;

[0257] dd) CDRH1 of SEQ ID NO:234, CDRH2 of SEQ ID NO:235, CDRH3 of SEQ ID NO:236, CDRL1 of SEQ ID NO:238, CDRL2 of SEQ ID NO:239, and CDRL3 of SEQ ID NO:240;

[0258] ee) CDRH1 of SEQ ID NO:242, CDRH2 of SEQ ID NO:243, CDRH3 of SEQ ID NO:244, CDRL1 of SEQ ID NO:246, CDRL2 of SEQ ID NO:247, and CDRL3 of SEQ ID NO:248;

[0259] ff) CDRH1 of SEQ ID NO:250, CDRH2 of SEQ ID NO:251, CDRH3 of SEQ ID NO:252, CDRL1 of SEQ ID NO:254, CDRL2 of SEQ ID NO:255, and CDRL3 of SEQ ID NO:256;

[0260] gg) CDRH1 of SEQ ID NO:258, CDRH2 of SEQ ID NO:259, CDRH3 of SEQ ID NO:260, CDRL1 of SEQ ID NO:262, CDRL2 of SEQ ID NO:263, and CDRL3 of SEQ ID NO:264;

[0261] hh) CDRH1 of SEQ ID NO:266, CDRH2 of SEQ ID NO:267, CDRH3 of SEQ ID NO:268, CDRL1 of SEQ ID NO:270, CDRL2 of SEQ ID NO:271, and CDRL3 of SEQ ID NO:272;

[0262] ii) CDRH1 of SEQ ID NO:274, CDRH2 of SEQ ID NO:275, CDRH3 of SEQ ID NO:276, CDRL1 of SEQ ID NO:278, CDRL2 of SEQ ID NO:279, and CDRL3 of SEQ ID NO:280;

[0263] jj) CDRH1 of SEQ ID NO:282, CDRH2 of SEQ ID NO:283, CDRH3 of SEQ ID NO:284, CDRL1 of SEQ ID NO:286, CDRL2 of SEQ ID NO:287, and CDRL3 of SEQ ID NO:288;

[0264] kk) CDRH1 of SEQ ID NO:290, CDRH2 of SEQ ID NO:291, CDRH3 of SEQ ID NO:292, CDRL1 of SEQ ID NO:294, CDRL2 of SEQ ID NO:295, and CDRL3 of SEQ ID NO:296;

[0265] 11) CDRH1 of SEQ ID NO:298, CDRH2 of SEQ ID NO:299, CDRH3 of SEQ ID NO:300, CDRL1 of SEQ ID NO:302, CDRL2 of SEQ ID NO:303, and CDRL3 of SEQ ID NO:304;

[0266] mm) CDRH1 of SEQ ID NO:306, CDRH2 of SEQ ID NO:307, CDRH3 of SEQ ID NO:308, CDRL1 of SEQ ID NO:310, CDRL2 of SEQ ID NO:311, and CDRL3 of SEQ ID NO:312;

[0267] nn) CDRH1 of SEQ ID NO:314, CDRH2 of SEQ ID NO:315, CDRH3 of SEQ ID NO:316, CDRL1 of SEQ ID NO:318, CDRL2 of SEQ ID NO:319, and CDRL3 of SEQ ID NO:320;

[0268] oo) CDRH1 of SEQ ID NO:322, CDRH2 of SEQ ID NO:323, CDRH3 of SEQ ID NO:324, CDRL1 of SEQ ID NO:326, CDRL2 of SEQ ID NO:327, and CDRL3 of SEQ ID NO:328;

[0269] pp) CDRH1 of SEQ ID NO:330, CDRH2 of SEQ ID NO:331, CDRH3 of SEQ ID NO:332, CDRL1 of SEQ ID NO:334, CDRL2 of SEQ ID NO:335, and CDRL3 of SEQ ID NO:336;

[0270] qq) CDRH1 of SEQ ID NO:338, CDRH2 of SEQ ID NO:339, CDRH3 of SEQ ID NO:340, CDRL1 of SEQ ID NO:342, CDRL2 of SEQ ID NO:343, and CDRL3 of SEQ ID NO:344;

[0271] rr) CDRH1 of SEQ ID NO:346, CDRH2 of SEQ ID NO:347, CDRH3 of SEQ ID NO:348, CDRL1 of SEQ ID NO:350, CDRL2 of SEQ ID NO:351, and CDRL3 of SEQ ID NO:352;

[0272] ss) CDRH1 of SEQ ID NO:354, CDRH2 of SEQ ID NO:355, CDRH3 of SEQ ID NO:356, CDRL1 of SEQ ID NO:358, CDRL2 of SEQ ID NO:359, and CDRL3 of SEQ ID NO:360;

[0273] tt) CDRH1 of SEQ ID NO:362, CDRH2 of SEQ ID NO:363, CDRH3 of SEQ ID NO:364, CDRL1 of SEQ ID NO:366, CDRL2 of SEQ ID NO:367, and CDRL3 of SEQ ID NO:368;

[0274] uu) CDRH1 of SEQ ID NO:370, CDRH2 of SEQ ID NO:371, CDRH3 of SEQ ID NO:372, CDRL1 of SEQ ID NO:374, CDRL2 of SEQ ID NO:375, and CDRL3 of SEQ ID NO:376;

[0275] vv) CDRH1 of SEQ ID NO:378, CDRH2 of SEQ ID NO:379, CDRH3 of SEQ ID NO:380, CDRL1 of SEQ ID NO:382, CDRL2 of SEQ ID NO:383, and CDRL3 of SEQ ID NO:384;

[0276] ww) CDRH1 of SEQ ID NO:386, CDRH2 of SEQ ID NO:387, CDRH3 of SEQ ID NO:388, CDRL1 of SEQ ID NO:390, CDRL2 of SEQ ID NO:391, and CDRL3 of SEQ ID NO:392;

[0277] xx) CDRH1 of SEQ ID NO:394, CDRH2 of SEQ ID NO:395, CDRH3 of SEQ ID NO:396, CDRL1 of SEQ ID NO:398, CDRL2 of SEQ ID NO:399, and CDRL3 of SEQ ID NO:400;

[0278] yy) CDRH1 of SEQ ID NO:402, CDRH2 of SEQ ID NO:403, CDRH3 of SEQ ID NO:404, CDRL1 of SEQ ID NO:406, CDRL2 of SEQ ID NO:407, and CDRL3 of SEQ ID NO:408;

[0279] zz) CDRH1 of SEQ ID NO:410, CDRH2 of SEQ ID NO:411, CDRH3 of SEQ ID NO:412, CDRL1 of SEQ ID NO:414, CDRL2 of SEQ ID NO:415, and CDRL3 of SEQ ID NO:416;

[0280] aaa) CDRH1 of SEQ ID NO:418, CDRH2 of SEQ ID NO:419, CDRH3 of SEQ ID NO:420, CDRL1 of SEQ ID NO:422, CDRL2 of SEQ ID NO:423, and CDRL3 of SEQ ID NO:424;

[0281] bbb) CDRH1 of SEQ ID NO:426, CDRH2 of SEQ ID NO:427, CDRH3 of SEQ ID NO:428, CDRL1 of SEQ ID NO:430, CDRL2 of SEQ ID NO:431, and CDRL3 of SEQ ID NO:432;

[0282] ccc) CDRH1 of SEQ ID NO:434, CDRH2 of SEQ ID NO:435, CDRH3 of SEQ ID NO:436, CDRL1 of SEQ ID NO:438, CDRL2 of SEQ ID NO:439, and CDRL3 of SEQ ID NO:440;

[0283] ddd) CDRH1 of SEQ ID NO:442, CDRH2 of SEQ ID NO:443, CDRH3 of SEQ ID NO:444, CDRL1 of SEQ ID NO:446, CDRL2 of SEQ ID NO:447, and CDRL3 of SEQ ID NO:448;

[0284] eee) CDRH1 of SEQ ID NO:450, CDRH2 of SEQ ID NO:451, CDRH3 of SEQ ID NO:452, CDRL1 of SEQ ID NO:454, CDRL2 of SEQ ID NO:455, and CDRL3 of SEQ ID NO:456;

[0285] fff) CDRH1 of SEQ ID NO:458, CDRH2 of SEQ ID NO:459, CDRH3 of SEQ ID NO:460, CDRL1 of SEQ ID NO:462, CDRL2 of SEQ ID NO:463, and CDRL3 of SEQ ID NO:464;

[0286] ggg) CDRH1 of SEQ ID NO:466, CDRH2 of SEQ ID NO:467, CDRH3 of SEQ ID NO:468, CDRL1 of SEQ ID NO:470, CDRL2 of SEQ ID NO:471, and CDRL3 of SEQ ID NO:472;

[0287] hhh) CDRH1 of SEQ ID NO:474, CDRH2 of SEQ ID NO:475, CDRH3 of SEQ ID NO:476, CDRL1 of SEQ ID NO:478, CDRL2 of SEQ ID NO:479, and CDRL3 of SEQ ID NO:480;

[0288] iii) CDRH1 of SEQ ID NO:482, CDRH2 of SEQ ID NO:483, CDRH3 of SEQ ID NO:484, CDRL1 of SEQ ID NO:486, CDRL2 of SEQ ID NO:487, and CDRL3 of SEQ ID NO:488; and

[0289] jjj) CDRH1 of SEQ ID NO:490, CDRH2 of SEQ ID NO:491, CDRH3 of SEQ ID NO:492, CDRL1 of SEQ ID NO:494, CDRL2 of SEQ ID NO:495, and CDRL3 of SEQ ID NO:496.

[0290] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises complementarity determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region VL comprises complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequence as described above containing substitutions, deletions or insertions of no more than 2 amino acids in one or more places.

[0291] In one aspect, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, wherein the isolated antibody or the antigen-binding fragment thereof specifically binds to HBsAg, characterized in that the isolated antibody or the antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences:

[0292] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0293] b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 13;

[0294] c) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 21;

[0295] d) VH of SEQ ID NO:25 and VL of SEQ ID NO:29;

[0296] e) VH of SEQ ID NO:33 and VL of SEQ ID NO:37;

[0297] f) VH of SEQ ID NO:41 and VL of SEQ ID NO:45;

[0298] g) VH of SEQ ID NO:49 and VL of SEQ ID NO:53;

[0299] h) VH of SEQ ID NO:57 and VL of SEQ ID NO:61;

[0300] i) VH of SEQ ID NO:65 and VL of SEQ ID NO:69;

[0301] j) VH of SEQ ID NO:73 and VL of SEQ ID NO:77;

[0302] k) VH of SEQ ID NO:81 and VL of SEQ ID NO:85;

[0303] 1) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0304] m) VH of SEQ ID NO:97 and VL of SEQ ID NO:101;

[0305] n) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0306] o) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0307] p) VH of SEQ ID NO: 121 and VL of SEQ ID NO: 125;

[0308] q) VH of SEQ ID NO: 129 and VL of SEQ ID NO: 133;

[0309] r) VH of SEQ ID NO: 137 and VL of SEQ ID NO: 141;

[0310] s) VH of SEQ ID NO: 145 and VL of SEQ ID NO: 149;

[0311] t) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0312] u) VH of SEQ ID NO: 161 and VL of SEQ ID NO: 165;

[0313] v) VH of SEQ ID NO: 169 and VL of SEQ ID NO: 173;

[0314] w) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0315] x) VH of SEQ ID NO: 185 and VL of SEQ ID NO: 189;

[0316] y) VH of SEQ ID NO: 193 and VL of SEQ ID NO: 197;

[0317] z) VH of SEQ ID NO: 201 and VL of SEQ ID NO: 205;

[0318] aa) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0319] bb) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0320] cc) VH of SEQ ID NO: 225 and VL of SEQ ID NO: 229;

[0321] dd) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0322] ee) VH of SEQ ID NO: 241 and VL of SEQ ID NO: 245;

[0323] ff) VH of SEQ ID NO: 249 and VL of SEQ ID NO: 253;

[0324] gg) VH of SEQ ID NO: 257 and VL of SEQ ID NO: 261;

[0325] hh) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0326] ii) VH of SEQ ID NO: 273 and VL of SEQ ID NO: 277;

[0327] jj) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285;

[0328] kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293;

[0329] 11) VH of SEQ ID NO:297 and VL of SEQ ID NO:301;

[0330] mm) VH of SEQ ID NO: 305 and VL of SEQ ID NO: 309;

[0331] nn) VH of SEQ ID NO:313 and VL of SEQ ID NO:317;

[0332] oo) VH of SEQ ID NO:321 and VL of SEQ ID NO:325;

[0333] pp) VH of SEQ ID NO: 329 and VL of SEQ ID NO: 333;

[0334] qq) VH of SEQ ID NO: 337 and VL of SEQ ID NO: 341;

[0335] rr) VH of SEQ ID NO:345 and VL of SEQ ID NO:349;

[0336] ss) VH of SEQ ID NO:353 and VL of SEQ ID NO:357;

[0337] tt) VH of SEQ ID NO:361 and VL of SEQ ID NO:365;

[0338] uu) VH of SEQ ID NO: 369 and VL of SEQ ID NO: 373;

[0339] vv) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0340] ww) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389;

[0341] xx) VH of SEQ ID NO: 393 and VL of SEQ ID NO: 397;

[0342] yy) VH of SEQ ID NO:401 and VL of SEQ ID NO:405;

[0343] zz) VH of SEQ ID NO:409 and VL of SEQ ID NO:413;

[0344] aaa) VH of SEQ ID NO:417 and VL of SEQ ID NO:421;

[0345] bbb) VH of SEQ ID NO:425 and VL of SEQ ID NO:429;

[0346] ccc) VH of SEQ ID NO:433 and VL of SEQ ID NO:437;

[0347] ddd) VH of SEQ ID NO:441 and VL of SEQ ID NO:445;

[0348] eee) VH of SEQ ID NO:449 and VL of SEQ ID NO:453;

[0349] fff) VH of SEQ ID NO:457 and VL of SEQ ID NO:461;

[0350] ggg) VH of SEQ ID NO: 465 and VL of SEQ ID NO: 469;

[0351] hhh) VH of SEQ ID NO:473 and VL of SEQ ID NO:477;

[0352] iii) VH of SEQ ID NO: 481 and VL of SEQ ID NO: 485; and

[0353] jjj) VH of SEQ ID NO:489 and VL of SEQ ID NO:493.

[0354] In certain embodiments, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) each independently have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above sequences.

[0355] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises CDRH1 of SEQ ID NO:50, CDRH2 of SEQ ID NO:51 and CDRH3 of SEQ ID NO:52, and the light chain variable region VL comprises CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55 and CDRL3 of SEQ ID NO:56.

[0356] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or an antigen-binding fragment thereof comprises a heavy chain variable region VH of SEQ ID NO:49 and a light chain variable region VL of SEQ ID NO:53.

[0357] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises CDRH1 of SEQ ID NO: 234, CDRH2 of SEQ ID NO: 235 and CDRH3 of SEQ ID NO: 236, and the light chain variable region VL comprises CDRL1 of SEQ ID NO: 238, CDRL2 of SEQ ID NO: 239 and CDRL3 of SEQ ID NO: 240.

[0358] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or an antigen-binding fragment thereof comprises a heavy chain variable region VH of SEQ ID NO: 233 and a light chain variable region VL of SEQ ID NO: 237.

[0359] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises CDRH1 of SEQ ID NO: 282, CDRH2 of SEQ ID NO: 283 and CDRH3 of SEQ ID NO: 284, and the light chain variable region VL comprises CDRL1 of SEQ ID NO: 286, CDRL2 of SEQ ID NO: 287 and CDRL3 of SEQ ID NO: 288.

[0360] In certain embodiments, the present invention relates to an isolated antibody or an antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or an antigen-binding fragment thereof comprises a heavy chain variable region VH of SEQ ID NO: 281 and a light chain variable region VL of SEQ ID NO: 285.

[0361] The sequence information of the antibody of the present invention is as follows:

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] Variants of the antibodies of the invention comprising the VH or VL amino acid sequences described above are within the scope of the invention. For example, the variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid substitutions in VH and / or VL that do not adversely affect the properties of the antibody. In some embodiments, the sequence identity relative to the VH or VL amino acid sequences of the invention may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Exemplary modifications are conservative amino acid substitutions, such as in the antigen binding site or in the framework, which do not adversely change the properties of the antibody. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity. Conservative substitutions are those that occur within a family of related amino acids in their side chains. The genetically encoded amino acids can be divided into four categories: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) non-charged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally each being grouped as hydroxyl-containing aliphatic; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine ​​and methionine) (Stryer (ed.), Biochemistry, 2nd ed., WH Freeman and Co., 1981). In addition, any native residue in the polypeptide can also be replaced with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al (1998) Acta Physiol. Scand. Suppl. 643: 55-67; ​​Sasaki et al (1998) Adv. Biophys. 35: 1-24). The desired amino acid replacement can be determined by those skilled in the art when such replacement is required. The characteristics of the resulting antibody variants can be tested using the assays described herein.

[0377] Amino acid substitutions can be carried out, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a variant library can be generated using known methods, such as using random (NNK) codons or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)), and then screening the library for variants with the desired properties.

[0378] Although the antibodies of the present invention include paired variable regions, one from the heavy chain and one from the light chain, those skilled in the art will recognize that alternative embodiments may include a single heavy chain variable region or a single light chain variable region. A single variable region can be used to screen for variable domains capable of forming two-domain specific antigen-binding fragments. The screening can be accomplished by phage display screening methods using, for example, the hierarchical double combinatorial method disclosed in International Patent Publication No. WO92 / 01047. In this combinatorial method, a single colony containing an H chain clone or an L chain clone is used to infect a complete library of clones encoding the other chain (L or H), and then the resulting two-chain specific antigen-binding domain is selected according to the described phage display technology. Therefore, separate VH polypeptide chains and VL polypeptide chains can be used to identify additional antibodies that specifically bind to the S protein domain, and the identification process uses the method disclosed in International Patent Publication No. WO92 / 01047.

[0379] The antibodies of the present invention can be prepared using a variety of techniques for producing antibodies. For example, the hybridoma method proposed by Kohler and Milstein in Nature 256:495, 1975 can be used to produce monoclonal antibodies. In the hybridoma method, mice or other host animals (such as hamsters, rats or monkeys) are immunized with S protein or fragments thereof, and then spleen cells from the immunized animals are fused with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Colonies produced by single immortalized hybridoma cells are screened for the preparation of antibodies with desired properties (such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, and affinity for antigens).

[0380] Various host animals can be used to prepare antibodies of the present invention. For example, Balb / c mice can be used to prepare antibodies. Various techniques can be used to humanize antibodies prepared in Balb / c mice and other non-human animals to produce more human-like sequences. Exemplary humanization techniques including selecting human receptor frameworks are known to those skilled in the art, including CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749), resurfacing (Padlan, Mol Immunol 28:489-499, 1991), resurfacing of specificity-determining residues (U.S. Patent Publication No. 20100261620), human remodeling (or human framework remodeling) (U.S. Patent Publication No. US2009 / 0118127), superhumanization (U.S. Patent No. 7,709,226) and directional selection (Osbourn et al (2005) Methods 36:61-68, 2005; U.S. Patent No. 5,565,332).

[0381] Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by introducing modified framework supporting residues to maintain binding affinity (back mutations) using disclosed techniques such as those described in International Patent Publication No. WO90 / 007861 and International Patent Publication No. WO92 / 22653.

[0382] Transgenic mice carrying human immunoglobulin (Ig) loci in their genomes can be used to generate human antibodies against target proteins as described, for example, in International Patent Publication No. WO90 / 04036, U.S. Pat. No. 6,150,584, International Patent Publication No. WO99 / 45962, International Patent Publication No. WO02 / 066630, International Patent Publication No. WO02 / 43478; Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93; Bruggemann et al (1991) Eur. J. Immunol. 21:1323-1326; Fishwild et al (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93; Bruggemann et al (1991) Eur. J. Immunol. 21:1323-1326; Fishwild et al (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93; Bruggemann et al (1991) Eur. J. Immunol. 21:1323-1326; Fishwild et al (1998) al (1996) Nat. Biotechnol. 14:845-851; Mendez et al (1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol. Methods 231:11-23; Yang et al (1999) Cancer Res. 59:1236-1243; Brüggemann and Taussig (1997) Curr. Opin. Biotechnol. 8:455-458; International Patent Publication No. WO02 / 043478). The endogenous immunoglobulin loci in such mice can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the mouse genome by homologous or nonhomologous recombination using a transchromosome or minigene.

[0383] Human antibodies can be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappiket al (2000) J. Mol. Biol. 296:57-86; Krebs et al (2001) J. Immunol. Meth. 254:67-84; Vaughan et al (1996) Nature Biotechnology 14:309-314; Sheets et al (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381; Marks et al (1991) J. Mol. Biol. 222:581). The antibodies of the present invention can be isolated from, for example, a phage display library that expresses the heavy chain variable region and light chain variable region of the antibody as a fusion protein with a phage pIX coat protein, as described in Shi et al (2010) J. Mol. Biol. 397: 385-96 and International Patent Publication No. WO09 / 085462. The library can be screened for phages that bind to the S protein, and the positive clones obtained can be further characterized, and Fabs are isolated from the clone lysates and expressed as full-length IgGs. Such phage display methods for isolating human antibodies are described, for example, in U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; 5,427,908 and 5,580,717 to Dower et al.; 5,969,108 and 6,172,197 to McCafferty et al.; and 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0384] Preparation of immunogenic antigens and generation of monoclonal antibodies can be performed with any suitable technology such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures (including whole cells, cell extracts or tissue extracts), or the antigens can be generated de novo in animals from nucleic acids encoding the antigens or portions thereof.

[0385] Another embodiment of the present invention is a separated polynucleotide encoding any one of the antibody heavy chain variable region and / or antibody light chain variable region of the present invention. In view of the genetic code degeneracy or codon priority in a given expression system, multiple polynucleotides encoding the same antibody of the present invention are also within the scope of the present invention. The polynucleotide sequence encoding the VH or VL of the antibody of the present invention or their fragments can be effectively connected to one or more regulatory elements, such as a promoter or enhancer, which allows the nucleotide sequence to be expressed in the intended host cell. The polynucleotide can be a cDNA.

[0386] Another embodiment of the present invention is a vector comprising a polynucleotide of the present invention. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, vectors based on transposons, or any other vectors suitable for introducing the synthetic polynucleotide of the present invention into a given organism or genetic background by any means. For example, a polynucleotide optionally connected to a constant region and encoding a light chain variable region and / or a heavy chain variable region of an antibody of the present invention is inserted into an expression vector. The light chain and / or the heavy chain can be cloned into the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain can be effectively connected to a control sequence that ensures the expression of immunoglobulin polypeptides in one or more expression vectors. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription terminator sequences, and such control sequences are selected to make them compatible with the host cell selected for expressing the antibody. Once the vector is incorporated into an appropriate host, the vector maintains the host under conditions suitable for high-level expression of proteins, which are encoded by the combined polynucleotides.

[0387] Suitable expression vectors can usually replicate in the host organism as a free gene or a part of the host chromosomal DNA. Usually, the expression vector comprises a selection marker, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance, so that cells transformed with the desired DNA sequence can be detected.

[0388] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, exemplary promoters include lacI, lacZ, T3, T7, gpt, λP and trc. For expression in eukaryotic cells, exemplary promoters include light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus very early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in the long terminal repeat sequence of retrovirus; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. For expression in yeast cells, exemplary promoters are constitutive promoters, such as ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulatable promoters, such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter and AOX1 (e.g., for Pichia). The selection of suitable vectors and promoters is within the capabilities of those of ordinary skill in the art.

[0389] A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for use in generating subject recombinant constructs. The following vectors are provided by way of example. Bacterial vectors: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic vectors: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0390] Another embodiment of the present invention is a host cell comprising one or more vectors of the present invention. The term "host cell" refers to a cell into which a vector has been introduced. It should be understood that the term "host cell" is intended to refer not only to the specific subject cell, but also to the progeny of such a cell, and also to stable cell lines produced by the specific subject cell. Due to mutation or due to environmental influences, certain modifications may occur in the offspring, so such progeny may be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells or archaeal cells.

[0391] Escherichia coli, bacilli (such as Bacillus subtilis) and other enterobacteriaceae (such as Salmonella, Serratia) and various Pseudomonas species are examples of prokaryotic host cells. Other microorganisms such as yeast can also be used for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells can be from mammals, insects, birds or other animal sources. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61) or DG44.

[0392] Another embodiment of the present invention is a method for preparing an antibody of the present invention, the method comprising culturing a host cell of the present invention under conditions that allow the antibody to be expressed, and then recovering the antibody produced by the host cell. Methods for preparing antibodies and purifying them are well known in the art. Once the whole antibody, its dimer, each light chain and / or heavy chain, or other antibody fragments (such as VH and / or VL) are synthesized (chemically or recombinantly), they can be purified according to standard procedures, including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The antibody can be substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more pure, for example, free of contaminants (such as cell debris, macromolecules other than the test antibody, etc.).

[0393] Another embodiment of the present invention is a method for preparing an antibody of the present invention, the method comprising:

[0394] combining a first polynucleotide encoding antibody VH and a second polynucleotide encoding antibody VL into an expression vector;

[0395] Transforming a host cell with the expression vector;

[0396] culturing the host cells in a culture medium under conditions that allow the VL and VH to be expressed and the antibody to be formed; then

[0397] The antibody is recovered from the host cells or culture medium.

[0398] The polynucleotide encoding the specific VH or VI sequence of the present invention is incorporated into the vector using standard molecular biological methods. Host cell transformation, culture, antibody expression and purification are accomplished using well-known methods.

[0399] III. Composition and Administration Method

[0400] The present invention provides pharmaceutical compositions comprising antibodies as described herein, and pharmaceutically acceptable carriers. For therapeutic purposes, the antibodies of the present invention can be prepared into pharmaceutical compositions comprising an effective amount of antibodies as active ingredients in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those derived from petroleum, animal, plant oils or synthetic oils, such as peanut oil, soybean oil, mineral oil and sesame oil. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional sterilization techniques (e.g., filtration) that are well known. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants and colorants. The concentration of the antibody of the invention in such pharmaceutical preparations can vary over a wide range, i.e., from less than about 0.5% by weight, typically to at least about 1% by weight to as much as 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight, and will be selected according to the particular mode of administration selected, primarily based on desired dose, fluid volume, viscosity, etc. Suitable vehicles and formulations (including other human proteins such as human serum albumin) are described, e.g., in Remington: The Science and Practice of Pharmacy, 21st edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, see especially pp. 958-989.

[0401] The manner of administering the antibodies of the invention in the methods of the invention described herein can be any suitable route, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, mucosal (oral, intranasal, vaginal, rectal) or other means known to the skilled person, which are well known in the art.

[0402] The antibodies in the methods of the invention described herein can be administered to the patient by any suitable route, such as parenteral administration by intravenous (iv) infusion or bolus, intramuscular administration, subcutaneous administration or intraperitoneal administration. The intravenous infusion can be given, for example, within 15, 30, 60, 90, 120, 180 or 240 minutes, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0403] In one embodiment, a therapeutically effective amount of an antibody of the present invention is administered to a subject in the methods of the present invention described herein. The "therapeutically effective amount" of an antibody can be determined by standard research techniques. For example, an in vitro assay can be used to help identify the optimal dose range. Optionally, a therapeutically effective amount of an antibody of the present invention can be determined by administering the antibody to a relevant animal model well known in the art. The selection of a specific effective dose can be determined by a person skilled in the art based on considerations of several factors (e.g., via clinical trials). Such factors include the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to the technician. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the doctor's judgment and the circumstances of each patient. The effective dose can be derived by a dose response curve from an in vitro or animal model test system. Any of the models described herein can be used to test the efficacy and effective dose of the antibodies of the present invention.

[0404] The antibodies in the methods of the invention can be administered repeatedly after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. The course of treatment can also be repeated, as in chronic administration. Repeated administration can be the same dose or different doses.

[0405] The antibodies of the methods of the invention may also be administered prophylactically to reduce the risk of a subject becoming infected with HBV, to delay the onset of HBV infection, and / or to reduce the risk of recurrence of HBV infection in a subject in remission.

[0406] The antibodies in the methods of the invention described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been demonstrated to be effective for conventional protein preparations, and well-known lyophilization and reconstitution techniques can be used.

[0407] In one embodiment, the pharmaceutical composition of the present disclosure comprises one or more antibodies described herein. It is easy for those skilled in the art to understand that when the HBV infected by the body has multiple mutations, or when the body produces multiple mutations under drug or immune pressure, the combination of one or more antibodies that bind to different mutation sites will have greater clinical significance and value in treatment and prevention.

[0408] In one embodiment, the pharmaceutical composition of the present disclosure further comprises one or more second agents or antibodies. The combined use of multiple agents or antibodies is well known in the art and is easily performed by those skilled in the art according to routine practice.

[0409] The present invention is now described in conjunction with the following specific non-limiting examples, which are only used to illustrate the present invention but not to limit the scope of the present invention.

[0410] Materials and Methods

[0411] The inventors used flow cytometry to sort out B cells specific for hepatitis B surface antibodies from peripheral blood PBMCs of donors who had recovered from hepatitis B infection. Then, the antibody sequence in the cells was cloned by PCR, obtained by sequencing, and constructed on an expression vector. The plasmid was introduced into the cells by transient transfection to express the protein, and then the cell supernatant was collected to purify and concentrate the antibody.

[0412] The human subjects involved in the present invention were approved by the Ethics Committee of Fudan University (Project Ethics Number: 2020-C013), and experiments involving all donor samples were conducted at the School of Basic Medical Sciences of Fudan University. Among the researchers, 325 were donors from recovered hepatitis B infection patients, and 16 were donors with high titers of anti-HBs after hepatitis B vaccine immunization. The serum specimens used to screen volunteers were heat-inactivated at 56°C for 60 minutes after specimen collection, and stored in a -80°C refrigerator. After extracting 200 ml of blood from AA14, BH2, CC7 and EB4, donors from recovered hepatitis B infection patients, and C10, C12 and C14, donors from hepatitis B vaccine recipients, human peripheral blood mononuclear cells (PBMCs) were isolated, and the isolated PBMCs were then resuspended in a freezing solution of 10% DMSO and 90% fetal bovine serum (inactivated), and frozen in liquid nitrogen.

[0413] Purification of B cells, dual fluorescent dye labeling of bait protein-bound B cells, and single cell sorting were as described previously (Y. Zhou et al (2020) STAR Protoc 1, 100129). PBMCs were thawed in a 37°C water bath and washed with RPMI medium. PBMCs were incubated with CD19 microbeads for positive selection of B lymphocytes. Cell surface molecules were blocked with human Fc block at 4°C, and bait protein-PE / APC (10ug / ml) and anti-CD20-PE-Cy7 (BD Bioscience) were incubated continuously. Single cell sorting was performed on a FACSAria II (BD Bioscience) flow cytometer, and single cells were sorted into 96-well plates and stored at -80°C.

[0414] As previously described (Y. Zhou et al (2020) STAR Protoc 1, 100129), the sorted single cells were reverse transcribed and antibody sequence PCR was performed, after the first round of PCR and the second round of nested PCR, electrophoresis and purification were performed in 2% agarose gel, and then Sanger sequencing was performed. The sequences of the heavy and light chains were analyzed by IMGT / V-QUEST (Brochet, X. et al (2008) Nucl. Acids Res. 36, W503-508) and IgBlast (J. Ye et al (2013) Nucl. Acids Res. 41, W34-40), and the V (D) J gene segment and CDR3 sequence of each antibody were determined. The selected antibodies were subjected to vector construction and antibody expression.

[0415] The antibody obtained by the present invention is subjected to ELISA detection. The HBsAg antigen is diluted to 10ug / ml with PBS, coated overnight at 4°C, and then blocked for 2 hours with 2% bovine serum albumin (BSA) dissolved in PBS. The antibody is diluted to 8 dilutions at a dilution ratio of 1:3 with a maximum concentration of 10ug / ml, and incubated at room temperature for 1 hour after addition. Incubate with goat anti-human IgG (Thermo Fisher Scientific) linked to HRP, and finally develop with ABTS color developing solution, and the BioRad plate reader reads the values ​​at 10 minutes, 20 minutes and 30 minutes respectively. Analyze by PRISM software, and calculate the area under the curve (AUC) for each antibody to evaluate the antigen-binding ability.

[0416] Competition between antibodies was detected by competitive ELISA, which has been reported previously (Q. Wang et al (2020) Cell Host & Microbe 28, 335-349). Briefly, the HBsAg antigen was diluted to 3ug / ml with PBS and coated overnight, the first blocking antibody concentration was 15ug / ml, and incubated at room temperature for 2 hours. Biotinylated secondary antibody (0.25ug / ml or 0.75ug / ml) was then added directly at room temperature for 30 minutes. Detection was performed with streptavidin-HRP (BD Bioscience). PBS buffer was used instead of the first blocking antibody as a standardization reference.

[0417] Neutralization experiments were performed using a cell model infected with HBV virus in vitro (Q. Wang et al (2020) Cell Host & Microbe 28, 335-349). There are two types of HBV virus preparation, one is the HBV virus derived from the cell line HepDE19, and the viral genotype is type D; by transfecting different genotype HBV plasmids into the HepG2 cell line, the transfection reagent is X-tremeGENEHP (Roche) to express HBV viruses of genotype A / B / C. Regardless of whether the virus is derived from a stable cell line or the virus obtained by transfection, it is obtained by ultrafiltration tube concentration. After the ultrafiltration is completed, the obtained virus is packaged, and a small amount of virus is taken out. After 10-fold gradient dilution, the HBV copy number is quantified by qPCR (Sansure Biotech) (F. Shen et al (2018) Hepatology 67, 1237-1252). HepG2-NTCP cells were seeded in 96-well plates and treated with DMEM medium containing 2% DMSO, 3% FBS, 1% NEAA and 1% penicillin-streptomycin mixture (infection medium) for 24 hours before infection. In order to detect the neutralizing ability of serum or antibodies, human serum (maximum concentration 10%) or monoclonal antibodies (maximum concentration 10ug / ml) were serially diluted in the infection medium at a ratio of 1:3, with a total of 9 dilutions. Subsequently, the diluted serum or monoclonal antibodies were incubated with HBV virus at 37°C for 30 minutes and then added to HepG2-NTCP cells. After 16 hours of infection, each well was washed 5 times with 200ul PBS and the infection medium was added. After 7 days of culture, the CLIA kit was used to detect the levels of HBsAg and HBeAg in the cell supernatant (Autobio), and the level of HBcAg in the cells was detected by immunofluorescence.

[0418] The present invention is used to detect the neutralization effect of antibodies on different hepatitis B virus mutants. The viruses used are transfected plasmids (pHBV1.3-G145R, pHBV1.3-K122R G145R and pHBV1.3-F134L) into HepG2 cells, and the supernatant is collected and concentrated to obtain HBV mutant viruses. The neutralization experiment operation steps are the same as the steps of the neutralization experiment of the cell model infected with HBV virus in vitro.

[0419] The antibody amino acid epitope screening (amino acid scanning), antibody binding to different viral genotypes ELISA, and antibody binding to different viral mutants were described previously (Q. Wang et al (2020) Cell Host & Microbe 28, 335-349). In brief, Huh7.5 cells were seeded in 6-well plates, and HBV1.3 plasmids with different mutation sites were transfected using lipofectamine 3000 (thermo fisher Scientific). 16 hours after transfection, the cell culture medium was replaced with DMEM medium containing 1% NEAA and 1% penicillin-streptomycin mixture. After 3 days of culture, the cell culture supernatant was collected, centrifuged to remove cell debris, and the cell culture medium was incubated in ELISA 96-well plates at 100ul per well at 4°C overnight. Then, the plate was blocked with 2% bovine serum albumin (BSA) dissolved in PBS for 2 hours, the antibody was incubated at a concentration of 1 ug / ml, and incubated with goat anti-human IgG (Thermo Fisher Scientific) linked to HRP, and finally developed with ABTS colorimetric solution. The BioRad plate reader read the values ​​at 10 minutes, 20 minutes, and 30 minutes. The readings of antibody binding to wild-type HBV were used as a standardized reference and analyzed by PRISM software.

[0420] For immunofluorescence, cells were fixed in 4% paraformaldehyde / PBS at room temperature for 20 minutes, neutralized with PBS containing 0.1M glycine for 15 minutes, and then with 0.1% TritonX-100 / PBS. Blocked with 5% goat serum / PBS at room temperature for 1 hour. Anti-HBc (Austral BIologicals) was diluted with 5% goat serum / PBS and incubated overnight at 4°C. Visualization was performed with goat anti-rabbit AlexaFluor594 (thermo Fisher Scientific), and cell nuclei were stained with DAPI. Cells were fluorescently photographed using a high-content cell imaging analyzer (Perkin Elmer, Operetta).

[0421] The detailed results of statistical analysis are shown in the Results and Figure Legends section. The area under the ELISA curve (AUC), the 50% inhibitory concentration (IC 50 ) values ​​were calculated in PRISM software.

[0422] Example 1. Antibody response of individual serum of hepatitis B recovered volunteers

[0423] Serum samples from 325 donors who were infected with HBV but recovered were collected from multiple hospitals in Shanghai. The results of two pairs of donor sera were all positive for anti-core antibodies, and the titers of anti-HBsAg antibodies were different. The neutralizing activity of the sera of recovered hepatitis B infection patients was tested. In HepG2 cells overexpressing the hepatitis B infection receptor protein sodium ion-taurocholic acid co-transporter (NTCP), the ability of donor serum samples to block HBV infection of HepG2-NTCP cells, i.e., neutralizing activity, was measured. We selected donors AA14, BH2, CC7, and EB4 with high anti-HBs titers and high neutralizing activity among recovered hepatitis B infection patients, and C10, C12, and C14 among hepatitis B vaccine recipients, and used donors with anti-HBs titers <2.0IU / ml as negative controls. The demographic information of the donors is shown in Table 2. We confirmed the neutralizing activity of the donors at different serum concentrations by diluting the serum specimens, such as Figure 2 .

[0424] Table 2. Demographic information of donors

[0425]

[0426] Example 2. Obtaining a fully human monoclonal antibody that specifically binds to HBsAg

[0427] The HBV surface protein is a four-transmembrane protein that can be divided into three regions: preS1, preS2, and S. The cloned antibodies of the present invention are mainly targeted at the S region, and the CHO purified HBsAg protein is used as the bait protein. In order to select B cells expressing HBsAg-specific IgG antibodies in individuals, a dual fluorescent dye labeling strategy is used to identify B cells that specifically bind to HBsAg ( Figure 3 ). Sample donors who were vaccinated with hepatitis B vaccine but had anti-HBs ELISA titers less than 2mIU / ml were recruited during donor recruitment. They were used as negative controls for the experiment. Their B cell staining was the background level of bait protein-specific B cells. However, donors AA14, BH2, CC7, EB4 with high serum neutralizing activity and vaccine-immunized donors C10, C12, and C14 showed bait protein-specific B cells ( Figure 4 ).

[0428] Flow cytometry gated double-positive cells (bait protein-PE+ and bait protein-APC+) were single-cell sorted, and single B cells were lysed, RNA was reverse transcribed, and the heavy and light chains of specific B cells were amplified by nested PCR reaction using immunoglobulin heavy and light chain gene-specific primers.

[0429] Example 3. ELISA to detect the binding of antibodies to HBsAg protein

[0430] After obtaining the antibody sequence, the antibody sequence was cloned into a vector, followed by protein expression and purification, and the obtained antibody was subjected to ELISA to detect the binding of the antibody to HBsAg. The data after ELISA detection were used to calculate the AUC value in PRISM. Among the antibodies numbered S001-S108, 62 antibodies can bind to HBsAg, and are displayed here in order of AUC values ​​from high to low, see Figure 5 .

[0431] Example 4. Evaluation of Antibody Neutralizing Activity in HBV Cell Infection Model

[0432] In order to determine whether antibodies that can bind to HBsAg can block HBV infection in vitro, HepG2-NTCP was infected with cell-derived concentrated and purified HBV virus for neutralization assay. During the experiment, the antibodies were diluted in a concentration gradient and then mixed with the same virus to infect HepG2-NTCP cells. Finally, the levels of HBsAg and HBeAg in the cell supernatant were detected. The infection rate was compared between the antibody-treated group and the non-antibody-treated group, and the 50% inhibition concentration of the antibody, i.e., the IC50 value, was calculated. The neutralizing activity of these antibodies varies significantly, ranging from effective neutralizing antibodies to almost no neutralizing activity ( Figure 6 ), specific IC50 values ​​are shown in Table 3.

[0433] Table 3. IC values ​​of antibodies tested in cell infection model 50 value

[0434]

[0435] Example 5. Antibody epitope analysis

[0436] The binding epitopes of the 23 antibodies S05, S12, S16, S17, S19, S20, S29, S33, S39, S41, S43, S64, S66, S81, S82, S89, S92, S95, S96, S101, S103, S105 and S106 with good neutralizing activity were characterized. First, determine whether the HBsAg binding antibodies bind to overlapping or non-overlapping epitopes, and perform a competitive ELISA. Since the S105 antibody binds weakly, its competition ELISA results are not shown here. Three mutually exclusive antibody groups (Group I, Group II and Group III) were identified by competition ELISA, see for details. Figure 7 , where Group I includes S33, S39, S64, S103 and S106, Group II includes S29, S43, S41, S66 and S81, and Group III includes S5, S12, S16, S17, S19, S20, S82, S89, S92, S95, S96 and S101.

[0437] The antibody recognizes the S protein mainly by recognizing the region of about 72 amino acids on the extracellular segment of HBsAg. A further alanine scanning method is performed, using the constructed HBsAg point mutation plasmid (the original amino acid is mutated to alanine), and after transfecting cells, HBsAg with different amino acid mutations is obtained. Subsequently, ELISA is used to detect whether the antibody can bind to the HBsAg amino acid mutant, thereby clarifying the specific antigenic amino acid site recognized by the obtained antibody ( Figure 8 ). Combined with the results of competitive ELISA, it is suggested that antibodies in different groups have obvious clustering in the key amino acid sites of antigen recognition. The antibodies from donors immunized with hepatitis B vaccine and those from donors recovered from hepatitis B infection are significantly different in recognition sites. The average number of amino acids that affect antibodies from hepatitis B vaccine is 12.6, and the average number of amino acids that affect antibodies from recovered hepatitis B infection is 22.1. The amino acid sites recognized by antibodies from hepatitis B vaccine donors are discontinuous, and the amino acid sites recognized by recovered donors from hepatitis B infection can be 3-5 consecutive amino acids, for example, S43 recognizes 143T-145G, S20 recognizes 133M-136S, and S17 recognizes 142P-146N. Subsequently, combined with the results of competitive ELISA, the antibody alanine scanning results were analyzed. The antibodies in the four groups all recognize the spatial epitope of the S protein, and 101Q, 102G, 103M, 105P, and 106V all have an effect on the binding of each antibody. The antibodies of the four groups showed their own specific amino acid mutant binding patterns, among which the amino acid sites concentrated in group I were 110I, 122K, 125T, 134F, 141K and 165W; the amino acid sites related to group II were 122K, 123T, 145G, 161Y and 165W; the amino acid sites related to group III were mainly 109L-111P, 120P-130G, 133M-136S, 144D, 146N, 153P, 161Y, 164E-167S. S64, S33 and S39 in group I antibodies were derived from two hepatitis B vaccine immunization donors, respectively, and S66, S29 and S41 in group II antibodies were derived from two hepatitis B vaccine immunization donors, respectively, indicating that the antibodies obtained by immunizing different individuals with hepatitis B vaccine have certain similarities, but the antibodies produced have diversity in recognizing epitopes and can recognize no less than two non-overlapping epitopes.

[0438] Example 6. Binding ability and neutralizing activity against different HBV genotypes

[0439] Hepatitis B virus is an ancient DNA virus. Currently, there are 9 confirmed genotypes, and there is one and one undetermined genotypes. A large number of studies have shown that the distribution of HBV genotypes varies in different regions. Among the people infected with HBV in my country, about 60% are genotype B and about 30% are genotype C.

[0440] The inventor's laboratory has successfully established an in vitro infection system for HepG2-NTCP cells infected with different genotypes of HBV, and used this system to detect whether antibodies that broadly bind to different genotypes of HBV can block the infection of different genotypes of HBV in vitro ( Fig. 9 ). After the antibody and virus were mixed in vitro, they were incubated at 37°C for 1 hour and then infected HepG2-NTCP cells. The levels of HBsAg and HBeAg in the cell supernatant were detected on the 7th day after infection. The 50% inhibition concentration of the antibody, i.e., the IC50 value, was calculated by comparing the antibody-treated group with the non-antibody-treated group.

[0441] The results showed that S12, S29, S33, S39, S41, S43, S66, S81 and S106 had neutralizing activity against the four genotypes A / B / C / D. Antibodies from donors who recovered from hepatitis B infection generally could not neutralize the virus of genotype A well, while antibodies from most donors immunized with hepatitis B vaccine had neutralizing activity against the four genotypes of HBV virus.

[0442] Example 7. Binding ability against naturally occurring HBV mutants

[0443] Studies have reported that there are many immune escapes in patients with chronic hepatitis B infection. In order to detect whether antibodies obtained from donors who have recovered from hepatitis B infection or donors who have been immunized with hepatitis B vaccine can bind to these mutants, common hepatitis B surface antigen amino acid mutation plasmids were constructed. The escape mutations selected by the present invention include: Q101R, Q101K, M103I, P105L, L109I, L109R, I110L, S113N, T114R, T115N, T116N, T118K, P120S, P120T, P120K-T123D, K122I, K122R-G145R, T123N, T123N-T143S, C124R, T125M, T126N, T127N-T128S, 26S, A128V, Q129H, G130N, G130R, M133T, F134L, F134V-D144G, C137Y, C137Y-D144V, C138Y, K141E, P142S, P142L-G145R, T143I, T143L, D144E, D144E-G145R, G145E, G145R, N146S, C149Y and Y161F. The above plasmids were transfected into cells, and the cell supernatant contained the antigen containing the HBsAg mutants. Then, the ELISA experiment was used to detect whether the antibody bound to the antigen ( Fig.10). Antibodies from donors immunized with hepatitis B vaccine can recognize an average of 31 mutation sites, while antibodies from donors recovered from hepatitis B infection can recognize an average of 37.4 mutation sites. The specific HBsAg mutant sites recognized by antibodies from the two immune backgrounds are also significantly different. Group I antibodies derived from donors immunized with hepatitis B vaccine do not recognize Q101R, P105L, C124R, C137Y, C137Y D144V, C138Y, K141E, and C149Y. S64 in group I can recognize K122R G145R, P142S, S142LG145R, D144E G 145R, G145E, and G145R sites. Group II antibodies derived mainly from donors immunized with hepatitis B vaccine do not recognize Q101R, P105L, P120K T123D, K122R G145R, T123N, T123N T143S, C124R, C137Y, C137Y D144V, C138Y, S142L G145R, and D144E G145R, G145E, G145R, S43 from donors who recovered from hepatitis B infection in group II cannot recognize the above mutation sites, nor can it recognize T143I, T143L and D144E. Group III antibodies from donors who recovered from hepatitis B infection cannot recognize Q101R, P105L, P120K T123D, T123N, T123N T143S, G130N, G130R, M133T, C138Y and C149Y, but can recognize C137Y, C137Y D144Y sites that cannot be recognized by group I and group II. The most common mutation site in clinical practice is G145R. ELISA results show that only S64 of antibodies from donors immunized with hepatitis B vaccine can recognize G145R.

[0444] Example 8. Neutralizing activity against naturally occurring HBV mutants

[0445] As shown in the results of Example 6, the present invention relates to antibodies with neutralizing activity binding to different HBV mutants. Here, we selected the most common clinical HBsAg G145R single site mutant and HBsAgK122R-G145R double mutant for neutralization experiments. In Example 6, it is suggested that antibodies derived from hepatitis B vaccine immune donors can better bind to HBsAg F134L mutants, and HBsAg F134L mutant viruses are also purified.

[0446] The experimental results showed that antibodies S12, S64, S82 and S92 could effectively neutralize HBV with G145R mutation, S12, S19, S20, S43 from donors recovered from HBV infection and S39, S82 and S92 from donors immunized with HBV vaccine could effectively neutralize HBV with K122R-G145R double mutation, S12, S43, S103 and S106 from donors recovered from HBV infection and S29, S33, S39, S41, S64, S66, S81 and S82 from donors immunized with HBV vaccine could effectively neutralize HBV with F134L mutation ( Fig.11 ).

[0447] References

[0448] 1.Hehle V, Beretta M, Bourgine M, Ait-Goughoulte M, Planchais C, MorisseS, Vesin B, et al. Potent humanbroadly neutralizing antibodies to hepatitis Bvirus from natural controllers. J Exp Med 2020; 217.

[0449] 2. Wang Q, Michailidis E, YuYP, Wang ZJ, Hurley AM, OrenDA, Mayer CT, et al. A Combination of Human BroadlyNeutralizing Antibodies against Hepatitis B VirusHBsAg with Distinct Epitopes Suppresses Escape Mutations. Cell Host&Microbe2020;28:335-+.

[0450] 3. Huang CH, Yuan Q, ChenPJ, Zhang YL, Chen CR, Zheng QB, Yeh SH, etal. Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occultHBV strains from blood donors. J Hepatol2012; 57:720-729.

[0451] 4.Ito K,QinY,Guarnieri M,Garcia T,Kwei K,Mizokami M,Zhang J,etal.Impairment ofhepatitis B virus virion secretionby single-amino-acidsubstitutions in the small envelope protein and rescue by a novelglycosylationsite.J Virol 2010;84:12850-12861.

[0452] 5.Carman WF,Zanetti AR,Karayiannis P,Waters J,Manzillo G,Tanzi E,Zuckerman AJ,et al.Vaccine-induced escape mutant ofhepatitis B virus.Lancet1990;336:325-329.

[0453] 6.Zhang M,Ge G,Yang Y,Cai X,Fu Q,Cai J,Huang Z.Decreasedantigenicityprofiles ofimmune-escaped and drug-resistant hepatitis B surfaceantigen(HBsAg)double mutants.Virol J 2013;10:292.

[0454] 7.Zanetti AR,Tanzi E,Manzillo G,Maio G,Sbreglia C,Caporaso N,ThomasH,et al.Hepatitis B variant in Europe.Lancet 1988;2:1132-1133.

[0455] 8.Roltgen K,Nielsen SCA,Silva O,Younes SF,Zaslavsky M,Costales C,YangF,et al.Immune imprinting,breadth ofvariant recognition,and germinalcenterresponse in human SARS-CoV-2 infection and vaccination.Cell2022;185:1025-1040e1014。

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to hepatitis B surface antigen (HBsAg), characterized in that The isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises complementarity determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region VL comprises complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences: a) CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, CDRH3 of SEQ ID NO:4, CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8; b) CDRH1 of SEQ ID NO: 10, CDRH2 of SEQ ID NO: 11, CDRH3 of SEQ ID NO: 12, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16; c) CDRH1 of SEQ ID NO: 18, CDRH2 of SEQ ID NO: 19, CDRH3 of SEQ ID NO: 20, CDRL1 of SEQ ID NO: 22, CDRL2 of SEQ ID NO: 23, and CDRL3 of SEQ ID NO: 24; d) CDRH1 of SEQ ID NO:26, CDRH2 of SEQ ID NO:27, CDRH3 of SEQ ID NO:28, CDRL1 of SEQ ID NO:30, CDRL2 of SEQ ID NO:31, and CDRL3 of SEQ ID NO:32; e) CDRH1 of SEQ ID NO:34, CDRH2 of SEQ ID NO:35, CDRH3 of SEQ ID NO:36, CDRL1 of SEQ ID NO:38, CDRL2 of SEQ ID NO:39, and CDRL3 of SEQ ID NO:40; f) CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:46, CDRL2 of SEQ ID NO:47, and CDRL3 of SEQ ID NO:48; g) CDRH1 of SEQ ID NO:50, CDRH2 of SEQ ID NO:51, CDRH3 of SEQ ID NO:52, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56; h) CDRH1 of SEQ ID NO:58, CDRH2 of SEQ ID NO:59, CDRH3 of SEQ ID NO:60, CDRL1 of SEQ ID NO:62, CDRL2 of SEQ ID NO:63, and CDRL3 of SEQ ID NO:64; i) CDRH1 of SEQ ID NO:66, CDRH2 of SEQ ID NO:67, CDRH3 of SEQ ID NO:68, CDRL1 of SEQ ID NO:70, CDRL2 of SEQ ID NO:71, and CDRL3 of SEQ ID NO:72; j) CDRH1 of SEQ ID NO:74, CDRH2 of SEQ ID NO:75, CDRH3 of SEQ ID NO:76, CDRL1 of SEQ ID NO:78, CDRL2 of SEQ ID NO:79, and CDRL3 of SEQ ID NO:80; k) CDRH1 of SEQ ID NO:82, CDRH2 of SEQ ID NO:83, CDRH3 of SEQ ID NO:84, CDRL1 of SEQ ID NO:86, CDRL2 of SEQ ID NO:87, and CDRL3 of SEQ ID NO:88; 1) CDRH1 of SEQ ID NO:90, CDRH2 of SEQ ID NO:91, CDRH3 of SEQ ID NO:92, CDRL1 of SEQ ID NO:94, CDRL2 of SEQ ID NO:95, and CDRL3 of SEQ ID NO:96; m) CDRH1 of SEQ ID NO:98, CDRH2 of SEQ ID NO:99, CDRH3 of SEQ ID NO:100, CDRL1 of SEQ ID NO:102, CDRL2 of SEQ ID NO:103, and CDRL3 of SEQ ID NO:104; n) CDRH1 of SEQ ID NO: 106, CDRH2 of SEQ ID NO: 107, CDRH3 of SEQ ID NO: 108, CDRL1 of SEQ ID NO: 110, CDRL2 of SEQ ID NO: 111, and CDRL3 of SEQ ID NO: 112; o) CDRH1 of SEQ ID NO: 114, CDRH2 of SEQ ID NO: 115, CDRH3 of SEQ ID NO: 116, CDRL1 of SEQ ID NO: 118, CDRL2 of SEQ ID NO: 119, and CDRL3 of SEQ ID NO: 120; p) CDRH1 of SEQ ID NO: 122, CDRH2 of SEQ ID NO: 123, CDRH3 of SEQ ID NO: 124, CDRL1 of SEQ ID NO: 126, CDRL2 of SEQ ID NO: 127, and CDRL3 of SEQ ID NO: 128; q) CDRH1 of SEQ ID NO: 130, CDRH2 of SEQ ID NO: 131, CDRH3 of SEQ ID NO: 132, CDRL1 of SEQ ID NO: 134, CDRL2 of SEQ ID NO: 135, and CDRL3 of SEQ ID NO: 136; r) CDRH1 of SEQ ID NO: 138, CDRH2 of SEQ ID NO: 139, CDRH3 of SEQ ID NO: 140, CDRL1 of SEQ ID NO: 142, CDRL2 of SEQ ID NO: 143, and CDRL3 of SEQ ID NO: 144; s) CDRH1 of SEQ ID NO: 146, CDRH2 of SEQ ID NO: 147, CDRH3 of SEQ ID NO: 148, CDRL1 of SEQ ID NO: 150, CDRL2 of SEQ ID NO: 151, and CDRL3 of SEQ ID NO: 152; t) CDRH1 of SEQ ID NO: 154, CDRH2 of SEQ ID NO: 155, CDRH3 of SEQ ID NO: 156, CDRL1 of SEQ ID NO: 158, CDRL2 of SEQ ID NO: 159, and CDRL3 of SEQ ID NO: 160; u) CDRH1 of SEQ ID NO: 162, CDRH2 of SEQ ID NO: 163, CDRH3 of SEQ ID NO: 164, CDRL1 of SEQ ID NO: 166, CDRL2 of SEQ ID NO: 167, and CDRL3 of SEQ ID NO: 168; v) CDRH1 of SEQ ID NO: 170, CDRH2 of SEQ ID NO: 171, CDRH3 of SEQ ID NO: 172, CDRL1 of SEQ ID NO: 174, CDRL2 of SEQ ID NO: 175, and CDRL3 of SEQ ID NO: 176; w) CDRH1 of SEQ ID NO: 178, CDRH2 of SEQ ID NO: 179, CDRH3 of SEQ ID NO: 180, CDRL1 of SEQ ID NO: 182, CDRL2 of SEQ ID NO: 183, and CDRL3 of SEQ ID NO: 184; x) CDRH1 of SEQ ID NO: 186, CDRH2 of SEQ ID NO: 187, CDRH3 of SEQ ID NO: 188, CDRL1 of SEQ ID NO: 190, CDRL2 of SEQ ID NO: 191, and CDRL3 of SEQ ID NO: 192; y) CDRH1 of SEQ ID NO: 194, CDRH2 of SEQ ID NO: 195, CDRH3 of SEQ ID NO: 196, CDRL1 of SEQ ID NO: 198, CDRL2 of SEQ ID NO: 199, and CDRL3 of SEQ ID NO: 200; z) CDRH1 of SEQ ID NO:202, CDRH2 of SEQ ID NO:203, CDRH3 of SEQ ID NO:204, CDRL1 of SEQ ID NO:206, CDRL2 of SEQ ID NO:207, and CDRL3 of SEQ ID NO:208; aa) CDRH1 of SEQ ID NO:210, CDRH2 of SEQ ID NO:211, CDRH3 of SEQ ID NO:212, CDRL1 of SEQ ID NO:214, CDRL2 of SEQ ID NO:215, and CDRL3 of SEQ ID NO:216; bb) CDRH1 of SEQ ID NO:218, CDRH2 of SEQ ID NO:219, CDRH3 of SEQ ID NO:220, CDRL1 of SEQ ID NO:222, CDRL2 of SEQ ID NO:223, and CDRL3 of SEQ ID NO:224; cc) CDRH1 of SEQ ID NO:226, CDRH2 of SEQ ID NO:227, CDRH3 of SEQ ID NO:228, CDRL1 of SEQ ID NO:230, CDRL2 of SEQ ID NO:231, and CDRL3 of SEQ ID NO:232; dd) CDRH1 of SEQ ID NO:234, CDRH2 of SEQ ID NO:235, CDRH3 of SEQ ID NO:236, CDRL1 of SEQ ID NO:238, CDRL2 of SEQ ID NO:239, and CDRL3 of SEQ ID NO:240; ee) CDRH1 of SEQ ID NO:242, CDRH2 of SEQ ID NO:243, CDRH3 of SEQ ID NO:244, CDRL1 of SEQ ID NO:246, CDRL2 of SEQ ID NO:247, and CDRL3 of SEQ ID NO:248; ff) CDRH1 of SEQ ID NO:250, CDRH2 of SEQ ID NO:251, CDRH3 of SEQ ID NO:252, CDRL1 of SEQ ID NO:254, CDRL2 of SEQ ID NO:255, and CDRL3 of SEQ ID NO:256; gg) CDRH1 of SEQ ID NO:258, CDRH2 of SEQ ID NO:259, CDRH3 of SEQ ID NO:260, CDRL1 of SEQ ID NO:262, CDRL2 of SEQ ID NO:263, and CDRL3 of SEQ ID NO:264; hh) CDRH1 of SEQ ID NO:266, CDRH2 of SEQ ID NO:267, CDRH3 of SEQ ID NO:268, CDRL1 of SEQ ID NO:270, CDRL2 of SEQ ID NO:271, and CDRL3 of SEQ ID NO:272; ii) CDRH1 of SEQ ID NO:274, CDRH2 of SEQ ID NO:275, CDRH3 of SEQ ID NO:276, CDRL1 of SEQ ID NO:278, CDRL2 of SEQ ID NO:279, and CDRL3 of SEQ ID NO:280; jj) CDRH1 of SEQ ID NO:282, CDRH2 of SEQ ID NO:283, CDRH3 of SEQ ID NO:284, CDRL1 of SEQ ID NO:286, CDRL2 of SEQ ID NO:287, and CDRL3 of SEQ ID NO:288; kk) CDRH1 of SEQ ID NO:290, CDRH2 of SEQ ID NO:291, CDRH3 of SEQ ID NO:292, CDRL1 of SEQ ID NO:294, CDRL2 of SEQ ID NO:295, and CDRL3 of SEQ ID NO:296; 11) CDRH1 of SEQ ID NO:298, CDRH2 of SEQ ID NO:299, CDRH3 of SEQ ID NO:300, CDRL1 of SEQ ID NO:302, CDRL2 of SEQ ID NO:303, and CDRL3 of SEQ ID NO:304; mm) CDRH1 of SEQ ID NO:306, CDRH2 of SEQ ID NO:307, CDRH3 of SEQ ID NO:308, CDRL1 of SEQ ID NO:310, CDRL2 of SEQ ID NO:311, and CDRL3 of SEQ ID NO:312; nn) CDRH1 of SEQ ID NO:314, CDRH2 of SEQ ID NO:315, CDRH3 of SEQ ID NO:316, CDRL1 of SEQ ID NO:318, CDRL2 of SEQ ID NO:319, and CDRL3 of SEQ ID NO:320; oo) CDRH1 of SEQ ID NO:322, CDRH2 of SEQ ID NO:323, CDRH3 of SEQ ID NO:324, CDRL1 of SEQ ID NO:326, CDRL2 of SEQ ID NO:327, and CDRL3 of SEQ ID NO:328; pp) CDRH1 of SEQ ID NO:330, CDRH2 of SEQ ID NO:331, CDRH3 of SEQ ID NO:332, CDRL1 of SEQ ID NO:334, CDRL2 of SEQ ID NO:335, and CDRL3 of SEQ ID NO:336; qq) CDRH1 of SEQ ID NO:338, CDRH2 of SEQ ID NO:339, CDRH3 of SEQ ID NO:340, CDRL1 of SEQ ID NO:342, CDRL2 of SEQ ID NO:343, and CDRL3 of SEQ ID NO:344; rr) CDRH1 of SEQ ID NO:346, CDRH2 of SEQ ID NO:347, CDRH3 of SEQ ID NO:348, CDRL1 of SEQ ID NO:350, CDRL2 of SEQ ID NO:351, and CDRL3 of SEQ ID NO:352; ss) CDRH1 of SEQ ID NO:354, CDRH2 of SEQ ID NO:355, CDRH3 of SEQ ID NO:356, CDRL1 of SEQ ID NO:358, CDRL2 of SEQ ID NO:359, and CDRL3 of SEQ ID NO:360; tt) CDRH1 of SEQ ID NO:362, CDRH2 of SEQ ID NO:363, CDRH3 of SEQ ID NO:364, CDRL1 of SEQ ID NO:366, CDRL2 of SEQ ID NO:367, and CDRL3 of SEQ ID NO:368; uu) CDRH1 of SEQ ID NO:370, CDRH2 of SEQ ID NO:371, CDRH3 of SEQ ID NO:372, CDRL1 of SEQ ID NO:374, CDRL2 of SEQ ID NO:375, and CDRL3 of SEQ ID NO:376; vv) CDRH1 of SEQ ID NO:378, CDRH2 of SEQ ID NO:379, CDRH3 of SEQ ID NO:380, CDRL1 of SEQ ID NO:382, CDRL2 of SEQ ID NO:383, and CDRL3 of SEQ ID NO:384; ww) CDRH1 of SEQ ID NO:386, CDRH2 of SEQ ID NO:387, CDRH3 of SEQ ID NO:388, CDRL1 of SEQ ID NO:390, CDRL2 of SEQ ID NO:391, and CDRL3 of SEQ ID NO:392; xx) CDRH1 of SEQ ID NO:394, CDRH2 of SEQ ID NO:395, CDRH3 of SEQ ID NO:396, CDRL1 of SEQ ID NO:398, CDRL2 of SEQ ID NO:399, and CDRL3 of SEQ ID NO:400; yy) CDRH1 of SEQ ID NO:402, CDRH2 of SEQ ID NO:403, CDRH3 of SEQ ID NO:404, CDRL1 of SEQ ID NO:406, CDRL2 of SEQ ID NO:407, and CDRL3 of SEQ ID NO:408; zz) CDRH1 of SEQ ID NO:410, CDRH2 of SEQ ID NO:411, CDRH3 of SEQ ID NO:412, CDRL1 of SEQ ID NO:414, CDRL2 of SEQ ID NO:415, and CDRL3 of SEQ ID NO:416; aaa) CDRH1 of SEQ ID NO:418, CDRH2 of SEQ ID NO:419, CDRH3 of SEQ ID NO:420, CDRL1 of SEQ ID NO:422, CDRL2 of SEQ ID NO:423, and CDRL3 of SEQ ID NO:424; bbb) CDRH1 of SEQ ID NO:426, CDRH2 of SEQ ID NO:427, CDRH3 of SEQ ID NO:428, CDRL1 of SEQ ID NO:430, CDRL2 of SEQ ID NO:431, and CDRL3 of SEQ ID NO:432; ccc) CDRH1 of SEQ ID NO:434, CDRH2 of SEQ ID NO:435, CDRH3 of SEQ ID NO:436, CDRL1 of SEQ ID NO:438, CDRL2 of SEQ ID NO:439, and CDRL3 of SEQ ID NO:440; ddd) CDRH1 of SEQ ID NO:442, CDRH2 of SEQ ID NO:443, CDRH3 of SEQ ID NO:444, CDRL1 of SEQ ID NO:446, CDRL2 of SEQ ID NO:447, and CDRL3 of SEQ ID NO:448; eee) CDRH1 of SEQ ID NO:450, CDRH2 of SEQ ID NO:451, CDRH3 of SEQ ID NO:452, CDRL1 of SEQ ID NO:454, CDRL2 of SEQ ID NO:455, and CDRL3 of SEQ ID NO:456; fff) CDRH1 of SEQ ID NO:458, CDRH2 of SEQ ID NO:459, CDRH3 of SEQ ID NO:460, CDRL1 of SEQ ID NO:462, CDRL2 of SEQ ID NO:463, and CDRL3 of SEQ ID NO:464; ggg) CDRH1 of SEQ ID NO:466, CDRH2 of SEQ ID NO:467, CDRH3 of SEQ ID NO:468, CDRL1 of SEQ ID NO:470, CDRL2 of SEQ ID NO:471, and CDRL3 of SEQ ID NO:472; hhh) CDRH1 of SEQ ID NO:474, CDRH2 of SEQ ID NO:475, CDRH3 of SEQ ID NO:476, CDRL1 of SEQ ID NO:478, CDRL2 of SEQ ID NO:479, and CDRL3 of SEQ ID NO:480; iii) CDRH1 of SEQ ID NO:482, CDRH2 of SEQ ID NO:483, CDRH3 of SEQ ID NO:484, CDRL1 of SEQ ID NO:486, CDRL2 of SEQ ID NO:487, and CDRL3 of SEQ ID NO:488; and jjj) CDRH1 of SEQ ID NO:490, CDRH2 of SEQ ID NO:491, CDRH3 of SEQ ID NO:492, CDRL1 of SEQ ID NO:494, CDRL2 of SEQ ID NO:495, and CDRL3 of SEQ ID NO:

496.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that The heavy chain variable region VH and light chain variable region VL are selected from the following amino acid sequences: a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5; b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 13; c) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 21; d) VH of SEQ ID NO:25 and VL of SEQ ID NO:29; e) VH of SEQ ID NO:33 and VL of SEQ ID NO:37; f) VH of SEQ ID NO:41 and VL of SEQ ID NO:45; g) VH of SEQ ID NO:49 and VL of SEQ ID NO:53; h) VH of SEQ ID NO:57 and VL of SEQ ID NO:61; i) VH of SEQ ID NO:65 and VL of SEQ ID NO:69; j) VH of SEQ ID NO:73 and VL of SEQ ID NO:77; k) VH of SEQ ID NO:81 and VL of SEQ ID NO:85; 1) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93; m) VH of SEQ ID NO:97 and VL of SEQ ID NO:101; n) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109; o) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117; p) VH of SEQ ID NO: 121 and VL of SEQ ID NO: 125; q) VH of SEQ ID NO: 129 and VL of SEQ ID NO: 133; r) VH of SEQ ID NO: 137 and VL of SEQ ID NO: 141; s) VH of SEQ ID NO: 145 and VL of SEQ ID NO: 149; t) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157; u) VH of SEQ ID NO: 161 and VL of SEQ ID NO: 165; v) VH of SEQ ID NO: 169 and VL of SEQ ID NO: 173; w) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181; x) VH of SEQ ID NO: 185 and VL of SEQ ID NO: 189; y) VH of SEQ ID NO: 193 and VL of SEQ ID NO: 197; z) VH of SEQ ID NO:201 and VL of SEQ ID NO:205; aa) VH of SEQ ID NO:209 and VL of SEQ ID NO:213; bb) VH of SEQ ID NO:217 and VL of SEQ ID NO:221; cc) VH of SEQ ID NO:225 and VL of SEQ ID NO:229; dd) VH of SEQ ID NO:233 and VL of SEQ ID NO:237; ee) VH of SEQ ID NO:241 and VL of SEQ ID NO:245; ff) VH of SEQ ID NO:249 and VL of SEQ ID NO:253; gg) VH of SEQ ID NO:257 and VL of SEQ ID NO:261; hh) VH of SEQ ID NO:265 and VL of SEQ ID NO:269; ii) VH of SEQ ID NO:273 and VL of SEQ ID NO:277; jj) VH of SEQ ID NO:283 and VL of SEQ ID NO:287; ) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285; kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293; ll) VH of SEQ ID NO: 297 and VL of SEQ ID NO: 301; mm) VH of SEQ ID NO: 305 and VL of SEQ ID NO: 309; nn) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317; oo) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325; pp) VH of SEQ ID NO: 329 and VL of SEQ ID NO: 333; qq) VH of SEQ ID NO: 337 and VL of SEQ ID NO: 341; rr) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349; ss) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357; tt) VH of SEQ ID NO: 354 and VL of SEQ ID NO: 357; NO:361 VH and SEQ ID NO:365 VL; uu) SEQ ID NO:369 VH and SEQ ID NO:373 VL; vv) SEQ ID NO:377 VH and SEQ ID NO:381 VL; ww) SEQ ID NO:385 VH and SEQ ID NO:389 VL; xx) SEQ ID NO:393 VH and SEQ ID NO:397 VL; yy) SEQ ID NO:401 VH and SEQ ID NO:405 VL; zz) SEQ ID NO:409 VH and SEQ ID NO:413 VL;aaa) VH of SEQ ID NO:417 and VL of SEQ ID NO:421; bbb) VH of SEQ ID NO:425 and VL of SEQ ID NO:429; ccc) VH of SEQ ID NO:433 and VL of SEQ ID NO:437; ddd) VH of SEQ ID NO:441 and VL of SEQ ID NO:445; eee) VH of SEQ ID NO:449 and VL of SEQ ID NO:453; fff) VH of SEQ ID NO:457 and VL of SEQ ID NO:461; ggg) VH of SEQ ID NO: 465 and VL of SEQ ID NO: 469; hhh) VH of SEQ ID NO:473 and VL of SEQ ID NO:477; iii) VH of SEQ ID NO: 481 and VL of SEQ ID NO: 485; and jjj) VH of SEQ ID NO:489 and VL of SEQ ID NO:

493.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The isolated antibody or antigen-binding fragment thereof specifically binds to an epitope in the S region of the extracellular domain of HBsAg.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The isolated antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acid residues 99-169 of the HBsAg protein shown in SEQ ID NO:

497.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The separated antibody or antigen-binding fragment thereof specifically binds to the HBsAg protein of wild-type HBV and its mutant strains.

6. The isolated antibody or antigen-binding fragment thereof according to claim 5, characterized in that The mutant strain comprises a mutation selected from the group consisting of G145R, K122R-G145R and F134L.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The isolated antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antibody fragment thereof.

8. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule.

9. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The isolated antibody or antigen-binding fragment thereof is of IgG1, IgG2, IgG3 or IgG4 type.

10. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9. An expression vector comprising the polynucleotide according to claim 10 . A host cell comprising the expression vector according to claim 11 .

13. A method for producing an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that The method comprises culturing the host cell according to claim 12 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof produced by the host cell.

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

15. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating or preventing HBV infection in a subject in need thereof.

16. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating or preventing HBV and its mutant strains infection in a subject in need thereof.

17. The use according to claim 16, characterized in that The mutant strain is a single or double site mutation contained in amino acid residues 99-169 of the HBsAg protein shown in SEQ ID NO:

497.

18. The use according to claim 16 or 17, characterized in that The mutant strain comprises a mutation selected from the group consisting of G145R, K122R-G145R and F134L.

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