Hepatitis b virus surface antibody and use thereof

Abstract

The present invention relates to an antibody against a hepatitis B surface antigen (HBsAg) and an antigen-binding fragment thereof, and methods for preparing and using the antibody and the antigen-binding fragment thereof. The present invention further relates to bispecific antibodies that specifically bind to different epitopes on HBsAg. The antibodies are derived from persons inoculated with hepatitis B vaccines and persons having recovered from hepatitis B infections, show superior effects in recognizing different hepatitis B surface antigens, and have greater advantages in neutralizing hepatitis B virus mutants and reducing hepatitis B mutant antigens.

Description

Hepatitis B virus surface antibody and its application Technical Field

[0001] The present invention relates to antibodies and antigen-binding fragments thereof targeting hepatitis B virus surface antigen (HBsAg), as well as methods for preparing and using the neutralizing antibodies and antigen-binding fragments thereof. Background Art

[0002] Acute and chronic infections caused by hepatitis B virus (HBV) are a global public health issue that poses a serious threat to human health. While the spread of the virus has been significantly controlled with the introduction of hepatitis B vaccination, my country still has the largest number of hepatitis B patients in the world. While current clinical treatments for chronic hepatitis B, including interferon and nucleoside / nucleotide analogs, can effectively inhibit HBV replication, they cannot completely eliminate the virus and thus cure chronic HBV infection, and drug resistance may also develop.

[0003] HBV is an enveloped DNA virus belonging to the Hepadnaviridae family. Currently, there are nine confirmed HBV genotypes (A-I) and one unidentified genotype (J), which are further subdivided into numerous subgenotypes with specific geographical distributions. The natural course of HBV infection varies depending on the HBV genotype, including the mode of transmission, the time to HBeAg seroconversion, the rate and severity of liver disease progression, such as hepatocellular carcinoma, and the likelihood of HBsAg clearance. HBsAg particles occur in three forms: large, medium, and small. Small HBsAg, encoded by 226 amino acids, is the primary component that elicits protective antibodies. The HBsAg protein has four transmembrane regions, including an extracellular domain of approximately 70 amino acids (amino acids 99-169). This extracellular domain is exposed on the surface of the viral particle and is crucial for viral infection of host cells. It also serves as a key site of recognition by the immune system, such as antibodies. Within this extracellular region, there is an immunodominant region called the "a" antigenic determinant, located between amino acids 124-147, between transmembrane regions 2 and 3. This is a key epitope recognized by the hepatitis B surface antibody (HBsAg). During chronic HBV infection, natural selection and the pressures of time and space for survival of the fittest lead to the emergence of a series of mutant strains. This "a" antigenic determinant is the region of HBsAg where mutations occur most frequently.

[0004] Hepatitis B surface antigen (HBsAg) can be derived from HBV cccDNA and / or integrated HBV DNA fragments. It not only participates in the process of forming complete viral particles as an HBV envelope protein, but also combines with host lipids to form non-infectious subviral particles, namely HBsAg particles, which exist freely in the blood of infected individuals, forming surface antigenemia. A growing number of studies have shown that the high presence of HBsAg in patients with chronic hepatitis B may be the cause of abnormal or exhausted immune responses and may affect the pathobiology of liver disease and the development of cancer. Therefore, reducing or eliminating HBsAg not only prevents HBV infection but also helps achieve a "functional cure," thereby reducing the incidence of complications (cirrhosis and hepatocellular carcinoma). Using monoclonal antibodies against the HBsAg protein (anti-HBs) to reduce or eliminate HBsAg in the human body is a clinical strategy for "functional cure" of HBV that is highly worthy of exploration and development.

[0005] Currently, the most cost-effective way to prevent and treat hepatitis B is vaccination. Hepatitis B surface antibodies (anti-HBs) produced after vaccination are protective antibodies against hepatitis B. Since China implemented the newborn vaccination policy in 1992, the HBV antigen-positivity rate in the population has declined annually. As of 2020, the HBV surface antigen-positivity rate in my country was approximately 6%. However, due to the large population base, there are still 86 million people infected with HBV in my country, with approximately one million new cases each year.

[0006] Antibodies are secreted by plasma cells differentiated from B lymphocytes. Each B lymphocyte can only produce antibodies specific to a specific antibody determinant, 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 continued to improve. Many studies have used different technologies to obtain anti-HBs monoclonal antibodies using peripheral blood B cells of vaccine-immunized individuals, such as 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 on the "a" determinant 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 potency against hepatitis B virus can produce anti-HBs monoclonal antibodies with high in vitro neutralization potency and can effectively inhibit the proliferation of hepatitis B virus in vivo (1,2). Multiple naturally humanized anti-HBs were recombinantly expressed, and most exhibited neutralizing efficacy in in vitro experiments. Notably, in in vivo infection experiments in human liver chimeric mice, significant viral escape occurred after several weeks of single-antibody treatment. Treatment with complementary monoclonal antibodies that recognize distinct epitopes more effectively suppressed viremia and prevented viral escape in mice. These results demonstrate that anti-HBs isolated from hepatitis B vaccine recipients block HBV infection and inhibit viral replication in both in vitro and in vivo experimental models.

[0007] Although hepatitis B virus is a DNA virus, its replication process involves an intermediate process through RNA reverse transcriptase, and DNA polymerase does not have the ability to self-correct errors. Compared with other DNA viruses, it is more prone to genomic nucleotide mutations, generating HBV mutants in this process. Most of these mutated new sequences cannot effectively compete with wild-type viruses or are non-viable, but when there is selective pressure, these new sequences provide a starting point for the emergence of mutants. When talking about viral mutants, we usually refer to RNA viruses such as the new coronavirus or influenza virus, which can produce large and rapid mutations. The difference is that HBV has four partially overlapping open reading frames (ORFs) on its circular DNA genome, which can encode seven proteins. Because gene overlap limits the variability of the virus, mutants or variant forms have been identified for four genes. HBV has a certain tendency to mutate. The "a" determinant cluster on HBsAg is a region with a high probability of mutation. The human immune response to HBsAg is also mainly targeted at 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 protein's spatial structure and characteristics (conformational epitope), it will cause the antibody to be unable to neutralize the viral infection and may also affect diagnostic detection. 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 that prevents 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 produce high-titer hepatitis B immunoglobulin. However, blood-derived HBIg also presents several challenges. For example, the raw material required for HBIg is high-titer anti-HBs plasma, limiting its production and scale of use. Furthermore, due to individual differences in plasma supply between batches, product quality and efficacy can vary between batches. In particular, only a portion of the antibodies in concentrated HBIg may be effective in neutralizing HBV. Despite strict national regulations on blood products, testing of various indicators for "healthy blood donors" remains limited. From a biosafety perspective, HBIg's safety profile is poor, and the clinical dose is limited to 200 IU. Finally, patients receiving HBIg injections may experience allergic reactions, such as IgG4 allergies. For immunosuppressed patients, such as those undergoing liver transplantation, adverse symptoms such as nausea, urticaria, and arthralgia may also occur. The clinical use of HBIg to block HBV transmission demonstrates the feasibility of anti-HBs in preventing HBV infection. However, these challenges highlight the urgent need for the development of highly effective and safe anti-HBs that can be engineered for mass production and ideally block mutant strains.

[0009] In recent years, many laboratories at home and abroad have used biotechnology to isolate peripheral blood cells from hepatitis B vaccine recipients and then clone human antibodies from these B cells. However, these methods suffer from a single source and a lack of coverage for diverse genotypes or clinical variants. Therefore, there is a continuing need in this field for hepatitis B surface antibodies with improved performance.

[0010] Summary of the Invention

[0011] In the present invention, the inventors screened individuals who had recovered from hepatitis B infection and those who had received the hepatitis B vaccine for high serum neutralizing activity and, by isolating single B cells from these individuals, obtained a series of monoclonal antibodies targeting HBsAg. Both in vitro and in vivo experiments demonstrated that these monoclonal antibodies possessed excellent antigen-binding capacity, in vitro neutralization potency, and broad recognition of various mutant strains. Furthermore, by comparing antibodies from hepatitis B vaccine recipients, the inventors discovered that antibodies from recovered hepatitis B infection possessed a wider range of mutant recognition and neutralization properties. In vivo experiments in mice further confirmed that this series of antibodies possessed excellent in vivo antiviral efficacy, thus completing the present invention.

[0012] The present invention provides 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:

[0013] 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;

[0014] 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;

[0015] 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;

[0016] 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;

[0017] 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;

[0018] 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;

[0019] 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;

[0020] 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;

[0021] 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;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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;

[0028] 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;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] 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;

[0040] 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;

[0041] 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;

[0042] 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;

[0043] 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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;

[0050] 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;

[0051] 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;

[0052] 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;

[0053] 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;

[0054] 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;

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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;

[0065] 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;

[0066] 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;

[0067] 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;

[0068] 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;

[0069] 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;

[0070] 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;

[0071] 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;

[0072] 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;

[0073] 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

[0074] 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.

[0075] In one embodiment, the heavy chain variable region VH and light chain variable region VL are selected from the following amino acid sequences:

[0076] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0077] b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 13;

[0078] c) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 21;

[0079] d) VH of SEQ ID NO: 25 and VL of SEQ ID NO: 29;

[0080] e) VH of SEQ ID NO: 33 and VL of SEQ ID NO: 37;

[0081] f) VH of SEQ ID NO: 41 and VL of SEQ ID NO: 45;

[0082] g) VH of SEQ ID NO: 49 and VL of SEQ ID NO: 53;

[0083] h) VH of SEQ ID NO: 57 and VL of SEQ ID NO: 61;

[0084] i) VH of SEQ ID NO: 65 and VL of SEQ ID NO: 69;

[0085] j) VH of SEQ ID NO: 73 and VL of SEQ ID NO: 77;

[0086] k) VH of SEQ ID NO: 81 and VL of SEQ ID NO: 85;

[0087] 1) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0088] m) VH of SEQ ID NO: 97 and VL of SEQ ID NO: 101;

[0089] n) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0090] o) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0091] p) VH of SEQ ID NO: 121 and VL of SEQ ID NO: 125;

[0092] q) VH of SEQ ID NO: 129 and VL of SEQ ID NO: 133;

[0093] r) VH of SEQ ID NO: 137 and VL of SEQ ID NO: 141;

[0094] s) VH of SEQ ID NO: 145 and VL of SEQ ID NO: 149;

[0095] t) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0096] u) VH of SEQ ID NO: 161 and VL of SEQ ID NO: 165;

[0097] v) VH of SEQ ID NO: 169 and VL of SEQ ID NO: 173;

[0098] w) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0099] x) VH of SEQ ID NO: 185 and VL of SEQ ID NO: 189;

[0100] y) VH of SEQ ID NO: 193 and VL of SEQ ID NO: 197;

[0101] z) VH of SEQ ID NO: 201 and VL of SEQ ID NO: 205;

[0102] aa) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0103] bb) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0104] cc) VH of SEQ ID NO: 225 and VL of SEQ ID NO: 229;

[0105] dd) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0106] ee) VH of SEQ ID NO: 241 and VL of SEQ ID NO: 245;

[0107] ff) VH of SEQ ID NO: 249 and VL of SEQ ID NO: 253;

[0108] gg) VH of SEQ ID NO: 257 and VL of SEQ ID NO: 261;

[0109] hh) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0110] ii) VH of SEQ ID NO: 273 and VL of SEQ ID NO: 277;

[0111] jj) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285;

[0112] kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293;

[0113] 11) VH of SEQ ID NO: 297 and VL of SEQ ID NO: 301;

[0114] mm) VH of SEQ ID NO: 305 and VL of SEQ ID NO: 309;

[0115] nn) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317;

[0116] oo) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325;

[0117] pp) VH of SEQ ID NO: 329 and VL of SEQ ID NO: 333;

[0118] qq) VH of SEQ ID NO: 337 and VL of SEQ ID NO: 341;

[0119] rr) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349;

[0120] ss) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357;

[0121] tt) VH of SEQ ID NO: 361 and VL of SEQ ID NO: 365;

[0122] uu) VH of SEQ ID NO: 369 and VL of SEQ ID NO: 373;

[0123] vv) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0124] ww) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389;

[0125] xx) VH of SEQ ID NO: 393 and VL of SEQ ID NO: 397;

[0126] yy) VH of SEQ ID NO: 401 and VL of SEQ ID NO: 405;

[0127] zz) VH of SEQ ID NO: 409 and VL of SEQ ID NO: 413;

[0128] aaa) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421;

[0129] bbb) VH of SEQ ID NO: 425 and VL of SEQ ID NO: 429;

[0130] ccc) VH of SEQ ID NO: 433 and VL of SEQ ID NO: 437;

[0131] ddd) VH of SEQ ID NO: 441 and VL of SEQ ID NO: 445;

[0132] eee) VH of SEQ ID NO: 449 and VL of SEQ ID NO: 453;

[0133] fff) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461;

[0134] ggg) VH of SEQ ID NO: 465 and VL of SEQ ID NO: 469;

[0135] hhh) VH of SEQ ID NO: 473 and VL of SEQ ID NO: 477;

[0136] iii) VH of SEQ ID NO: 481 and VL of SEQ ID NO: 485; and

[0137] jjj) VH of SEQ ID NO: 489 and VL of SEQ ID NO: 493.

[0138] 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.

[0139] 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.

[0140] In one embodiment, the isolated antibodies or antigen-binding fragments thereof described herein are expressed at 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 methods described herein. 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] In one embodiment, the isolated antibody or antigen-binding fragment thereof described herein is of the IgG1, IgG2, IgG3 or IgG4 type.

[0145] In one embodiment, the isolated antibodies or antigen-binding fragments thereof described herein are bispecific or multispecific.

[0146] The present invention also provides a bispecific antibody comprising a first domain that specifically binds to a first epitope on hepatitis B surface antigen (HBsAg) and a second domain that specifically binds to a second epitope on HBsAg, wherein the first epitope and the second epitope are each independently selected from:

[0147] a) an epitope comprising amino acid residues 110I, 122K, 125T, 134F, 141K, and 165W;

[0148] b) an epitope comprising amino acid residues 122K, 123T, 145G, 161Y, and 165W; and

[0149] c) an epitope comprising amino acid residues 109L-111P, 120P-130G, 133M-136S, 144D, 146N, 153P, 161Y and 164E-167S,

[0150] The amino acid residue numbers are based on the amino acid positions shown in SEQ ID NO: 497.

[0151] wherein the first epitope is different from the second epitope.

[0152] In one embodiment, the first domain 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:

[0153] a) 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;

[0154] b) 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;

[0155] c) 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;

[0156] d) 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; and

[0157] e) 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;

[0158] The second domain 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:

[0159] a) 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;

[0160] b) 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;

[0161] c) 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;

[0162] d) 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; and

[0163] e) 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.

[0164] In one embodiment, the first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences:

[0165] a) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0166] b) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0167] c) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0168] d) VH of SEQ ID NO: 433 and VL of SEQ ID NO: 437; and

[0169] e) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461,

[0170] or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences,

[0171] The second domain 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:

[0172] a) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0173] b) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0174] c) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0175] d) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285; and

[0176] e) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317,

[0177] Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences.

[0178] In one embodiment, the first domain 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:

[0179] a) 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;

[0180] b) 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;

[0181] c) 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;

[0182] d) 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; and

[0183] e) 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;

[0184] The second domain 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:

[0185] 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;

[0186] b) 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;

[0187] c) 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;

[0188] d) 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;

[0189] e) 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;

[0190] f) 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;

[0191] g) 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;

[0192] j) 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;

[0193] i) 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;

[0194] j) 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;

[0195] k) 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; and

[0196] 1) 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.

[0197] In one embodiment, the first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences:

[0198] a) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0199] b) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0200] c) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0201] d) VH of SEQ ID NO: 433 and VL of SEQ ID NO: 437; and

[0202] e) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461,

[0203] or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences,

[0204] The second domain 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:

[0205] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0206] b) VH of SEQ ID NO: 49 and VL of SEQ ID NO: 53;

[0207] c) VH of SEQ ID NO: 81 and VL of SEQ ID NO: 85;

[0208] d) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0209] e) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0210] f) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0211] g) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325;

[0212] h) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349;

[0213] i) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357;

[0214] j) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0215] k) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389; and

[0216] 1) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421,

[0217] Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences.

[0218] In one embodiment, the first domain 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:

[0219] a) 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;

[0220] b) 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;

[0221] c) 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;

[0222] d) 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; and

[0223] e) 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;

[0224] The second domain 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:

[0225] 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;

[0226] b) 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;

[0227] c) 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;

[0228] d) 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;

[0229] e) 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;

[0230] f) 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;

[0231] g) 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;

[0232] h) 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;

[0233] i) 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;

[0234] j) 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;

[0235] k) 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; and

[0236] 1) 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.

[0237] In one embodiment, the first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences:

[0238] a) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0239] b) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0240] c) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0241] d) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285; and

[0242] e) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317,

[0243] or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences,

[0244] The second domain 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:

[0245] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0246] b) VH of SEQ ID NO: 49 and VL of SEQ ID NO: 53;

[0247] c) VH of SEQ ID NO: 81 and VL of SEQ ID NO: 85;

[0248] d) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0249] e) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0250] f) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0251] g) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325;

[0252] h) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349;

[0253] i) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357;

[0254] j) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0255] k) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389; and

[0256] 1) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421,

[0257] Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the above groups of sequences.

[0258] In a specific embodiment, the first domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises 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; and the second domain comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises 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.

[0259] In another specific embodiment, the first domain comprises the VH of SEQ ID NO:281 and the VL of SEQ ID NO:285, and the second domain comprises the VH of SEQ ID NO:49 and the VL of SEQ ID NO:53.

[0260] In one embodiment, the first domain and / or the second domain is of the IgG1, IgG2, IgG3 or IgG4 isotype.

[0261] In one embodiment, the first domain is an IgG full-length antibody and the second domain is a scFv; or the first domain is a scFv and the second domain is an IgG full-length antibody.

[0262] In one embodiment, the N-terminus of the scFv is linked to the C-terminus of at least one heavy chain of an IgG full-length antibody; or the N-terminus of the scFv is linked to the C-terminus of at least one light chain of an IgG full-length antibody.

[0263] In one embodiment, the first domain and the second domain are connected by a flexible peptide linker, preferably the flexible peptide linker has a length of no more than about 30 amino acids, more preferably the flexible peptide linker is (G4S) 3. In one embodiment, scFv is connected to the IgG full-length antibody via a flexible peptide linker.

[0264] In one embodiment, the bispecific antibody is CrossMAb-Fab.

[0265] The present invention also provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof described herein.

[0266] The present invention also provides an expression vector comprising the polynucleotide described herein.

[0267] The present invention also provides a host cell, comprising the expression vector described herein.

[0268] 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.

[0269] The present invention also provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof or bispecific antibody described herein and a pharmaceutically acceptable carrier.

[0270] The present invention also provides use of the isolated antibody or antigen-binding fragment thereof or bispecific antibody in the preparation of a medicament for treating or preventing HBV infection in a subject in need thereof.

[0271] The present invention also provides use of the isolated antibody or antigen-binding fragment thereof or bispecific antibody in the preparation of a medicament for treating or preventing HBV and its mutant strains infection in a subject in need thereof.

[0272] In certain aspects, the present invention provides methods for treating or preventing HBV infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one isolated antibody or antigen-binding fragment thereof or bispecific antibody disclosed herein.

[0273] In certain aspects, the present invention provides a method for treating or preventing HBV and its mutants infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one isolated antibody or antigen-binding fragment thereof or bispecific antibody disclosed herein.

[0274] In certain aspects, the present invention provides an isolated antibody or antigen-binding fragment thereof or a bispecific antibody disclosed herein for use in treating or preventing HBV infection in a subject in need thereof.

[0275] In certain aspects, the present invention provides an isolated antibody or antigen-binding fragment thereof or a bispecific antibody disclosed herein for use in treating or preventing HBV and its mutants infection in a subject in need thereof.

[0276] In one embodiment of the above aspects, the HBV includes genotype A, genotype B, genotype C and genotype D.

[0277] In certain aspects, the present invention provides a hepatitis B virus surface antibody or an antigen-binding fragment thereof, which 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. In one embodiment, the epitope is located in amino acid residues 124-147 of the S region of HBsAg. In one embodiment, the HBsAg has the amino acid sequence shown in SEQ ID NO: 497. In one embodiment, the above amino acid residue numbering is based on the amino acid positions 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

[0278] The present invention will be better understood with reference to the accompanying drawings.

[0279] FIG1 shows a schematic diagram of the structure of the extracellular domain of HBsAg, illustrating the configuration of amino acid residues 99-169.

[0280] Figure 2 shows the antibody responses of recovered hepatitis B virus (HBV) patients and recipients of the HBV vaccine. Neutralization of HBV was assessed in an HBV cell infection model in the presence of different serum dilutions.

[0281] Figure 3 shows the gating strategy for cell sorting. Double-positive (bait protein-APC+ and bait protein-PE+) cells were single-cell sorted for antibody cloning.

[0282] Figure 4 shows the results of flow cytometry for B cell sorting using different donor specificities, showing the frequency of B lymphocytes recognizing HBsAg in a control donor (low anti-HBs level) from a hepatitis B vaccine recipient and selected recovered and vaccinated individuals. Representative flow cytometric results show binding to biotinylated HBsAg labeled with phycocyanin (APC) and phycoerythrin (PE), respectively, for the dual fluorescence-dye sorting strategy.

[0283] FIG5 shows the area under the curve (AUC) values ​​of antibody binding to HBsAg protein ELISA.

[0284] Figure 6 shows the neutralizing activity of antibodies evaluated in a HBV cell infection model. In this model based on in vitro HBV infection in HepG2-NTCP cells, the levels of HBV antigens—HBsAg and HBeAg—in the cell supernatant represent the neutralizing potency of human monoclonal antibodies.

[0285] Figure 7 shows a competitive ELISA and the three major hepatitis B surface antibody groupings. This competitive ELISA validates three non-competing epitopes on HBsAg. The primary antibody is an unlabeled HBsAg-binding antibody and is used to block the epitope on the coated HBsAg. The secondary antibody is a biotinylated HBsAg antibody for streptavidin-HRP-dependent detection. H019 serves as the control antibody for Group I, H017 as the control antibody for Group III, and H007 as the control antibody for Group II. PBS buffer was used as a normalization reference in place of the primary blocking antibody.

[0286] Figure 8 shows the results of the antibody amino acid scan. Consistent with the competitive ELISA results, the four groups of antibodies each showed binding levels to HBsAg at specific mutation sites. The level of binding to wild-type HBsAg for each antibody served as a normalization reference for binding to other HBsAg mutants, with PBS serving as a negative control.

[0287] Figure 9 shows the activity of antibodies against different genotypes (genotype A, genotype B, and genotype C) in an in vitro model of HBV neutralization. The level of HBsAg, the HBV antigen, in the cell supernatant represents the neutralizing potency of the human monoclonal antibody. Antibodies that can neutralize HBV are shown here.

[0288] Figure 10 shows the results of antibody binding to different HBV mutants. The binding of antibodies to different HBsAg mutants produced by transfected cells was tested by ELISA. The level of each antibody binding to wild-type HBsAg was used as a reference for normalization of its binding to other HBsAg mutants. PBS was used as a negative control.

[0289] FIG11 shows the neutralizing activity against HBV mutant strains (HBV-G145R, HBV-K122R G145R and F134L) viruses.

[0290] FIG12 shows an exemplary design scheme for a bispecific antibody.

[0291] FIG13 shows the results of physicochemical property testing (A) and SDS PAGE (B) of exemplary bispecific antibodies.

[0292] FIG14 shows the OD values ​​of exemplary bispecific antibodies binding to HBsAg protein ELISA.

[0293] Figure 15 shows the evaluation of the neutralizing activity of exemplary bispecific antibodies in an HBV cell infection model. In a cell model based on in vitro HBV infection in HepG2-NTCP cells, the levels of HBV antigens - HBsAg and HBeAg in the cell supernatant represent the neutralizing potency of the antibody. DETAILED DESCRIPTION

[0294] Disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to HBsAg. In certain embodiments, the antibodies or antigen-binding fragments thereof bind to an epitope within the extracellular domain of HBsAg. In certain embodiments, the antibodies or antigen-binding fragments thereof have excellent antigen-binding capacity, in vitro neutralization potency, and broad recognition of various mutant strains. In certain embodiments, the antibodies are human monoclonal antibodies.

[0295] 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.

[0296] 1. Definition

[0297] It should be understood that the foregoing general description and the following detailed description are illustrative and explanatory only 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. Furthermore, the use of the term "include," as well as other forms such as "comprising," is not limiting and encompasses the term "consisting of."

[0298] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 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 for any purpose.

[0299] The term "hepatitis B surface antigen (HBsAg)" refers to the surface antigen contained within the outer shell of the hepatitis B virus (HBV). This antigen is crucial for viral infection of host cells and serves as a key site of recognition by the immune system (e.g., antibodies). The HBsAg protein contains four transmembrane regions, including an extracellular domain of approximately 70 amino acids within the S region, the structure of which is shown in Figure 1. The full-length amino acid sequence of the HBsAg protein is shown in SEQ ID NO:497, with the extracellular domain of amino acid residues 99-169 underlined.

[0300] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such 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, diselenphosphates, and phosphoramidates. The term "oligonucleotide" refers to a polynucleotide comprising 200 or fewer nucleotides. In certain embodiments, an oligonucleotide is 10-60 bases long. In other embodiments, an oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. An oligonucleotide can be single-stranded or double-stranded, such as for constructing a mutant gene. An oligonucleotide can 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.

[0301] The term "vector" is intended to refer to any molecule or entity (eg, a nucleic acid, plasmid, phage, or virus) used to transfer protein coding information into a host cell.

[0302] The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing nucleic acid sequences that direct and / or regulate the expression of one or more operably linked heterologous coding regions. Expression constructs may include, but are not limited to, sequences that affect or regulate transcription and translation, and, if introns are present, sequences that affect RNA splicing of the operably linked coding region.

[0303] As used herein, "operably linked" means that the components to which the term is applied 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.

[0304] The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a gene of interest. Regardless of whether the offspring is identical in morphology or genetic makeup to the original mother cell, the term includes the offspring of the mother cell as long as the gene of interest is present in the offspring.

[0305] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell. A cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Various 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.

[0306] The term "transformation" refers to a change in the genetic characteristics of a cell. A cell is transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed when its native state is genetically modified by the introduction of new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombinate with the cell's DNA by physically integrating into the cell's chromosomes, may be temporarily retained as an extrachromosomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA replicates as the cell divides.

[0307] The term "amino acid" includes its ordinary meaning in the art. Conventional single-letter and three-letter amino acid codes are used herein, as shown in Table 1.

[0308] Table 1

[0309] 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-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, 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.

[0310] 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.

[0311] 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 the sequences. "Percent identity" 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, eds.), 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., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0312] When calculating 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 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 for which percent identity is to be determined. 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 typically 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).

[0313] 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, supra; Gap penalty: 12 (but no penalty for terminal gaps); Gap length penalty: 4; Similarity threshold: 0.

[0314] Certain alignment designs for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and even if there is no significant relationship between the two full-length sequences, this small alignment region may have a very high sequence identity. Therefore, if it is desired to generate an alignment that spans at least 50 or other numbers of contiguous amino acids of the target polypeptide, the selected alignment method (GAP program) may be adjusted.

[0315] The term "derivative" refers to a molecule that has been chemically modified, including but not limited to insertions, deletions, or substitutions 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 moieties. In certain embodiments, chemically modified antigen-binding proteins may have a longer circulating half-life than antigen-binding proteins that have not been chemically modified. In certain embodiments, chemically modified antigen-binding proteins may improve their ability to target desired cells, tissues, and / or organs. In certain embodiments, the derivatized antigen-binding proteins are covalently modified to include one or more water-soluble polymeric linkers, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. In certain embodiments, the derivatized 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-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., ethylene glycol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0316] In certain embodiments, derivatives are covalently modified with polyethylene glycol (PEG) subunits. 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 these methods are discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0317] 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, fully human antibodies, and bispecific antibodies. "Antibody" is a class of antigen-binding proteins. A complete antibody will typically contain at least two full-length heavy chains and two full-length light chains, but may include fewer chains in certain cases, for example, antibodies naturally occurring in camels may contain only heavy chains. Antibodies may be derived from only a single source, or may be "chimeric", meaning that 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, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, and fragments thereof. Furthermore, unless expressly 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.

[0318] Naturally occurring antibody structural units typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having a full-length "light chain" (in certain embodiments, approximately 25 kDa) and a full-length "heavy chain" (in certain embodiments, approximately 50-70 kDa). The amino-terminal portion of each chain typically comprises a variable region of approximately 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 isotype of the antibody is 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 further divided 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, for example, 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 the antigen-binding site.

[0319] 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.

[0320] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from each pair of chains are typically positioned by the framework regions, which allow for binding to specific epitopes. The two light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus. The amino acid assignments to each domain are generally 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 for 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, the CDR sequences referred to in this disclosure are numbered and defined according to IMGT.

[0321] 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.

[0322] 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 of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0323] 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, and 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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 form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0328] "F(ab')2 fragment" contains two light chains and a C H 1 and C H The F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

[0329] An "Fv fragment" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0330] "Single-chain antibodies (scFv)" are Fv molecules in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain, thereby forming the 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.

[0331] "Monoclonal antibody" refers to a homogeneous population of antibodies with a single molecular composition. Monoclonal antibodies can be nonspecific or polyspecific.

[0332] "Domain antibodies" are immunologically functional immunoglobulin fragments containing 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 with a peptide linker to form a bivalent domain antibody. H The regions may target the same or different antigens.

[0333] "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. Bispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, for example cynomolgus (cyno) or chimpanzees, or may bind to epitopes shared between two or more different antigens. Bispecific antibodies can be monovalent or divalent with respect to their binding to different antigens or epitopes.

[0334] "Valency" refers to the presence of a specified number of binding sites in a molecule that are specific for an antigen. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites in a molecule that are specific for an antigen, respectively. "Multivalent" refers to the presence of two or more binding sites in a molecule that are specific for an antigen.

[0335] An antibody is "selective" when it binds one target more tightly than it binds a second target.

[0336] "Recombinant antibodies" are antibodies produced using recombinant technology. Methods and techniques for producing recombinant antibodies are well known in the art.

[0337] 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 linking Fab' fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0338] Each individual immunoglobulin chain is typically composed of several "immunoglobulin domains," each of which consists 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 typically contains one or more domains that may 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 heavy chains called C H 1. C H 2 and C H The provided antibodies may be of any of these isotypes and subtypes.

[0339] The term "neutralize" 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 antigen-binding fragment thereof, 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 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 an in vitro competitive binding assay). In certain embodiments, neutralizing ability is characterized and / or described via competition assays. In certain embodiments, neutralizing ability is measured as a 50% inhibitory concentration (IC 50 ) value to describe.

[0340] The term "target" refers to a molecule or portion of a molecule that can be bound by an antibody or antigen-binding fragment thereof. 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 that the protein sequence comprises an antigen that can be bound by an antibody. In this case, the protein is not necessarily foreign or capable of inducing an immune response.

[0341] 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 an assay in which 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 a domain thereof). 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 (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), 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 label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Manual).

[0014] The present invention relates to a method for the determination of the competitive inhibition of antigens by direct solid-phase labeling of antigens with the aid of a direct solid-phase labeling of antigens (e.g., Immunol., 1984, pp. 106-114); direct solid-phase biotin-avidin EIA (e.g., Cheung, et al., 1990, Virology 176:546-552); and direct 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 cells bearing either an unlabeled detection antibody or a labeled reference antibody. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antibody being tested. Typically, the antibody being tested is present in excess. Typically, when a competing antibody is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antibody to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0342] 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.

[0343] 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 targeting the antigen. When the antigen is a protein, it contains specific amino acids that are in direct contact with the antibody. In most cases, the epitope is located on the protein, but in some cases, it 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. An epitope can be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are close in three-dimensional space due to the folding of the protein molecule. The antibody "epitope" depends on the method used to identify the epitope. Unless otherwise specified, the present application uses a competitive ELISA combined with alanine scanning to identify antibody epitopes.

[0344] The term "therapeutically effective amount" refers to the amount of the antibody or antigen-binding fragment thereof of the present invention that is determined to produce a therapeutic response in a mammal. The therapeutically effective amount can be easily determined by a person skilled in the art.

[0345] 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.

[0346] 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.

[0347] The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring is reduced in the presence of the antibody or method.

[0348] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and conversion (e.g., electroporation, lipofection). Enzyme reactions and purification techniques can be implemented according to manufacturer specifications, or can be completed or carried out as described herein according to methods generally used in the art. Conventionally, conventional methods well known in the art, as well as various general and more specific references cited and discussed in this specification, can be used to implement the aforementioned techniques and methods. 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 incorporated herein by reference for any purpose. Unless clearly defined, the terms and experimental techniques and techniques used with analytical chemistry, synthetic organic chemistry, medicine, and pharmaceutical chemistry as 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, delivery, and treatment of patients.

[0349] 2. Antibodies of the present invention and methods for preparing the same

[0350] 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 discovered that antibodies cloned from donors who have recovered from HBV infection demonstrate superior efficacy in recognizing different HBV surface antigens, and are more advantageous in neutralizing HBV mutants and reducing HBV mutant antigen activity, compared to those from donors who have received the HBV vaccine.

[0351] In some embodiments, the antibodies described herein are expressed at a 50% inhibitory concentration (IC) of less than 0.5 μg / ml. 50 ) values ​​for in vitro neutralization of 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 ) values ​​for in vitro neutralization of 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.

[0352] The present invention relates to isolated antibodies or antigen-binding fragments thereof against HBV and its mutants. In one embodiment, the present invention relates to isolated antibodies or antigen-binding fragments thereof that bind to HBsAg protein. In another embodiment, the present invention relates to isolated antibodies or antigen-binding fragments thereof that bind to an epitope in the extracellular domain of HBsAg. In another embodiment, the present invention relates to isolated antibodies or antigen-binding fragments thereof that bind to an epitope in amino acid residues 99-169 of HBsAg. In another embodiment, the present invention relates to isolated antibodies or antigen-binding fragments thereof that bind to an epitope in amino acid residues 124-147 of HBsAg.

[0353] In one embodiment, the HBsAg protein comprises a single or double mutation. In one embodiment, the mutation is selected from the group consisting of 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.

[0354] In one aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to 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:

[0355] 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;

[0356] 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;

[0357] 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;

[0358] 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;

[0359] 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;

[0360] 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;

[0361] 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;

[0362] 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;

[0363] 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;

[0364] 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;

[0365] 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;

[0366] 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;

[0367] 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;

[0368] 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;

[0369] 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;

[0370] 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;

[0371] 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;

[0372] 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;

[0373] 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;

[0374] 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;

[0375] 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;

[0376] 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;

[0377] 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;

[0378] 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;

[0379] 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;

[0380] 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;

[0381] 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;

[0382] 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;

[0383] 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;

[0384] 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;

[0385] 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;

[0386] 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;

[0387] 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;

[0388] 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;

[0389] 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;

[0390] 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;

[0391] 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;

[0392] 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;

[0393] 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;

[0394] 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;

[0395] 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;

[0396] 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;

[0397] 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;

[0398] 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;

[0399] 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;

[0400] 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;

[0401] 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;

[0402] 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;

[0403] 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;

[0404] 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;

[0405] 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;

[0406] 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;

[0407] 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;

[0408] 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;

[0409] 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;

[0410] 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;

[0411] 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;

[0412] 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;

[0413] 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;

[0414] 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;

[0415] 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

[0416] 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.

[0417] 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.

[0418] In one aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to 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 and the light chain variable region VL are selected from the following amino acid sequences:

[0419] a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 5;

[0420] b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 13;

[0421] c) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 21;

[0422] d) VH of SEQ ID NO: 25 and VL of SEQ ID NO: 29;

[0423] e) VH of SEQ ID NO: 33 and VL of SEQ ID NO: 37;

[0424] f) VH of SEQ ID NO: 41 and VL of SEQ ID NO: 45;

[0425] g) VH of SEQ ID NO: 49 and VL of SEQ ID NO: 53;

[0426] h) VH of SEQ ID NO: 57 and VL of SEQ ID NO: 61;

[0427] i) VH of SEQ ID NO: 65 and VL of SEQ ID NO: 69;

[0428] j) VH of SEQ ID NO: 73 and VL of SEQ ID NO: 77;

[0429] k) VH of SEQ ID NO: 81 and VL of SEQ ID NO: 85;

[0430] 1) VH of SEQ ID NO: 89 and VL of SEQ ID NO: 93;

[0431] m) VH of SEQ ID NO: 97 and VL of SEQ ID NO: 101;

[0432] n) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109;

[0433] o) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117;

[0434] p) VH of SEQ ID NO: 121 and VL of SEQ ID NO: 125;

[0435] q) VH of SEQ ID NO: 129 and VL of SEQ ID NO: 133;

[0436] r) VH of SEQ ID NO: 137 and VL of SEQ ID NO: 141;

[0437] s) VH of SEQ ID NO: 145 and VL of SEQ ID NO: 149;

[0438] t) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157;

[0439] u) VH of SEQ ID NO: 161 and VL of SEQ ID NO: 165;

[0440] v) VH of SEQ ID NO: 169 and VL of SEQ ID NO: 173;

[0441] w) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181;

[0442] x) VH of SEQ ID NO: 185 and VL of SEQ ID NO: 189;

[0443] y) VH of SEQ ID NO: 193 and VL of SEQ ID NO: 197;

[0444] z) VH of SEQ ID NO: 201 and VL of SEQ ID NO: 205;

[0445] aa) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213;

[0446] bb) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221;

[0447] cc) VH of SEQ ID NO: 225 and VL of SEQ ID NO: 229;

[0448] dd) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237;

[0449] ee) VH of SEQ ID NO: 241 and VL of SEQ ID NO: 245;

[0450] ff) VH of SEQ ID NO: 249 and VL of SEQ ID NO: 253;

[0451] gg) VH of SEQ ID NO: 257 and VL of SEQ ID NO: 261;

[0452] hh) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269;

[0453] ii) VH of SEQ ID NO: 273 and VL of SEQ ID NO: 277;

[0454] jj) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285;

[0455] kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293;

[0456] 11) VH of SEQ ID NO: 297 and VL of SEQ ID NO: 301;

[0457] mm) VH of SEQ ID NO: 305 and VL of SEQ ID NO: 309;

[0458] nn) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317;

[0459] oo) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325;

[0460] pp) VH of SEQ ID NO: 329 and VL of SEQ ID NO: 333;

[0461] qq) VH of SEQ ID NO: 337 and VL of SEQ ID NO: 341;

[0462] rr) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349;

[0463] ss) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357;

[0464] tt) VH of SEQ ID NO: 361 and VL of SEQ ID NO: 365;

[0465] uu) VH of SEQ ID NO: 369 and VL of SEQ ID NO: 373;

[0466] vv) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381;

[0467] ww) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389;

[0468] xx) VH of SEQ ID NO: 393 and VL of SEQ ID NO: 397;

[0469] yy) VH of SEQ ID NO: 401 and VL of SEQ ID NO: 405;

[0470] zz) VH of SEQ ID NO: 409 and VL of SEQ ID NO: 413;

[0471] aaa) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421;

[0472] bbb) VH of SEQ ID NO: 425 and VL of SEQ ID NO: 429;

[0473] ccc) VH of SEQ ID NO: 433 and VL of SEQ ID NO: 437;

[0474] ddd) VH of SEQ ID NO: 441 and VL of SEQ ID NO: 445;

[0475] eee) VH of SEQ ID NO: 449 and VL of SEQ ID NO: 453;

[0476] fff) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461;

[0477] ggg) VH of SEQ ID NO: 465 and VL of SEQ ID NO: 469;

[0478] hhh) VH of SEQ ID NO: 473 and VL of SEQ ID NO: 477;

[0479] iii) VH of SEQ ID NO: 481 and VL of SEQ ID NO: 485; and

[0480] jjj) VH of SEQ ID NO: 489 and VL of SEQ ID NO: 493.

[0481] In certain embodiments, the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) are each independently at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the above sequences.

[0482] 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 antigen-binding fragment thereof 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.

[0483] 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 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.

[0484] 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 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 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.

[0485] 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 the 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.

[0486] 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 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 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.

[0487] In certain embodiments, the present invention relates to an isolated antibody or antigen-binding fragment thereof, which specifically binds to HBsAg, characterized in that the isolated antibody or 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.

[0488] The sequence information of the antibodies of the present invention is shown in Table 2 below.

[0489] Table 2: Antibodies of the present invention

[0490] 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, a 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, that do not adversely alter 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. 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) noncharged 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 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 one skilled in the art when such replacement is desired. The characteristics of the resulting antibody variants can be tested using the assays described herein.

[0491] Amino acid replacement can be carried out, for example, by PCR mutagenesis (U.S. Patent number 4,683,195). Alternatively, a variant library can be generated using known methods, for example 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.

[0492] 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 dual combinatorial method disclosed in International Patent Publication No. WO92 / 01047. In this combinatorial method, a single colony containing either 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 the resulting two-chain specific antigen-binding domains are then selected according to the described phage display technology. Thus, separate VH and VL polypeptide chains can be used to identify additional antibodies that specifically bind to the S protein domain using the methods disclosed in International Patent Publication No. WO92 / 01047.

[0493] 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, a mouse or other host animal (such as a hamster, rat, or monkey) is immunized with an S protein or a fragment thereof, and then spleen cells from the immunized animal 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 a single immortalized hybridoma cell are screened for the production of antibodies having the desired properties (such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, and affinity for the antigen).

[0494] Various host animals can be used to prepare the 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 directed selection (Osbourn et al (2005) Methods 36:61-68, 2005; U.S. Patent No. 5,565,332).

[0495] Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by introducing modified framework support 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.

[0496] Transgenic mice carrying human immunoglobulin (Ig) loci in their genomes can be used to generate human antibodies against a protein of interest 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 ...

[0015] In some embodiments, the present invention provides a method for producing a human immunoglobulin gene that is expressed in a mouse model of a human immunoglobulin gene. The method comprises the steps of: (i) expressing a human immunoglobulin gene in a mouse model of a human immunoglobulin gene that is expressed ...

[0497] 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, for example, from a phage display library that expresses the heavy and light chain variable regions of an antibody as a fusion protein with the coat protein of phage pIX, 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 phage binding to the S protein, and positive clones obtained can be further characterized, and Fabs can be isolated from clone lysates and expressed as full-length IgG. 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.

[0498] Preparation of immunogenic antigens and generation of monoclonal antibodies can be performed using 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.

[0499] The antibodies of the present invention may be bispecific or multispecific antibodies. Exemplary antibodies that can be used to engineer bispecific and multispecific antibodies comprising a first domain that specifically binds to a first epitope on hepatitis B surface antigen (HBsAg) and a second domain that specifically binds to a second epitope on HBsAg include those selected from the V domains shown in Table 2. H / V L In one embodiment, the antibodies for engineering the first domain and the second domain and possibly more domains are identical, or bind to the same epitope. In this case, bispecific and multispecific antibodies are also referred to as bivalent or multivalent antibodies.

[0500] In one embodiment, the antibodies used to engineer the first domain and the second domain, and possibly further domains, are different. For example, the antibodies used to engineer the first domain and the second domain are each independently selected from the following different groups of antibodies:

[0501] Group I: S33, S39, S64, S103, and S106;

[0502] Group II: S29, S43, S41, S66, and S81; and

[0503] Group III: S5, S12, S16, S17, S19, S20, S82, S89, S92, S95, S96 and S101.

[0504] The bispecific antibodies provided herein include antibodies having the structure of a full-length antibody.

[0505] A "Fab arm" or "half molecule" refers to a heavy chain-light chain pair that specifically binds an antigen.

[0506] Full-length bispecific antibodies as described herein can be produced, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half-molecule to promote heterodimer formation of two antibody half-molecules with different specificities in a cell-free environment in vitro or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each of which binds to a different epitope.

[0507] "Homodimerization" refers to the interaction of two heavy chains with the same CH3 amino acid sequence. "Homodimer" refers to an antibody with two heavy chains containing the same CH3 amino acid sequence.

[0508] "Heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. "Heterodimer" refers to an antibody with two heavy chains containing different CH3 amino acid sequences.

[0509] In some embodiments, bispecific antibodies include bispecific antibodies such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), buttons (Genentech), CrossMAbs (Roche) and electrostatic partners (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), SEEDbodies (EMD Serono), Biclonic (Merus), and Design of technology (Genmab A / S).

[0510] Triomab quadroma technology can be used to generate full-length bispecific antibodies that incorporate the VH and VL of the antibodies of the invention. Triomab technology facilitates the exchange of Fab arms between two parent chimeric antibodies, one parent mAb having IgG2a and the second parent mAb having rat IgG2b constant regions, thereby generating chimeric bispecific antibodies.

[0511] The "knob" strategy (see, e.g., International Publication WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the junction of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains, thereby promoting heterodimer formation. Amino acids with small side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "knob" with the heavy chain with the "knob." Exemplary CH3 substitution pairs that form a knob and buckle (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0512] CrossMAb technology can be used to generate full-length bispecific antibodies. In addition to utilizing a "button" strategy to promote Fab wall exchange, CrossMAb has an exchanged CH1 domain and CL domain in one of the half-arms to ensure the correct light chain pairing of the resulting bispecific antibody (see, e.g., U.S. Patent 8,242,247).

[0513] Other exchange strategies can be used to generate full-length bispecific antibodies by exchanging variable or constant domains, or both domains, between the heavy and light chains or within the heavy chain in one or both arms of the bispecific antibody. These exchanges include, for example, VH-CH1 with VL-CL, VH with VL, CH3 with CL, and CH3 with CH1, as described in International Patent Publications WO2009 / 080254, WO2009 / 080251, WO2009 / 018386, and WO2009 / 080252.

[0514] Other strategies can also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in US Patent Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y 407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0515] LUZ-Y technology can be used to generate bispecific antibodies. In this technology, a leucine zipper is added to the C-terminus of the CH3 domain to drive the assembly of heterodimers from the parent mAb, which is removed by post-purification, as described in Wranik et al., 2012, J Biol Chem, Vol. 287, No. 52, pp. 42221-42229.

[0516] SEEDbody technology can be used to generate bispecific antibodies. SEEDbodies have selected IgG residues substituted with IgA residues in their constant domains to promote heterodimerization, as described in US Pat. No. 20070287170.

[0517] According to the methods described in International Patent Publication No. WO2011 / 131746, the bispecific antibodies described herein can be produced in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization. In these methods, a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of cysteines in the hinge region; thereby producing a bispecific antibody by Fab arm exchange. The incubation conditions are returned to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. For example, the following conditions can be used: incubation in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a pH of 5-8, such as pH 7.0 or pH 7.4, at a temperature of at least 20°C, for at least 90 minutes.

[0518] In some embodiments described herein, a bispecific antibody comprising a first domain that specifically binds a first epitope on hepatitis B surface antigen (HBsAg) and a second domain that specifically binds a second epitope on HBsAg comprises at least one substitution in the antibody CH3 constant domain.

[0519] In some embodiments described herein, at least one substitution in the antibody CH3 constant domain is a K409R, F405L, or F405L and R409K substitution, where the residues are numbered according to the EU index.

[0520] Antibody domains and position numbering are well known. "Asymmetric" refers to different substitutions within the two CH3 domains of two independent heavy chains of an antibody. The IgG1 CH3 region typically consists of residues 341-446 on IgG1 (residues are numbered according to the EU index).

[0521] In some embodiments described herein, the bispecific antibody is of the IgG1 isotype and comprises a F405L substitution in the antibody first heavy chain (HC1) and a K409R substitution in the antibody second heavy chain (HC2) when compared to wild-type IgG1.

[0522] In some embodiments described herein, the bispecific antibody is of the IgG1 isotype and comprises a K409R substitution in the antibody first heavy chain (HC1) and a F405L substitution in the antibody second heavy chain (HC2) when compared to wild-type IgG1.

[0523] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and comprises an S228P substitution in HC1 and S228P, F405L, and R409K substitutions in HC2 when compared to wild-type IgG4.

[0524] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and comprises S228P, F405L, and R409K substitutions in HCl and an S228P substitution in HC2 when compared to wild-type IgG4.

[0525] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and comprises S228P, F234A, and L235A substitutions in HC1 and S228P, F234A, L235A, F405L, and R409K substitutions in HC2 when compared to wild-type IgG4.

[0526] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and comprises S228P, F234A, L235A, F405L, and R409K substitutions in HCl and S228P, F234A, and L235A substitutions in HC2 when compared to wild-type IgG4.

[0527] In some embodiments described herein, the bispecific antibodies of the invention comprise at least one, two, three, four, five, six, seven, or eight asymmetric substitutions at residue positions 350, 366, 368, 370, 399, 405, 407, or 409 in HCl and HC2, when the residues are numbered according to the EU index.

[0528] In some embodiments described herein, the bispecific antibodies of the invention comprise at least one, two, three, or four asymmetric substitutions at residue positions 350, 370, 405, or 409 in HCl and HC2, when the residues are numbered according to the EU index.

[0529] In some embodiments described herein, the bispecific antibodies of the invention comprise at least one asymmetric substitution at residue position 405 or 409 in HC1 and HC2, when the residues are numbered according to the EU index.

[0530] Replacements are generally made at the DNA level to the molecular level (such as the constant domains of an antibody) using standard methods.

[0531] The antibodies of the present invention can be engineered into various known antibody formats.

[0532] In some embodiments, the bispecific antibodies of the invention are chiasmatic.

[0533] In some embodiments, the bispecific antibodies of the present invention include recombinant IgG-like dual-targeting molecules, wherein the two sides of the molecule each contain a Fab fragment or a portion of a Fab fragment of at least two different antibodies; an IgG fusion molecule, wherein a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; an Fc fusion molecule, wherein a scFv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; a Fab fusion molecule, wherein different Fab fragments are fused together; and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and diabodies, wherein different scFv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0534] ScFv-based and diabody-based domain antibodies include bispecific T cell engagers (BITE) (Micromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting molecules (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, Receptor Logics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and dual-targeting heavy chain domain antibodies only. Various forms of bispecific antibodies have been described, for example, in Chames and Baty, 2009, Curr Opin Drug Disc Dev, Vol. 12, p. 276 and Nunez-Prado et al., 2015, Drug Discovery Today, Vol. 20, No. 5, pp. 588-594.

[0535] In some embodiments, the first domain and the second domain of the bispecific antibody of the present invention are fused to each other (i.e., covalently linked). As used herein, the term "fusion" refers to that the first domain and the second domain of the bispecific antibody of the present invention are directly or indirectly linked together by covalent linkage. In some embodiments, fusion includes but is not limited to direct fusion via a peptide bond or a non-peptide bond or indirect fusion via a linker such as a peptide linker. It will be understood by those skilled in the art that any direct or indirect connection method known in the art or that may be found in the future for linking polypeptides together can be used for "fusion" of the present disclosure.

[0536] The first domain and the second domain can be directly connected by a single chemical bond (such as a peptide bond) or via a peptide linker. The first domain can be fused at the N-terminus or C-terminus of any (including each) polypeptide of the second domain, or can be fused at an internal position of any (including each) polypeptide of the second domain, such as at the N-terminus of the Fc region in the heavy chain of the second domain. The fusion polypeptide can be recombinantly obtained or chemically obtained.

[0537] The bispecific antibodies of the present invention may comprise one or more peptide linkers between the first domain and the second domain. In some embodiments, the first domain and the second domain are directly fused to each other without providing a peptide linker.

[0538] In some embodiments, the peptide linkers connecting different domains can be identical or different. Each peptide linker can be optimized separately. The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acids long. In some embodiments, the length of the peptide linker is any one of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids or about 10 amino acids to about 30 amino acids long. The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived only from the hinge region of an antibody heavy chain can be used as a linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n (n is an integer of at least 1), glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, (GGGGS)n, wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGS or GGGGSGGGGSGGGGS (also known as (G4S)3). In some embodiments, the peptide linker comprises the hinge region of IgG, such as the hinge region of human IgG1. In some embodiments, the peptide linker comprises the amino acid sequence EPKSCDKTHTCPPCP. In some embodiments, the peptide linker comprises a modified sequence derived from the hinge region of IgG, such as the hinge region of human IgG1. For example, one or more cysteines in the hinge region of IgG can be replaced by serine. In some embodiments, the peptide linker comprises the amino acid sequence EPKSSDKTHTSPPSP.

[0539] In some embodiments, the first domain and / or the second domain comprises a single-chain antibody fragment scFv, which comprises VH and VL. In some embodiments, the bispecific antibody of the present invention further comprises an Fc region.

[0540] In some embodiments, the first domain comprises a heavy chain containing a VH domain and a light chain containing a VL domain. In some embodiments, the heavy chain further comprises one or more heavy chain constant domains, such as CH1, CH2, CH3 and CH4, and / or a hinge region (HR). In some embodiments, the light chain further comprises a light chain constant domain (CL). In some embodiments, the N-terminus of the second domain is fused to the C-terminus of the heavy chain. In some embodiments, the C-terminus of the second domain is fused to the N-terminus of the heavy chain. In some embodiments, the N-terminus of the second domain is fused to the C-terminus of the light chain. In some embodiments, the C-terminus of the second domain is fused to the N-terminus of the light chain. In some embodiments, the C-terminus of the second domain is fused to the N-terminus of the light chain.

[0541] In some embodiments, the first domain comprises a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the full-length antibody is a full-length monoclonal antibody consisting of two identical heavy chains and two identical light chains. In some embodiments, the second domain is a scFv.

[0542] It should be understood that the first domain and the second domain are only used for illustration and are not intended to limit the relative positions of the various domains of the bispecific antibody. In some cases, the features of the first domain may also be applicable to the second domain, and vice versa.

[0543] Another embodiment of the present invention is a kind of isolated polynucleotide, it encodes 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 promoters or enhancers, which allow the nucleotide sequence to be expressed in the intended host cell.The polynucleotide can be cDNA.

[0544] Another embodiment of the present invention is a carrier comprising the polynucleotide of the present invention. Such carriers can be plasmid vectors, viral vectors, carriers for baculovirus expression, carriers based on transposons or any other carriers suitable for introducing the synthetic polynucleotide of the present invention into a given organism or genetic background by any means. For example, polynucleotides that are optionally connected to a constant region and encoding the light chain variable region and / or the heavy chain variable region of an antibody of the present invention are inserted into an expression vector. The light chain and / or heavy chain can be cloned into the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain can be effectively connected to the control sequence that ensures the expression of immunoglobulin polypeptides in one or more expression vectors. Such control sequences include signal sequences, promoters (for example, naturally associated or heterologous promoters), enhancer elements and transcription terminator sequences, and such control sequences are selected so that they are compatible with the host cell selected for expressing the antibody. Once the carrier is incorporated into an appropriate host, the carrier maintains the host under conditions suitable for high-level expression of protein, and the protein is encoded by the combined polynucleotides.

[0545] Suitable expression vectors can usually be replicated 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.

[0546] 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 and / or heavy chain immunoglobulin gene promoters and enhancer elements; the cytomegalovirus very early promoter; the herpes simplex virus thymidine kinase promoter; the early and late SV40 promoters; promoters present in the long terminal repeat of retroviruses; the 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 ability of those of ordinary skill in the art.

[0547] 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).

[0548] 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 progeny, and thus such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells can be eukaryotic, prokaryotic, plant, or archaeal cells.

[0549] 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) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC 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.

[0550] Another embodiment of the present invention is a method for preparing an antibody of the present invention, 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 synthesized (chemically or recombinantly), all antibodies, dimers thereof, light chains and / or heavy chains, or other antibody fragments (such as VH and / or VL) can be purified according to standard procedures, including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The antibody can be substantially pure, e.g., 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 even purer, e.g., free of contaminants (such as cell debris, macromolecules other than the test antibody, etc.).

[0551] Another embodiment of the present invention is a method for preparing an antibody of the present invention, comprising:

[0552] combining a first polynucleotide encoding antibody VH and a second polynucleotide encoding antibody VL into an expression vector;

[0553] transforming a host cell with the expression vector;

[0554] The host cells are cultured in a culture medium under conditions that allow the expression of VL and VH and the formation of the antibody; and then

[0555] The antibodies are recovered from the host cells or culture medium.

[0556] The polynucleotide encoding the specific VH or VI sequence of the present invention is incorporated into a vector using standard molecular biological methods. Host cell transformation, culture, antibody expression and purification are accomplished using well-known methods.

[0557] III. Composition and Administration Method

[0558] The present invention provides pharmaceutical compositions comprising the antibodies described herein and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the present invention can be prepared into pharmaceutical compositions comprising an effective amount of the antibody as an active ingredient 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, 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 known in the art (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of the antibodies of the invention in such pharmaceutical formulations can vary widely, 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 chosen, primarily based on desired dose, fluid volume, viscosity, etc. Suitable vehicles and formulations (including other human proteins such as human serum albumin) are described, for example, 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.

[0559] 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, intravaginal, rectal), or other means known to the skilled artisan, which are well known in the art.

[0560] The antibodies of the methods of the invention described herein can be administered to a patient by any suitable route, for example, parenterally by intravenous (iv) infusion or bolus, intramuscularly, subcutaneously, or intraperitoneally. Intravenous infusion can be given, for example, within 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0561] 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 as described herein. The "therapeutically effective amount" of an antibody can be determined by standard research techniques. For example, in vitro assays 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 relevant animal models well known in the art. The selection of a specific effective dose can be determined by those 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 based on the doctor's judgment and the circumstances of each patient. The effective dose can be derived from 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.

[0562] The antibodies of the methods of the present 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 longer. Treatment courses can also be repeated, as with chronic administration. Repeated administrations can be of the same dose or of different doses.

[0563] The antibodies of the methods of the present 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.

[0564] The antibodies used 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 employed.

[0565] In one embodiment, the pharmaceutical composition of the present disclosure comprises one or more antibodies described herein. It will be readily understood by those skilled in the art that when the HBV infection in the body has multiple mutations, or when the body produces multiple mutations under drug or immune pressure, a combination of one or more antibodies that bind to different mutation sites will have greater clinical significance and value in treatment and prevention.

[0566] 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 can be easily performed by those skilled in the art according to routine practice.

[0567] The present invention will now be described with reference to the following specific non-limiting examples, which are intended only to illustrate the present invention but not to limit the scope of the present invention.

[0568] Materials and Methods

[0569] The inventors used flow cytometry to isolate B cells specific for hepatitis B surface antibodies from peripheral blood mononuclear cells (PBMCs) of donors recovering from hepatitis B infection. They then cloned the antibody sequence within the cells using PCR, obtained it by sequencing, and constructed it into an expression vector. The plasmid was then introduced into the cells via transient transfection for protein expression. The cell supernatant was then collected and the antibodies purified and concentrated.

[0570] The human subjects involved in the present invention were all 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 were aliquoted 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 separated, and the separated PBMCs were then resuspended in a freezing solution of 10% DMSO and 90% fetal bovine serum (inactivated), and frozen in liquid nitrogen.

[0571] B cell purification, dual fluorescent dye labeling of bait proteins with bound B cells, and single-cell sorting were performed as previously described (Y. Zhou et al (2020) STAR Protoc 1, 100129). PBMCs were rapidly 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 a human Fc block at 4°C, and bait proteins (PE / APC) (10 μg / ml) and anti-CD20-PE-Cy7 (BD Bioscience) were incubated sequentially. Single-cell sorting was performed on a FACSAria II (BD Bioscience) flow cytometer, sorted into 96-well plates, and stored at −80°C.

[0572] As previously described (Y. Zhou et al (2020) STAR Protoc 1, 100129), the sorted single cells were reverse transcribed and the antibody sequence PCR was performed. After the first round of PCR and the second round of nested PCR, electrophoresis and purification were performed on a 2% agarose gel, followed by Sanger sequencing. 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 constructed and expressed in vectors.

[0573] The antibodies obtained by the present invention were subjected to ELISA detection. The HBsAg antigen was diluted to 10ug / ml with PBS, coated at 4°C overnight, and then blocked with 2% bovine serum albumin (BSA) dissolved in PBS for 2 hours. The antibody was diluted to 8 dilutions at a dilution ratio of 1:3 with a maximum concentration of 10ug / ml. After addition, the cells were incubated at room temperature for 1 hour. Incubation was performed with HRP-linked goat anti-human IgG (Thermo Fisher Scientific), and finally color development was performed with ABTS color development solution. The BioRad plate reader read the values ​​at 10 minutes, 20 minutes, and 30 minutes, respectively. Analysis was performed using PRISM software, and the area under the curve (AUC) was calculated for each antibody to evaluate the antigen-binding ability.

[0574] Competition between antibodies was detected by competitive ELISA, as previously reported (Q. Wang et al (2020) Cell Host & Microbe 28, 335-349). Briefly, HBsAg antigen was diluted to 3 μg / ml in PBS and coated overnight with the first blocking antibody at a concentration of 15 μg / ml and incubated at room temperature for 2 hours. Biotinylated secondary antibody (0.25 μg / ml or 0.75 μg / ml) was then added directly at room temperature for 30 minutes. Detection was performed using streptavidin-HRP (BD Bioscience). PBS buffer was used instead of the first blocking antibody as a standard reference.

[0575] 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 HBV virus derived from the cell line HepDE19, with a viral genotype of D; the other is HBV virus of genotype A / B / C expressed by transfecting HBV plasmids of different genotypes into the HepG2 cell line using X-tremeGENE HP (Roche) as the transfection reagent. Both viruses derived from stable cell lines and transfected viruses were concentrated by ultrafiltration tubes. After ultrafiltration, the obtained viruses were packaged, and a small amount of virus was taken out. After 10-fold gradient dilution, the HBV copy number was 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 containing 2% DMSO, 3% FBS, 1% NEAA, and 1% penicillin-streptomycin (infection medium) for 24 hours before infection. To test the neutralizing ability of serum or antibodies, human serum (maximum concentration 10%) or monoclonal antibodies (maximum concentration 10 μg / ml) were serially diluted in infection medium at a ratio of 1:3 for a total of nine dilutions. Subsequently, the diluted serum or monoclonal antibodies were incubated with HBV virus at 37°C for 30 minutes before being added to HepG2-NTCP cells. After 16 hours of infection, the cells were washed five times with 200 μl of PBS per well, and infection medium was added. After 7 days of culture, HBsAg and HBeAg levels in the cell supernatant were measured using CLIA kits (Autobio), and HBcAg levels in the cells were measured by immunofluorescence.

[0576] The present invention tests the neutralization activity of antibodies against different HBV mutants. HepG2 cells were transfected with plasmids (pHBV1.3-G145R, pHBV1.3-K122R G145R, and pHBV1.3-F134L). The supernatant was collected and concentrated to obtain the HBV mutants. The neutralization assay procedures were identical to those used in the aforementioned in vitro HBV-infected cell model.

[0577] The methods for antibody amino acid epitope screening (amino acid scanning), antibody binding to different viral genotypes ELISA, and antibody binding to different viral mutants were as described previously (Q. Wang et al (2020) Cell Host & Microbe 28, 335-349). Briefly, Huh7.5 cells were seeded in 6-well plates and transfected with HBV1.3 plasmids with different mutation sites using Lipofectamine 3000 (Thermo Fisher Scientific). 16 hours after transfection, the cell culture medium was replaced with DMEM medium containing a mixture of 1% NEAA and 1% penicillin and streptomycin. After culturing for 3 days, 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. The plates were then blocked with 2% bovine serum albumin (BSA) dissolved in PBS for 2 hours. Antibodies were then incubated at a concentration of 1 μg / ml, followed by incubation with HRP-linked goat anti-human IgG (Thermo Fisher Scientific). Finally, the plates were developed with ABTS colorimetric solution and read on a BioRad plate reader at 10, 20, and 30 minutes. The antibody-bound wild-type HBV readings were used as a normalized reference and analyzed using PRISM software.

[0578] For immunofluorescence, cells were fixed in 4% paraformaldehyde / PBS at room temperature for 20 minutes, neutralized with PBS containing 0.1 M glycine for 15 minutes, and then treated with 0.1% Triton X-100 / PBS. Blocking was performed with 5% goat serum / PBS at room temperature for 1 hour. Anti-HBc (Austral Biologicals) was diluted in 5% goat serum / PBS and incubated overnight at 4°C. Visualization was performed with goat anti-rabbit AlexaFluor 594 (Thermo Fisher Scientific), and cell nuclei were stained with DAPI. Fluorescence images were taken using a high-content cell imaging analyzer (Perkin Elmer, Operetta).

[0579] The detailed results of statistical analysis are shown in the results and figure legends. The area under the ELISA curve (AUC), the 50% inhibitory concentration (IC 50 ) values ​​were calculated in PRISM software.

[0580] Example 1. Antibody response in serum of recovered hepatitis B patients

[0581] Serum samples from 325 recovered HBV donors were collected from multiple hospitals in Shanghai. Two pairs of donor sera were collected, all positive for anti-HBV antibodies, while anti-HBsAg antibody titers varied. Neutralizing activity was measured in HepG2 cells overexpressing the sodium-taurocholate cotransporter (NTCP), a receptor for HBV infection. We selected donors AA14, BH2, CC7, and EB4 with high anti-HBs titers and neutralizing activity from recovered HBV patients, as well as C10, C12, and C14 from HBV vaccine recipients. Donors with anti-HBs titers <2.0 IU / ml served as negative controls. Demographic information of the donors is shown in Table 3. Neutralizing activity was confirmed by diluting the serum samples at different serum concentrations, as shown in Figure 2.

[0582] Table 3. Demographic information of donors

[0583] Example 2. Obtaining a fully human monoclonal antibody that specifically binds to HBsAg

[0584] 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 HBsAg-specific binding B cells (Figure 3). During donor recruitment, sample donors who were vaccinated with hepatitis B vaccine but had an anti-HBs ELISA titer of less than 2mIU / ml were recruited. They were used as negative controls for the experiment, and their B cells stained as bait protein-specific B cells at the background level. 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).

[0585] Double-positive cells (bait protein-PE+ and bait protein-APC+) gated by flow cytometry 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 using immunoglobulin heavy and light chain gene-specific primers.

[0586] Example 3. ELISA detection of antibody binding to HBsAg protein

[0587] After obtaining the antibody sequence, the antibody sequence was cloned into a vector, followed by protein expression and purification. The resulting antibodies were then subjected to ELISA to detect their binding to HBsAg. The ELISA data were used to calculate the AUC value in PRISM. Among the antibodies numbered S001-S108, 62 antibodies were found to bind to HBsAg. These antibodies are presented here in descending order of AUC value, as shown in Figure 5.

[0588] Example 4. Evaluation of Antibody Neutralizing Activity in HBV Cell Infection Model

[0589] To determine whether antibodies that can bind to HBsAg can block HBV infection in vitro, neutralization assays were performed using cell-derived concentrated and purified HBV virus (genotype D) to infect HepG2-NTCP. 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 measured. The infection rate was compared between the antibody-treated group and the non-antibody-treated group, and the 50% inhibitory concentration of the antibody, i.e., the IC50 value, was calculated. The neutralizing activity of these antibodies varied significantly, ranging from effective neutralizing antibodies to almost no neutralizing activity (Figure 6). The specific IC50 values ​​are shown in Table 4.

[0590] Table 4. IC values ​​of antibodies tested in cell infection models 50 value

[0591] Example 5. Antibody epitope analysis

[0592] The binding epitopes of the 23 antibodies with strong neutralizing activity (S05, S12, S16, S17, S19, S20, S29, S33, S39, S41, S43, S64, S66, S81, S82, S89, S92, S95, S96, S101, S103, S105, and S106) were characterized. First, competition ELISAs were performed to determine whether the HBsAg-binding antibodies bound to overlapping or non-overlapping epitopes. Because the S105 antibody bound weakly, its competition ELISA results are not presented here. Three mutually exclusive antibody groups (Group I, Group II, and Group III) were identified by competition ELISA, as shown in Figure 7 , where Group I included S33, S39, S64, S103, and S106, Group II included S29, S43, S41, S66, and S81, and Group III included S5, S12, S16, S17, S19, S20, S82, S89, S92, S95, S96, and S101.

[0593] Antibodies that recognize the S protein primarily recognize a region of approximately 72 amino acids on the extracellular segment of HBsAg. A further alanine scanning method was performed, using a constructed HBsAg point mutation plasmid (the original amino acid was mutated to alanine) to transfect cells to obtain HBsAg with different amino acid mutations. ELISA was then used to detect whether the antibody could bind to the HBsAg amino acid mutant, thereby clarifying the specific antigenic amino acid sites recognized by the obtained antibodies (Figure 8). Combined with the results of competitive ELISA, it was suggested that antibodies in different groups had obvious clustering in terms of recognizing key amino acid sites on the antigen. Antibodies from donors immunized with the hepatitis B vaccine and antibodies from donors recovered from hepatitis B infection differed significantly in their recognition sites. The average number of amino acids affecting antibodies derived from the hepatitis B vaccine was 12.6, and the average number of amino acids affecting antibodies from recovered hepatitis B infection was 22.1. Antibodies derived from hepatitis B vaccine donors recognize discontinuous amino acid sites, while those from recovered donors can recognize a continuous sequence of three to five amino acids. For example, S43 recognizes 143T-145G, S20 recognizes 133M-136S, and S17 recognizes 142P-146N. Subsequently, combined with the results of a competitive ELISA, analysis of antibody alanine scanning revealed that all four groups of antibodies recognize spatial epitopes on the S protein, with 101Q, 102G, 103M, 105P, and 106V all having an impact on binding. The antibodies in each of the four groups displayed specific binding patterns for amino acid variants. Group I recognized amino acid sites 110I, 122K, 125T, 134F, 141K, and 165W; Group II recognized amino acid sites 122K, 123T, 145G, 161Y, and 165W; and Group III recognized amino acid sites 109L-111P, 120P-130G, 133M-136S, 144D, 146N, 153P, 161Y, and 164E-167S. In Group I, S64, S33, and S39 were derived from two donors immunized with hepatitis B vaccine, respectively. In Group II, S66, S29, and S41 were derived from two donors immunized with hepatitis B vaccine, respectively. This suggests that while antibodies obtained from different individuals immunized with hepatitis B vaccine have some similarity, the resulting antibodies exhibit epitope diversity, recognizing at least two non-overlapping epitopes.

[0594] Example 6. Binding ability and neutralizing activity against different HBV genotypes

[0595] Hepatitis B virus (HBV) is an ancient DNA virus with nine confirmed genotypes, one unidentified genotype, and one unidentified genotype. Numerous studies have shown that the distribution of HBV genotypes varies across regions. Among people infected with HBV in my country, approximately 60% have genotype B and approximately 30% have genotype C.

[0596] The inventors' laboratory has successfully established an in vitro infection system for HepG2-NTCP cells infected with different HBV genotypes. This system was used to test whether antibodies that broadly bind to different HBV genotypes can block infection by different HBV genotypes in vitro (Figure 9). After mixing the antibodies with the virus in vitro, they were incubated at 37°C for 1 hour and then infected with HepG2-NTCP cells. HBsAg and HBeAg levels in the cell supernatant were measured on day 7 after infection. The 50% inhibitory concentration (IC50) of the antibody-treated group was calculated compared with the non-antibody-treated group.

[0597] The results showed that S12, S29, S33, S39, S41, S43, S66, S81, and S106 all had neutralizing activity against genotypes A, B, C, and D (see Figure 9 and Table 4). Antibodies from recovered donors generally did not effectively neutralize genotype A, whereas antibodies from most hepatitis B vaccine-immunized donors had neutralizing activity against all four genotypes of HBV.

[0598] Example 7. Binding ability against naturally occurring HBV mutants

[0599] Studies have reported that patients with chronic hepatitis B infection exhibit a wide range of immune escape mechanisms. To test whether antibodies obtained from recovered hepatitis B infected donors or those immunized with hepatitis B vaccine could bind to these mutant strains, plasmids harboring common hepatitis B surface antigen amino acid mutations 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, T126S, 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 antigens containing HBsAg mutants. ELISA was then used to detect whether the antibodies bound to the antigen (Figure 10). Antibodies from donors immunized with the hepatitis B vaccine recognized an average of 31 mutation sites, while antibodies from donors recovered from hepatitis B infection recognized an average of 37.4 mutation sites. The specific HBsAg mutant sites recognized by antibodies from the two immune backgrounds also showed significant differences. Group I antibodies derived from hepatitis B vaccine-immunized donors do not recognize Q101R, P105L, C124R, C137Y, C137Y D144V, C138Y, K141E, and C149Y. S64 in group I can recognize K122R G145R, P142S, S142L G145R, D144E G 145R, G145E, and G145R sites. Group II antibodies derived mainly from hepatitis B vaccine-immunized donors 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 recovered from hepatitis B infection in group II could not recognize the above mutation sites, and also could not recognize T143I, T143L and D144E.Group III antibodies from recovered hepatitis B donors fail to recognize Q101R, P105L, P120K, T123D, T123N, T123N, T143S, G130N, G130R, M133T, C138Y, and C149Y. However, they recognize C137Y and C137Y, which are not recognized by either group I or group II. G145R is the most common mutation clinically. ELISA results show that only S64 among antibodies from hepatitis B vaccine-immunized donors recognizes G145R.

[0600] Example 8. Neutralizing activity against naturally occurring HBV mutants

[0601] As shown in the ELISA results of Example 6, the present invention relates to antibodies with neutralizing activity that bind to different HBV mutants. Here, we selected the most common clinically detected HBsAg G145R single-site mutant and the HBsAg K122R-G145R double mutant for neutralization experiments. Example 6 demonstrates that antibodies derived from hepatitis B vaccine-immunized donors can better bind to the HBsAg F134L mutant, and HBsAg F134L mutant viruses were also purified.

[0602] The experimental results suggest that antibodies S12, S64, S82 and S92 can effectively neutralize the G145R mutant HBV virus, S12, S19, S20, S43 from donors recovered from hepatitis B infection and S39, S82, S92 from donors immunized with hepatitis B vaccine can effectively neutralize the K122R-G145R double mutation HBV virus, and S12, S43, S103 and S106 from donors recovered from hepatitis B infection and S29, S33, S39, S41, S64, S66, S81 and S82 from donors immunized with hepatitis B vaccine can effectively neutralize the F134L mutant HBV virus (Figure 11).

[0603] Example 9. Construction and analysis of bispecific antibodies

[0604] In this example, two antibodies, S66 and S12, were selected as parental antibody sources for bispecific antibody engineering, resulting in five constructs, as shown in Figure 12 . Preparation was completed by Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., which also provides physical and chemical property testing and SDS PAGE results for the bispecific antibodies, as shown in Figure 13 .

[0605] ELISA was performed on the bispecific antibodies to detect their binding to HBsAg, following the method of Example 3. All constructed bispecific antibodies, P74, P75, P76, P77, and P78, bound to HBsAg with similar affinities to those of the parent antibodies, S66 and S12, as shown in Figure 14 .

[0606] To determine whether bispecific antibodies that bind to HBsAg can block HBV infection in vitro, neutralization assays were performed using purified, concentrated cell-derived HBV virus (genotype D) to infect HepG2-NTCP cells according to the method of Example 4. The antibodies were diluted in a concentration gradient and then mixed with the same virus to infect HepG2-NTCP cells. HBsAg and HBeAg levels in the cell supernatant were measured. The infection rates of the antibody-treated and non-antibody-treated groups were compared, and the 50% inhibitory concentration (IC50) of the antibodies was calculated. The results are shown in Figure 15.

[0607] The results showed that all constructed bispecific antibodies P74, P75, P76, P77 and P78 had better ability to block HBV infection than the parental antibodies S66 and S12, among which p76 and p77 had IC50 values ​​lower than the two parental monoclonal antibodies by about 1 log.

[0608] According to the antibody epitope analysis of Example 5, a variety of bispecific antibodies were further constructed from the monoclonal antibodies of Group I: S33, S39, S64, S103 and S106, Group II: S29, S43, S41, S66 and S81, and Group III: S5, S12, S16, S17, S19, S20, S82, S89, S92, S95, S96 and S101, and it was found that they obtained similar results as the S66 / S12 bispecific antibody (data not shown).

[0609] References

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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: 281 and VL of SEQ ID NO: 285; kk) VH of SEQ ID NO: 289 and VL of SEQ ID NO: 293; 11) 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:361 and VL of SEQ ID NO:365; uu) VH of SEQ ID NO: 369 and VL of SEQ ID NO: 373; vv) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381; ww) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389; xx) VH of SEQ ID NO: 393 and VL of SEQ ID NO: 397; yy) VH of SEQ ID NO:401 and VL of SEQ ID NO:405; zz) VH of SEQ ID NO:409 and VL of SEQ ID NO:413; 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, Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences.

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 isolated antibody or antigen-binding fragment thereof specifically binds to HBsAg proteins 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 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. A bispecific antibody, characterized in that The bispecific antibody comprises a first domain that specifically binds to a first epitope on hepatitis B surface antigen (HBsAg) and a second domain that specifically binds to a second epitope on HBsAg, wherein the first epitope and the second epitope are each independently selected from: a) an epitope comprising amino acid residues 110I, 122K, 125T, 134F, 141K and 165W; b) an epitope comprising amino acid residues 122K, 123T, 145G, 161Y, and 165W; and c) an epitope comprising amino acid residues 109L-111P, 120P-130G, 133M-136S, 144D, 146N, 153P, 161Y and 164E-167S, The amino acid residue numbers are based on the amino acid positions shown in SEQ ID NO:

497. wherein the first epitope is different from the second epitope.

11. The bispecific antibody according to claim 10, characterized in that: The first domain 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: 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; b) 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; c) 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; d) 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; and e) 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; The second domain 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: 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; b) 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; c) 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; d) 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; and e) 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.

12. The bispecific antibody according to claim 11, characterized in that: The first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences: a) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181; b) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213; c) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269; d) VH of SEQ ID NO:433 and VL of SEQ ID NO:437; and e) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences, The second domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences: a) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157; b) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221; c) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237; d) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285; and e) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317, Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences.

13. The bispecific antibody according to claim 10, characterized in that: The first domain 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: 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; b) 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; c) 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; d) 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; and e) 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; The second domain 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: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; c) 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; d) 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; e) 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; f) 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; g) 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; j) 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; i) 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; j) 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; k) 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; and 1) 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.

14. The bispecific antibody according to claim 13, characterized in that: The first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences: a) VH of SEQ ID NO: 177 and VL of SEQ ID NO: 181; b) VH of SEQ ID NO: 209 and VL of SEQ ID NO: 213; c) VH of SEQ ID NO: 265 and VL of SEQ ID NO: 269; d) VH of SEQ ID NO:433 and VL of SEQ ID NO:437; and e) VH of SEQ ID NO: 457 and VL of SEQ ID NO: 461, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences, The second domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the 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:49 and VL of SEQ ID NO:53; c) VH of SEQ ID NO:81 and VL of SEQ ID NO:85; d) VH of SEQ ID NO:89 and VL of SEQ ID NO:93; e) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109; f) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117; g) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325; h) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349; i) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357; j) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381; k) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389; and 1) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421, Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences.

15. The bispecific antibody according to claim 10, characterized in that: The first domain 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: 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; b) 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; c) 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; d) 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; and e) 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; The second domain 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: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; c) 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; d) 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; e) 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; f) 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; g) 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; h) 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; i) 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; j) 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; k) 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; and 1) 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.

16. The bispecific antibody according to claim 15, characterized in that: The first domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the light chain variable region VL are selected from the following amino acid sequences: a) VH of SEQ ID NO: 153 and VL of SEQ ID NO: 157; b) VH of SEQ ID NO: 217 and VL of SEQ ID NO: 221; c) VH of SEQ ID NO: 233 and VL of SEQ ID NO: 237; d) VH of SEQ ID NO: 281 and VL of SEQ ID NO: 285; and e) VH of SEQ ID NO: 313 and VL of SEQ ID NO: 317, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences, The second domain comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region VH and the 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:49 and VL of SEQ ID NO:53; c) VH of SEQ ID NO:81 and VL of SEQ ID NO:85; d) VH of SEQ ID NO:89 and VL of SEQ ID NO:93; e) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 109; f) VH of SEQ ID NO: 113 and VL of SEQ ID NO: 117; g) VH of SEQ ID NO: 321 and VL of SEQ ID NO: 325; h) VH of SEQ ID NO: 345 and VL of SEQ ID NO: 349; i) VH of SEQ ID NO: 353 and VL of SEQ ID NO: 357; j) VH of SEQ ID NO: 377 and VL of SEQ ID NO: 381; k) VH of SEQ ID NO: 385 and VL of SEQ ID NO: 389; and 1) VH of SEQ ID NO: 417 and VL of SEQ ID NO: 421, Or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the above groups of sequences.

17. The bispecific antibody according to claim 10, characterized in that: a) the first domain comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprising 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; and the second domain comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprising 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; or b) the first domain comprises the VH of SEQ ID NO:281 and the VL of SEQ ID NO:285, and the second domain comprises the VH of SEQ ID NO:49 and the VL of SEQ ID NO:

53.

18. The bispecific antibody according to any one of claims 10 to 17, characterized in that The first domain and / or the second domain is of IgG1, IgG2, IgG3 or IgG4 isotype.

19. The bispecific antibody according to any one of claims 10 to 18, characterized in that The first domain is a full-length IgG antibody, and the second domain is a scFv; or the first domain is a scFv, and the second domain is a full-length IgG antibody.

20. The bispecific antibody according to claim 19, characterized in that The N-terminus of the scfv is linked to the C-terminus of at least one heavy chain of a full-length IgG antibody; or the N-terminus of the scfv is linked to the C-terminus of at least one light chain of a full-length IgG antibody.

21. The bispecific antibody according to any one of claims 10 to 20, characterized in that The first domain and the second domain are connected by a flexible peptide linker, preferably the flexible peptide linker has a length of no more than about 30 amino acids, more preferably the flexible peptide linker is (G4S)3.

22. The bispecific antibody according to any one of claims 10 to 18, characterized in that The bispecific antibody is CrossMAb-Fab.

23. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9.

24. An expression vector comprising the polynucleotide according to claim 23.

25. A host cell comprising the expression vector according to claim 24.

26. 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 25 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.

27. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22, and a pharmaceutically acceptable carrier.

28. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22, for use in treating or preventing HBV infection in a subject in need thereof.

29. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22, for use in treating or preventing HBV and its mutant strains infection in a subject in need thereof.

30. The isolated antibody or antigen-binding fragment thereof or bispecific antibody for use according to claim 28 or 29, characterized in that The HBV includes genotype A, genotype B, genotype C and genotype D.

31. The isolated antibody or antigen-binding fragment thereof or bispecific antibody for use according to claim 29, 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.

32. The isolated antibody or antigen-binding fragment thereof or bispecific antibody for use according to claim 29, characterized in that The mutant strain comprises a mutation selected from the group consisting of G145R, K122R-G145R and F134L.

33. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22, in the preparation of a medicament for treating or preventing HBV infection in a subject in need thereof.

34. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22, in the preparation of a medicament for treating or preventing HBV and its mutant strains infection in a subject in need thereof.

35. The use according to claim 33 or 34, characterized in that The HBV includes genotype A, genotype B, genotype C and genotype D.

36. The use according to claim 34, 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.

37. The use according to claim 34, characterized in that The mutant strain comprises a mutation selected from the group consisting of G145R, K122R-G145R and F134L.

38. A method of treating or preventing HBV infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22.

39. A method for treating or preventing HBV and its mutant strains infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the bispecific antibody according to any one of claims 10-22.

40. The method according to claim 38 or 39, characterized in that The HBV includes genotype A, genotype B, genotype C and genotype D.

41. The method according to claim 39, 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.

42. The method according to claim 39, characterized in that The mutant strain comprises a mutation selected from the group consisting of G145R, K122R-G145R and F134L.

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