Antibodies binding to hepatitis b virus surface antigen and uses thereof

Abstract

The present application belongs to the technical field of antibodies, and provides an antibody or an antigen-binding fragment thereof specifically binding to hepatitis B virus surface antigen (HBsAg), a pharmaceutical composition comprising the antibody or the antigen-binding fragment, and uses thereof. In addition, the present application also provides a nucleic acid molecule encoding the antibody, a vector and a host cell comprising the nucleic acid molecule, and a method for preparing the antibody.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese application 202010659026.2, filed on July 9, 2020, and Chinese application 202010659828.3, filed on July 10, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of antibody technology and relates to an antibody that binds to hepatitis B virus surface antigen, and the use of the antibody in treating diseases related to hepatitis B virus infection. Background Technology

[0004] Hepatitis B virus (HBV) infection is a global epidemic, but the prevalence varies greatly across different regions. According to the World Health Organization, approximately 2 billion people worldwide have been infected with HBV, of whom 240 million are chronic HBV carriers. About 650,000 people die annually from liver failure, cirrhosis, and hepatocellular carcinoma (HCC) caused by HBV infection. Globally, HBV infection accounts for 30% and 45% of cirrhosis and HCC patients, respectively. In my country, the proportions of cirrhosis and HCC patients caused by HBV infection are 60% and 80%, respectively. my country has a large population with chronic hepatitis B; epidemiological surveys show that more than 7% of the population is currently infected with HBV, totaling nearly 100 million people. As of the end of 2015, only 9% of HBV-infected individuals were tested and diagnosed; among those diagnosed, only 8% received treatment (data from the China Hepatitis Prevention and Control Foundation). They not only require long-term or even lifelong treatment, placing a huge burden on their families and society, but also face the risk of developing cirrhosis and liver cancer. It is estimated that there are 28 million chronic hepatitis B patients in my country, with nearly one million new cases of cirrhosis and approximately 300,000 new cases of liver cancer each year. Based on a 5% consultation rate for chronic hepatitis patients and a 95% consultation rate for cirrhosis and liver cancer patients, the direct medical expenses related to the treatment of hepatitis B in my country reach 80-120 billion yuan annually.

[0005] While antiviral treatment can reduce viral load and slow disease progression in hepatitis B patients, many do not completely eliminate the hepatitis B virus. Hepatitis B patients require long-term medication, placing a heavy financial burden on them. According to data released by the Chinese Center for Disease Control and Prevention, the average annual treatment cost for chronic hepatitis patients accounts for 56.24% of the average annual family income, compensated cirrhosis accounts for 81.53%, decompensated cirrhosis accounts for 157.21%, and liver cancer accounts for 96.76%. This heavy economic burden forces many patients to abandon treatment or causes their families to fall into poverty. Furthermore, the evolution of liver disease treatment costs over the past decade shows a year-on-year increase of approximately 10% to 20%, with the individual's share also increasing accordingly.

[0006] Studies show that the large number of subviral particles composed of HBsAg in the blood of hepatitis B patients is a major reason why the immune system is suppressed and unable to clear infected liver cells. Reducing blood HBsAg levels by 10-100 times using certain technologies can activate the body's own T-cell immunity, enabling the body's immune system to effectively kill infected liver cells. Therefore, effectively reducing blood HBsAg levels is not only one of the important criteria for curing hepatitis B, but also a prerequisite for clearing infected cells in hepatitis B treatment.

[0007] The latest clinical research results show that patients who have cleared viral DNA after nucleoside analog treatment have a greater than 50% chance of clearing HBsAg with interferon if their HBsAg level is below a certain level (e.g., HBsAg < 1500 IU / mL), which is much higher than the 5-10% cure rate that we usually think of with interferon.

[0008] However, due to the current lack of effective drugs to lower HBsAg, the vast majority of patients (over 90%) have HBsAg levels exceeding this threshold, making interferon ineffective in curing these patients (which explains why the cure rate of interferon is only 5-10%). This new clinical evidence highlights the more realistic and urgent need for treatments to lower HBsAg.

[0009] Current research has identified three main pathways to reduce hepatitis B virus surface antigen: inhibiting HBsAg expression (siRNA), preventing HBsAg release from cells (NAPs), and HBsAg antibody.

[0010] We have identified antibody technology as a research direction for reducing HBsAg levels. After screening, we have obtained multiple antibodies. In vitro studies have shown that the antibodies of this invention can specifically bind to HBsAg, and animal model studies have shown that they can effectively reduce HBsAg levels. Invention Overview

[0011] One of the objectives of this application is to provide an antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg). In vitro antigen affinity assays have demonstrated that the antibody of this application has the ability to specifically bind to HBsAg. In vivo assays have further demonstrated that the antibody of this application can act as a neutralizing antibody against hepatitis B virus (HBV), effectively reducing HBsAg levels and inhibiting HBV proliferation.

[0012] Specifically, this application relates to:

[0013] 1. An antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg), comprising a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein:

[0014] The heavy chain variable region contains a complementarity-determining region (CDR) amino acid sequence as shown below:

[0015] HCDR1:X1YX3FX5X6X7Y (SEQ ID NO: 33),

[0016] HCDR2: X11NX13X14X15X16X17X18 (SEQ ID NO: 34),

[0017] HCDR3: ARDX21WX23X24X25X26DX28YGMDX33 (SEQ ID NO: 35);

[0018] The light chain variable region contains the following CDR amino acid sequence:

[0019] LCDR1:X34X35X36SX38X39 (SEQ ID NO: 36),

[0020] LCDR2: X40X41X42 (SEQ ID NO: 37),

[0021] LCDR3: QQSYSTPLX51 (SEQ ID NO: 38);

[0022] Among them, X1, X3, X5, X6, X7, X11, X13, X14, X15, X16, X17, X18, X21, X23, X24, X25, X26, X28, X33, X34, X35, X36, X38, X39, X40, X41, X42, and X51 are each selected from any one amino acid.

[0023] 2. The antibody or antigen-binding fragment of HBsAg specifically binding to item 1, wherein:

[0024] X1 is selected from G or A, X3 is selected from T, A or S, X5 is selected from T, A or I, X6 is selected from G, A, Y or D, X7 is selected from Y or A, X11 is selected from I or A, X13 is selected from P or A, X14 is selected from N, A or Y, X15 is selected from S, A or N, X16 is selected from G or A, X17 is selected from G or A, X18 is selected from T or A, X21 is selected from L, V or A, X23 is selected from N, Q or A, X2 4 is selected from D, Q, or A; X25 is selected from D, G, or A; X26 is selected from V, G, or A; X28 is selected from Y or A; X33 is selected from V or A; X34 is selected from Q or A; X35 is selected from S or A; X36 is selected from I, A, or V; X38 is selected from T, A, or S; X39 is selected from Y or A; X40 is selected from A, G, D, T, or S; X41 is selected from A or S; X42 is selected from A or S; X51 is selected from T or A.

[0025] 3. The antibody or antigen-binding fragment of HBsAg specifically binding to item 2, wherein:

[0026] In HCDR1, X1, X3, X5, X6, and X7 are selected from any of the following combinations in sequence:

[0027] G, T, T, G, Y; A, T, T, G, Y; G, A, T, G, Y; G, T, A, G, Y; G, T, T, A, Y; G, T, T, G, A; G, S, I, G, Y; G, T, T, D, Y; G, T, T, Y, Y;

[0028] In HCDR2, X11, X13, X14, X15, X16, X17, and X18 are selected from any of the following combinations in sequence: I, P, N, S, G, G, T; A, P, N, S, G, G, T; I, A, N, S, G, G, T; I, P, A, S, G, G, T; I, P, N, A, G, G, T; I, P, N, S, A, G, T; I, P, N, S, G, G, A; I, P, Y, N, G, G, T;

[0029] In HCDR3, X21, X23, X24, X25, X26, X28, and X33 are selected from any of the following combinations in sequence: L, N, D, D, V, Y, V; A, ​​N, D, D, V, Y, V; L, A, D, D, V, Y, V; L, N, A, D, V, Y, V; L, N, D, A, V, Y, V; L, N, D, D, A, Y, V; L, N, D, D, V, A, V; L, N, D, D, V, Y, A; V, Q, Q, G, G, Y, V;

[0030] In LCDR1, X34, X35, X36, X38, and X39 are selected from any of the following combinations in sequence:

[0031] Q, S, I, T, Y; A, S, I, T, Y; Q, A, I, T, Y; Q, S, A, T, Y; Q, S, I, A, Y; Q, S, I, T, A; Q, S, I, S, Y; Q, S, V, S, Y;

[0032] In LCDR2, X40, X41, and X42 are selected from any of the following combinations in sequence:

[0033] A,A,S;S,A,S;A,S,S;A,A,A;G,A,S;D,A,S;T,A,S;and,

[0034] X51 in LCDR3 is selected from: T or A

[0035] 4. The antibody or antigen-binding fragment of HBsAg specifically binding to item 3, wherein X1, X3, X5, X6, X7, X11, X13, X14, X15, X16, X17, X18, X21, X23, X24, X25, X26, X28, and X33 are selected sequentially from any combination in Table 1 below:

[0036] Table 1:

[0037]

[0038]

[0039] 5. The antibody or antigen-binding fragment of HBsAg specifically binding to item 3 or 4, wherein X34, X35, X36, X38, X39, X40, X41, X42, and X51 are selected sequentially from any combination in Table 2 below:

[0040] Table 2:

[0041]

[0042] 6. The antibody or antigen-binding fragment of HBsAg specifically binding to item 3, wherein X1, X3, X5, X6, X7, X11, X13, X14, X15, X16, X17, X18, X21, X23, X24, X25, X26, X28, X33, X34, X35, X36, X38, X39, X40, X41, X42, and X51 are selected sequentially from any combination of those in Table 3 below:

[0043] Table 3:

[0044]

[0045]

[0046]

[0047]

[0048] In some embodiments, in the antibody or antigen-binding fragment thereof that specifically binds to HBsAg, the VH comprises a complementarity-determining region (CDR) amino acid sequence as shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, HCDR3 as shown in SEQ ID NO: 23, and the VL comprises LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; LCDR3 as shown in SEQ ID NO: 32; or

[0049] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0050] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 20, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 24, and the VL contains LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0051] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 18, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0052] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 29; and LCDR3 as shown in SEQ ID NO: 32; or

[0053] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 30; and LCDR3 as shown in SEQ ID NO: 32; or

[0054] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 30; and LCDR3 as shown in SEQ ID NO: 32; or

[0055] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 31; and LCDR3 as shown in SEQ ID NO: 32.

[0056] 7. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of items 1-6, comprising a human universal framework region (FR). In some embodiments, the VH comprises a human subgroup III universal framework region (FR). In some embodiments, the VL comprises a human subgroup III universal framework region (FR). Universal framework. In some implementations, it includes a human universal framework region (FR) and one or more amino acid substitutions based on the human universal framework region (FR).

[0057] In some embodiments, the framework region is a human universal framework region and contains one or more (e.g., 1-20, 1-15, 1-10, 1-5, 1-4, 1-3) amino acid substitutions. In some embodiments, the framework region is a human universal framework region or has 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity with a human universal framework region. In some exemplary embodiments of the invention, the VH contains the following FR amino acid sequence:

[0058] HFR1: Z1Z2Z3LZ4Z5SGAEVKKPGASZ6KVSCKAS (SEQ ID NO: 39)

[0059] HFR2: Z7HWVRQAPGQGZ8EWMGW (SEQ ID NO: 40)

[0060] HFR3: NYAQKFQGRVTZ9TZ10DZ11SZ12STAYMELSZ13LRSZ14DTAVYYC (SEQ ID NO: 41)

[0061] HFR4: WGZ15GTZ16VTVSS (SEQ ID NO: 42)

[0062] VL contains the following FR amino acid sequence:

[0063] LFR1: Z17Z18Z19LTQSPZ20Z21LSZ22SZ23GZ24RZ25TZ26Z27CRAS (SEQ ID NO: 43)

[0064] LFR2: LZ28WYQQKPGZ29APZ30LLIZ31 (SEQ ID NO: 44)

[0065] LFR3: Z32Z33Z34Z35GZ36PZ37RFSGGSGTZ38FTLTIZ39SLZ40Z41Z42DZ43ATYYC (SEQID NO: 45)

[0066] LFR4: FGZ44GTZ45Z46Z47IKR (SEQ ID NO: 46)

[0067] Z1-Z47 are each selected from any one amino acid.

[0068] 8. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg, as specified in item 7, wherein...

[0069] Z1 is selected from Q or E, Z2 from V or I, Z3 from Q or T, Z4 from V or K, Z5 from E or Q, Z6 from V or M, Z7 from M, I or L, Z8 from L or P, Z9 from M or I, Z10 from R or A, Z11 from T or K, Z12 from I or T, Z13 from R or S, Z14 from D or E, Z15 from K or Q, Z16 from L or M, Z17 from D or E, Z18 from I or T, Z19 from Q, T or V, Z20 from S, A or G, Z21 from S or T, Z22 from A or L, Z23 from V or P, Z24 from D or E, Z 25 is selected from V or A, Z26 is selected from I or L, Z27 is selected from T or S, Z28 is selected from N or A, Z29 is selected from K or Q, Z30 is selected from K, Q or R, Z31 is selected from Y or S, Z32 is selected from S or N, Z33 is selected from L or R, Z34 is selected from Q or A, Z35 is selected from S or T, Z36 is selected from V or I, Z37 is selected from S or A, Z38 is selected from D or E, Z39 is S or R, Z40 is selected from Q or E, Z41 is selected from P or S, Z42 is selected from E or G, Z43 is selected from F or L, Z44 is selected from G, Q or P, Z45 is selected from K or R, Z46 is selected from V or L, Z47 is selected from D or E.

[0070] 9. The antibody or antigen-binding fragment of HBsAg specifically binding to Item 8, wherein:

[0071] In HFR1, Z1, Z2, Z3, Z4, Z5, and Z6 are selected from any of the following combinations in sequence: Q, V, Q, V, E, V; E, V, Q, V, Q, V; E, V, Q, V, E, V; Q, I, T, K, E, V; E, V, Q, V, Q, M;

[0072] In HFR2, Z7 and Z8 are selected from any of the following combinations in sequence: M, L; L, L; I, P;

[0073] The combinations of Z9, Z10, Z11, Z12, Z13, and Z14 in HFR3 are: M, R, T, I, R, D; or I, A, K, T, S, E.

[0074] In HFR4, Z15 and Z16 are selected from any of the following combinations in sequence: K, L; Q, M; Q, L; or K, M;

[0075] 10. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg, as specified in item 8 or 9, wherein:

[0076] In LFR1, Z17, Z18, Z19, Z20, Z21, Z22, Z23, Z24, Z25, Z26, and Z27 are selected sequentially from any of the following combinations: D, I, Q, S, S, A, V, D, V, I, T; E, I, V, G, T, L, P, E, A, L, S; E, I, V, A, T, L, P, E, A, L, S; E, T, T, S, T, A, V, D, V, I, T;

[0077] In LFR2, Z28, Z29, Z30, and Z31 are selected from any of the following combinations in sequence: N, K, K, Y; A, Q, R, Y; N, K, K, S; N, K, Q, Y;

[0078] In LFR3, Z32, Z33, Z34, Z35, Z36, Z37, Z38, Z39, Z40, Z41, Z42, and Z43 are selected from any of the following combinations in sequence: S, L, Q, S, V, S, D, S, Q, P, E, F; S, R, A, T, I, A, E, S, Q, S, E, F; N, R, A, T, I, A, D, S, E, P, E, F; S, L, Q, S, V, S, E, R, Q, P, E, F; S, L, Q, S, V, S, D, S, Q, P, G, L;

[0079] In LFR4, Z44, Z45, Z46, and Z47 are selected from any of the following combinations in sequence: G, K, V, D; G, K, L, E; P, K, V, E; G, K, V, E; Q, R, L, E;

[0080] 11. The antibody or antigen-binding fragment of HBsAg specifically bound to item 10, wherein Z1-Z16 are selected sequentially from any combination in Table 4 below:

[0081] Table 4:

[0082]

[0083] 12. An antibody or antigen-binding fragment of any one of items 8-11 that specifically binds to HBsAg, wherein Z17-Z47 are selected sequentially from any combination in Table 5 below:

[0084] Table 5:

[0085]

[0086] 13. The antibody or antigen-binding fragment of HBsAg specifically binding to item 8, Z1-Z47, are selected sequentially from any combination in Table 6 below:

[0087] Table 6:

[0088]

[0089]

[0090] 14. The antibody or antigen-binding fragment of the antibody that specifically binds to HBsAg in item 1, wherein the VH sequence is selected from any sequence in SEQ ID NO: 1-6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, or 99% identity with any sequence in SEQ ID NO: 1-6.

[0091] 15. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg, as described in item 1 or 14, wherein the VL sequence is selected from any sequence in SEQ ID NO: 7-13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, or 99% identity with any sequence in SEQ ID NO: 7-13.

[0092] 16. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg, as specified in any one of items 1-15, wherein the VH sequence and VL sequence are selected from any one of the following groups:

[0093] The amino acid sequences that have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with the above-mentioned VH or VL sequences. SEQ ID NO:1 and SEQ ID NO:7, SEQ ID NO:1 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:12, SEQ ID NO:2 and SEQ ID NO:13, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:9, SEQ ID NO:5 and SEQ ID NO:10, SEQ ID NO:5 and SEQ ID NO:11, SEQ ID NO:6 and SEQ ID NO:7.

[0094] 17. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg from any of items 1-16, selected from: Fab, F(ab')2, Fab', scFv, Fv, Fd, dAb, diabody, or multibody.

[0095] 18. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg in any of items 1-17, wherein the antibody is a fully human antibody, a humanized antibody, a murine antibody, a chimeric antibody, or a nanobody.

[0096] 19. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of items 1-18, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG, and the light chain constant region is selected from the κ chain or the λ chain.

[0097] 20. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg as described in item 19, wherein the heavy chain constant region is the heavy chain constant region of human IgG1. In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 14, or comprises an amino acid sequence having 70% or more sequence identity with SEQ ID NO: 14 (e.g., amino acid sequences with 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity).

[0098] 21. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to item 19 or 20, wherein the light chain constant region is derived from the human κ chain. In some embodiments, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 15, or comprises an amino acid sequence having 70% or more sequence identity with SEQ ID NO: 15 (e.g., an amino acid sequence with 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity).

[0099] 22. An antibody or antigen-binding fragment thereof that specifically binds to HBsAg in any of items 1-21, which is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0100] 23. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to HBsAg, any one of items 1-22.

[0101] 24. A construct comprising the nucleic acid molecule in the term, said construct being selected from: plasmids, phage particles, viral vectors, or linear nucleic acids.

[0102] 25. A virus, bacteriophage, or cell that expresses an antibody or antigen-binding fragment thereof that specifically binds to HBsAg, any one of items 1-22, or a nucleic acid contained in item 23 or a construct contained in item 24.

[0103] 26. A pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of items 1-22, a nucleic acid according to item 23, a construct according to item 24, or a virus, bacteriophage, or cell according to item 25.

[0104] 27. A kit comprising: an antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of items 1-22, a nucleic acid according to item 23, a construct according to item 24, or a virus, bacteriophage, or cell according to item 25.

[0105] 28. A method for treating or preventing hepatitis B virus (HBV) infection, or for relieving hepatitis B symptoms, comprising administering to a patient an effective amount of the pharmaceutical composition of item 26.

[0106] 29. A method for detecting HBV, comprising contacting an antibody or antigen-binding fragment thereof that specifically binds to HBsAg, any one of items 1-22, a nucleic acid, any construct, any virus, or any phage from item 25, with bodily fluids from a subject.

[0107] 30. The method of item 29, wherein the bodily fluid is the subject's plasma or serum. Attached Figure Description

[0108] Figure 1 This study demonstrates the effects of tail vein injection of antibodies 005, 062, 079, and 083 (selected from the constant region of murine IgG1) on plasma HBV DNA in AAV / HBV mice.

[0109] Figure 2 This study demonstrates the effects of tail vein injection of murine IgG1 constant region antibodies 005, 062, 079, and 083 on plasma HBsAg in AAV / HBV mice.

[0110] Figure 3 This study demonstrates the effects of tail vein injection of antibodies 005, 021, 062, and 088 (selected from the constant region of murine IgG1) on plasma HBV DNA in AAV / HBV mice.

[0111] Figure 4 This study demonstrates the effects of tail vein injection of murine IgG1 constant region antibodies 005, 021, 062, and 088 on plasma HBsAg in AAV / HBV mice.

[0112] Figure 5 The effects of tail vein injection of antibodies 021, 090, 091, and 093 selected from the constant region of human IgG1, and intraperitoneal injection of antibody 021 selected from the constant region of human IgG1, on plasma HBV DNA in AAV / HBV mice were shown.

[0113] Figure 6 This study demonstrates the effects of tail vein injection of antibodies selected from the human IgG1 constant region (021, 090, 091, and 093) and intraperitoneal injection of antibody selected from the human IgG1 constant region on plasma HBsAg in AAV / HBV mice.

[0114] Figure 7 This study showed the effects of tail vein injection of antibodies 021, 095, and 096 selected from the constant region of human IgG1, and intraperitoneal injection of antibody 021 selected from the constant region of human IgG1, on plasma HBV DNA in AAV / HBV mice.

[0115] Figure 8 The effects of tail vein injection of antibodies 021, 095, and 096 selected from the human IgG1 constant region, and intraperitoneal injection of 021 selected from the mouse IgG1 constant region, on plasma HBsAg in AAV / HBV mice were shown.

[0116] Figure 9 This study demonstrates the effect of continuous administration of the antibody of this application to mice on plasma HBsAg in AAV / HBV mice. Invention Details

[0117] This application aims to provide a novel antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg). Through this binding, it reduces the expression level of HBsAg in the patient's blood, activates the body's own T-cell immunity, and enables the body's immune system to effectively kill infected hepatocytes and inhibit HBV proliferation. Through this specifically binding antibody or antigen-binding fragment, this application provides new options and approaches for the prevention and treatment of HBV, and also provides new tools for the detection and diagnosis of HBV.

[0118] definition

[0119] As used herein, the term "antibody," also known as "immunoglobulin," encompasses antibodies that have the structural features of natural antibodies and antibody-like molecules that have structural features different from those of natural antibodies but exhibit specificity for binding to antigen molecules. The term antibody is intended to include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0120] In this paper, the “antigen-binding fragment” of an antibody can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include, in particular, Fab, Fab', F(ab')2, FV, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, bispecific antibodies, and peptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the peptide. Examples of antigen-antibody fragments described herein include, but are not limited to: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments, i.e., Fab fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains of an antibody single arm; and (vi) dAb fragments consisting of a VH domain (Ward et al., Nature). 341, 544-546 (1989)); (vii) isolated CDR region; (viii) F(ab')2 fragment, a bivalent fragment containing two Fab' fragments bridged by disulfide bonds in the hinge region; (ix) single-chain antibody molecule (e.g., single-chain Fv; scFv) (Bird et al., Science 242: 423-426 (1988); and Huston et al., PNAS (USA) 85: 5879-5883 (1988)); (x) "biantibody" having two antigen-binding sites, which Includes a heavy chain variable region (VH) linked to a light chain variable region (VL) in the same polypeptide chain (see, for example, EP404,097; WO93 / 11161; and Hollinger et al., Pr℃.Natl.Acad.Sci.USA, 90:6444-6448(1993)); (xi) "linear antibody" which includes a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062(1995); and U.S. Patent No. 5,641,870).

[0121] The terms “heavy chain” (“CH”), “light chain” (“CL”), “light chain variable region” (“VL”), “heavy chain variable region” (“VH”), and “framework region” (“FR”) refer to the domains in naturally occurring immunoglobulins and their corresponding domains in synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a tetramer with two light chains and two heavy chains. The amino-terminal (“N”) portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl terminus (the "C" portion of each chain) defines a constant region; light chains have a single constant region, while heavy chains typically have three constant regions and a hinge region. Therefore, the naturally occurring light chain structure of the IgG molecule is N'-VL-CL-C', and the heavy chain structure is N'-VH-CH1-H-CH2-CH3-C' (where H is the hinge region). The variable region of the IgG molecule contains the complementarity-determining region (CDR), which is responsible for recognizing and contacting antigens. The non-CDR fragments, i.e., the frame region (FR), maintain the variable region structure and determine the CDR. The position of the rings. Therefore, the VL and VH domains have the structure N'-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C'. In this application, the three CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3; the four frame regions of VH are referred to as HFR1, HFR2, HFR3, and HFR4; the three CDRs of VL are referred to as LCDR1, LCDR2, and LCDR3; and the four frame regions of VL are referred to as LFR1, LFR2, LFR3, and LFR4.

[0122] In this application, the CDR and FR are determined according to the ImMunoGeneTics (IMGT) numbering system (see, for example, Lefranc MP The IMGT unique numbering for immunoglobulins, T-cell receptors, and Ig-like domains. The immunologist 7, 132-136, 1999 (1999)).

[0123] In this paper, the term "nanobody" refers to a single-domain antibody composed solely of the variable region of the antibody heavy chain. It is called a nanobody because of its relative molecular size, which is on the nanometer scale. Nanobodies can bind tightly to antigens like normal antibodies, but they do not easily aggregate into clumps like single-chain antibodies. The term "single-domain antibody" refers to an independent antigen-binding unit consisting of a variable region or an engineered constant domain that only assists in target binding. The term "dimeric antibody" refers to a dimer of scFvs, formed by the co-expression of two scFv fragments through interchain pairing (cross-pairing) of the VH and VL domains. Similarly, multimers formed by multiple scFvs are called multimeric antibodies. For example, trimeric antibodies formed by three scFvs, tetrameric antibodies formed by four scFvs, etc. The scFvs can have the same antigen specificity or different antigen specificities.

[0124] As used in this article, "monospecific antibody," "bispecific antibody," and "multispecific antibody" refer to a single antibody molecule that can bind to one, two, or more different antigens or one, two, or more different antigenic epitopes of the same antigen, respectively.

[0125] As used herein, the term "chimeric antibody" refers to an antibody that combines antibody fragments from different species. Specifically, for example, a monoclonal antibody from one species (e.g., mouse) whose Fc constant region is replaced by an Fc constant region from another species (e.g., human) via DNA recombination technology. See, for example, patent applications PCT / US86 / 02269; EP / 173,494.

[0126] As used herein, the term "humanized antibody" refers to an antibody that comprises the human immunoglobulin framework region and one or more core-residue domains (CDRs) derived from a non-human (e.g., mouse, rat, rabbit, or synthetic) immunoglobulin. Apart from the CDRs, all other portions of a humanized antibody are substantially identical to the corresponding portions of the natural human immunoglobulin sequence. Methods for constructing humanized antibodies through genetic engineering are illustrated, for example, in patent application US / 5,585,089.

[0127] As used herein, the term "fully human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Fully human antibodies of this technology may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, as used herein, the term "fully human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., rabbit) has been grafted onto a human framework sequence. Therefore, as used herein, the term "fully human antibody" refers to an antibody whose protein molecule is virtually non-immunogenic in humans, with only minor sequence variations or alterations relative to human native immunoglobulins, and whose almost all parts (e.g., CDR, FR, CL, HC domains (e.g., CH1, CH2, CH3), hinge, VL, VH) exhibit only minor sequence variations or alterations relative to human native immunoglobulins. Therefore, fully human antibodies differ from chimeric or humanized antibodies. It should be noted that fully human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells that are capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes.

[0128] As used herein, the term "specific binding" refers to the property of complementary binding with high affinity, determined by the spatial conformation of the antigenic determinants and the variable regions of the antibody molecule. This high affinity determines that once the antibody molecule binds to the antigen, it can perform its corresponding physiological function, for example, in some embodiments of this application, the antibody binds to and helps clear the antigen.

[0129] The term "human universal framework" refers to the framework containing the most frequently occurring amino acid residues in the selected human immunoglobulin VL or VH framework sequence. Generally, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. Typically, this subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In some embodiments, for VL, the subgroup is as described in Kabat et al. (ibid.). In some implementations, for VH, the subgroup is subgroup III as described by Kabat et al. (ibid.).

[0130] The "homology" or "identity" between two amino acid sequences or nucleotide sequences refers to the percentage of amino acid residues or nucleotide residues that are completely identical between the two sequences. If the two sequences to be compared differ in length, the "homology" or "identity" preferably refers to the percentage of nucleotide residues in the shorter sequence that are completely identical to the amino acid residues or nucleotide residues in the longer sequence. Sequence identity can be routinely determined using sequence analysis software commonly used in the art, such as the Wisconsin sequence analysis package.

[0131] In this application, the terms "polynucleotide" or "nucleic acid" and "nucleic acid molecule" are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0132] As used in this article, "construction" refers to a vector that can introduce a multinucleotide sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence, including plasmids, phage particles, viral vectors, etc.

[0133] As used herein, the term "phageparticle" refers to a vector derived from filamentous phages. Its basic components primarily include the plasmid's origin of replication, selection markers, and intergenic spacer (IG region). It typically also contains negative and positive strand packaging sequences and origins of replication, the gene for the phage coat protein, restriction endonuclease recognition sites, a promoter, and a DNA fragment encoding a signal peptide. Furthermore, phageparticles may contain a molecular tag to facilitate the screening of phageparticle-based libraries. Phageparticles cannot independently assemble progeny phage particles; other structural and functional proteins required for their life cycle are provided by helper phages. Helper phages are mutant filamentous phages with extremely low DNA replication efficiency. Therefore, when helper phages co-infect the host bacterium with phageparticle-packaged phages, a large number of phageparticle-containing phages are packaged, while only a small number of helper phages are packaged.

[0134] As used in this article, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast, and actinomycetes. It exists in the cytoplasm or nucleus, has the ability to replicate autonomously, and maintains a constant copy number in daughter cells, while expressing the genetic information it carries.

[0135] As used in this article, "viral vector" refers to a virus-based gene vector, which is a genetically engineered viral genome that can carry foreign genes and related gene elements and is packaged into viral particles. The foreign genes carried by the virus are introduced into cells through viral infection.

[0136] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0137] Antibody or its antigen-binding fragment

[0138] On the one hand, this application provides an antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg). Typically, the antibody of this application comprises a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein:

[0139] The heavy chain variable region comprises the complementarity-determining region (CDR) amino acid sequence of the following general formula:

[0140] HCDR1:X1YX3FX5X6X7Y (SEQ ID NO: 33),

[0141] HCDR2: X11NX13X14X15X16X17X18 (SEQ ID NO: 34),

[0142] HCDR3: ARDX21WX23X24X25X26DX28YGMDX33 (SEQ ID NO: 35);

[0143] The light chain variable region contains the following CDR amino acid sequence:

[0144] LCDR1:X34X35X36SX38X39 (SEQ ID NO: 36),

[0145] LCDR2: X40X41X42 (SEQ ID NO: 37),

[0146] LCDR3: QQSYSTPLX51 (SEQ ID NO: 38);

[0147] X1, X3, X5, X6, X7, X11, X13, X14, X15, X16, X17, X18, X21, X23, X24, X25, X26, X28, X33, X34, X35, X36, X38, X39, X40, X41, X42, and X51 can all be selected from any one of the 20 natural amino acids. The selected amino acid can be any one of the 20 natural amino acids, or it can be an artificially synthesized or modified amino acid.

[0148] HCDR1, HCDR2, and HCDR3 represent three CDRs in the heavy chain variable region, arranged sequentially from the N' end to the C' end of the heavy chain variable region. Similarly, LCDR1, LCDR2, and LCDR3 represent three CDRs in the light chain variable region, arranged sequentially from the N' end to the C' end of the light chain variable region.

[0149] In the embodiments of this application, a selection of amino acids were used to detect the CDR provided by the general formula, including: when X1 is G or A, X3 is T, A or S, X5 is T, A or I, X6 is G, A, Y or D, X7 is Y or A, X11 is I or A, X13 is P or A, X14 is N, A or Y, X15 is S, A or N, X16 is G or A, X17 is G or A, X18 is T or A, and X21 is L. When A or V, X23 is N, Q, or A; X24 is D, Q, or A; X25 is D, G, or A; X26 is V, G, or A; X28 is Y or A; X33 is V or A; X34 is Q or A; X35 is S or A; X36 is I, A, or V; X38 is T, A, or S; X39 is Y or A; X40 is A, G, D, T, or S; X41 is A or S; X42 is A or S; and X51 is T or A. The detection results confirm that the antibody of this application containing the above general formula can specifically bind to HBsAg.

[0150] Those skilled in the art should know that the values ​​of each Xn (where n represents the integer subscript of each X in the general formula) in the general formula can be combined with each other. For example, in some embodiments, X1, X3, X5, X6 and X7 in HCDR1 are selected from any of the following combinations in sequence: G, T, T, G, Y; A, T, T, G, Y; G, A, T, G, Y; G, T, A, G, Y; G, T, T, A, Y; G, T, T, G, A; G, S, I, G, Y; G, T, T, D, Y; G, T, T, Y, Y. In some specific embodiments, HCDR1 is selected from GYTFTGYY (SEQ ID NO: 17); GYSFIGYY (SEQ ID NO: 18); GYTFTDYY (SEQ ID NO: 19); GYTFTYYY (SEQ ID NO: 20).

[0151] In HCDR2, X11, X13, X14, X15, X16, X17, and X18 are selected sequentially from any of the following combinations: I, P, N, S, G, G, T; A, P, N, S, G, G, T; I, A, N, S, G, G, T; I, P, A, S, G, G, T; I, P, N, A, G, G, T; I, P, N, S, A, G, T; I, P, N, S, G, G, A; I, P, Y, N, G, G, T. In some specific embodiments, HCDR2 is selected from INPNSGGT (SEQ ID NO: 21); INPYNGGT (SEQ ID NO: 22).

[0152] In HCDR3, X21, X23, X24, X25, X26, X28, and X33 are selected sequentially from any of the following combinations: L, N, D, D, V, Y, V; A, ​​N, D, D, V, Y, V; L, A, D, D, V, Y, V; L, N, A, D, V, Y, V; L, N, D, A, V, Y, V; L, N, D, D, A, Y, V; L, N, D, D, V, A, V; L, N, D, D, V, Y, A; V, Q, Q, G, G, Y, V. In some specific embodiments, HCDR3 is ARDLWNDDVDYYGMDV (SEQ ID NO: 23) or ARDLWQQGGYYYYMDV (SEQ ID NO: 24).

[0153] In LCDR1, X34, X35, X36, X38, and X39 are selected sequentially from any of the following combinations: Q, S, I, T, Y; A, S, I, T, Y; Q, A, I, T, Y; Q, S, A, T, Y; Q, S, I, A, Y; Q, S, I, T, A; Q, S, I, S, Y; Q, S, V, S, Y. In some specific embodiments, LCDR1 is selected from QSISTY (SEQ ID NO: 25); QSISSY (SEQ ID NO: 26); and QSVSSY (SEQ ID NO: 27).

[0154] In LCDR2, X40, X41, and X42 are selected from any of the following combinations in sequence: A, A, S; S, A, S; A, S, S; A, A, A; G, A, S; D, A, S; T, A, S; In some specific embodiments, LCDR2 is selected from AAS (SEQ ID NO: 28); GAS (SEQ ID NO: 29); DAS (SEQ ID NO: 30); TAS (SEQ ID NO: 31).

[0155] X51 in LCDR3 is selected from T or A. In some specific embodiments, LCDR3 is QQSYSTPLT (SEQ ID NO: 32).

[0156] In some implementations, the VH includes:

[0157] HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, HCDR3 as shown in SEQ ID NO: 23; or

[0158] HCDR1 as shown in SEQ ID NO: 20, HCDR2 as shown in SEQ ID NO: 21, HCDR3 as shown in SEQ ID NO: 24; or

[0159] HCDR1 as shown in SEQ ID NO: 18, HCDR2 as shown in SEQ ID NO: 22, HCDR3 as shown in SEQ ID NO: 23; or

[0160] HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23.

[0161] In some implementations, the VL includes:

[0162] LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; LCDR3 as shown in SEQ ID NO: 32; or

[0163] LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 28; LCDR3 as shown in SEQ ID NO: 32; or

[0164] LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 29; LCDR3 as shown in SEQ ID NO: 32; or

[0165] LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 30; LCDR3 as shown in SEQ ID NO: 32; or

[0166] LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 30; LCDR3 as shown in SEQ ID NO: 32; or

[0167] LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 31; and LCDR3 as shown in SEQ ID NO: 32.

[0168] In some embodiments, the antibody that specifically binds to hepatitis B virus surface antigen (HBsAg) or its antigen-binding fragment comprises a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein:

[0169] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0170] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0171] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 20, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 24, and the VL contains LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0172] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 18, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO: 32; or

[0173] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 29; and LCDR3 as shown in SEQ ID NO: 32; or

[0174] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 27; LCDR2 as shown in SEQ ID NO: 30; and LCDR3 as shown in SEQ ID NO: 32; or

[0175] The VH contains the complementarity-determining region (CDR) amino acid sequences as shown below: HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 30; and LCDR3 as shown in SEQ ID NO: 32; or

[0176] The VH contains the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23, and the VL contains LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 31; and LCDR3 as shown in SEQ ID NO: 32.

[0177] After selecting any one of the combinations, a specific sequence of CDRs can be determined according to the above general formula. The specific sequences of HCDR1, HCDR2, and HCDR3 can be further combined to form specific heavy chain CDR combinations, such as the combinations in Table 1 above. Similarly, the specific sequences of LCDR1, LCDR2, and LCDR3 can be further combined to form specific light chain CDR combinations, such as the combinations in Table 2 above. The groups of HCDRs and LCDRs can also be randomly combined. For example, in some specific embodiments, the CDR combinations of light and heavy chains are shown in Table 3 above.

[0178] Furthermore, in some embodiments, the antibody that specifically binds to HBsAg also includes frame regions (FRs) flanking each CDR region. These frame regions are universal human frame regions, or universal human frame regions containing at least one amino acid substitution, deletion, or insertion. In one exemplary embodiment, VH contains the following FR amino acid sequence:

[0179] HFR1: Z1Z2Z3LZ4Z5SGAEVKKPGASZ6KVSCKAS (SEQ ID NO: 39)

[0180] HFR2: Z7HWVRQAPGQGZ8EWMGW (SEQ ID NO: 40)

[0181] HFR3: NYAQKFQGRVTZ9TZ10DZ11SZ12STAYMELSZ13LRSZ14DTAVYYC (SEQ ID NO: 41)

[0182] HFR4: WGZ15GTZ16VTVSS (SEQ ID NO: 42)

[0183] VL contains the following FR amino acid sequence:

[0184] LFR1: Z17Z18Z19LTQSPZ20Z21LSZ22SZ23GZ24RZ25TZ26Z27CRAS (SEQ ID NO: 43)

[0185] LFR2: LZ28WYQQKPGZ29APZ30LLIZ31 (SEQ ID NO: 44)

[0186] LFR3: Z32Z33Z34Z35GZ36PZ37RFSGGSGTZ38FTLTIZ39SLZ40Z41Z42DZ43ATYYC (SEQID NO: 45)

[0187] LFR4: FGZ44GTZ45Z46Z47IKR (SEQ ID NO: 46), wherein Z1-Z47 are each selected from any one amino acid. The amino acid can be any one of the 20 natural amino acids, or it can be an artificially synthesized or modified amino acid.

[0188] HFR1, HFR2, HFR3, and HFR4 represent the four FRs in the heavy chain variable region, arranged sequentially from the N' end to the C' end. Similarly, LFR1, LFR2, LFR3, and LFR4 represent the four FRs in the light chain variable region, arranged sequentially from the N' end to the C' end.

[0189] In the embodiments of this application, a subset of amino acids were selected to constitute a specific FR sequence. Random matching with the aforementioned CDR was performed to detect the supporting effect of the general formula of the FR amino acid sequence on the CDR conformation. The selected amino acids include, for example:

[0190] Z1 is selected from Q or E, Z2 from V or I, Z3 from Q or T, Z4 from V or K, Z5 from E or Q, Z6 from V or M, Z7 from M, I or L, Z8 from L or P, Z9 from M or I, Z10 from R or A, Z11 from T or K, Z12 from I or T, Z13 from R or S, Z14 from D or E, Z15 from K or Q, Z16 from L or M, Z17 from D or E, Z18 from I or T, Z19 from Q, T or V, Z20 from S, A or G, Z21 from S or T, Z22 from A or L, Z23 from V or P, Z24 from D or E, Z 25 is selected from V or A, Z26 is selected from I or L, Z27 is selected from T or S, Z28 is selected from N or A, Z29 is selected from K or Q, Z30 is selected from K, Q or R, Z31 is selected from Y or S, Z32 is selected from S or N, Z33 is selected from L or R, Z34 is selected from Q or A, Z35 is selected from S or T, Z36 is selected from V or I, Z37 is selected from S or A, Z38 is selected from D or E, Z39 is S or R, Z40 is selected from Q or E, Z41 is selected from P or S, Z42 is selected from E or G, Z43 is selected from F or L, Z44 is selected from G, Q or P, Z45 is selected from K or R, Z46 is selected from V or L, Z47 is selected from D or E.

[0191] In some implementation schemes, Z1, Z2, Z3, Z4, Z5, and Z6 in HFR1 are selected from any of the following combinations in sequence: Q, V, Q, V, E, V; E, V, Q, V, Q, V; E, V, Q, V, E, V; Q, I, T, K, E, V; E, V, Q, V, Q, M; Z7 and Z8 in HFR2 are selected from any of the following combinations in sequence: M, L; L, L; I, P; Z9, Z10, Z11, Z12, Z13, and Z14 in HFR3 are combined as follows: M, R, T, I, R, D; or I, A, K, T, S, E; Z15 ​​and Z16 in HFR4 are combined as follows: K, L; Q, M; Q, L; or K, M. In some embodiments, Z17, Z18, Z19, Z20, Z21, Z22, Z23, Z24, Z25, Z26, and Z27 in LFR1 are selected sequentially from any of the following combinations: D, I, Q, S, S, A, V, D, V, I, T; E, I, V, G, T, L, P, E, A, L, S; E, I, V, A, T, L, P, E, A, L, S; E, T, T, S, T, A, V, D, V, I, T. In some embodiments, Z28, Z29, Z30, and Z31 in LFR2 are selected sequentially from any of the following combinations: N, K, K, Y; A, Q, R, Y; N, K, K, S; N, K, Q, Y. In some implementations, Z32, Z33, Z34, Z35, Z36, Z37, Z38, Z39, Z40, Z41, Z42, and Z43 in LFR3 are selected sequentially from any of the following combinations: S, L, Q, S, V, S, D, S, Q, P, E, F; S, R, A, T, I, A, E, S, Q, S, E, F; N, R, A, T, I, A, D, S, E, P, E, F; S, L, Q, S, V, S, E, R, Q, P, E, F; S, L, Q, S, V, S, D, S, Q, P, G, L. In some implementations, Z44, Z45, Z46, and Z47 in LFR4 are selected from any of the following combinations in sequence: G, K, V, D; G, K, L, E; P, K, V, E; G, K, V, E; Q, R, L, E.

[0192] In some implementations, the variable parameter Zn of FR in the variable region of the heavy chain (where n represents the integer subscript of each Z in the general formula) can be selected from any combination of those in Table 4 above. Similarly, the variable parameter Zn of FR in the variable region of the light chain can be selected from any combination of those in Table 5 above. In some implementations, the CDR combination of the light chain and the heavy chain is shown in Table 6 above.

[0193] It should be clarified here that in all tables in this application, such as Tables 1 to 6, all parameter values ​​(e.g., parameter values ​​of Zn or Xn) in each row of cells constitute one or a combination.

[0194] In some specific embodiments, the VH sequence is selected from SEQ ID NO: 1-6. In some specific embodiments, the VL sequence is selected from SEQ ID NO: 7-13. In some specific embodiments, the VH sequence of the antibody is selected from SEQ ID NO: 1-6 and the VL sequence is selected from SEQ ID NO: 7-13. In some specific embodiments, the VH sequence and VL sequence are selected from any of the following groups: SEQ ID NO: 1 and SEQ ID NO: 7, SEQ ID NO: 1 and SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 12, SEQ ID NO: 2 and SEQ ID NO: 13, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 9, SEQ ID NO: 5 and SEQ ID NO: 10, SEQ ID NO: 5 and SEQ ID NO: 11, SEQ ID NO: 6 and SEQ ID NO: 7.

[0195] However, those skilled in the art should recognize that not all antibodies or their antigen-binding fragments contain complete heavy chain variable regions and light chain variable regions. Furthermore, in many cases, protein molecules containing only antibody-antigen-binding fragments still possess the ability to specifically bind and / or neutralize antigens. In some embodiments, the antibody may contain only the antibody's VH and / or VL, such as dAb, single-domain antibodies containing only the light chain variable region, nanobodies, etc. In some embodiments, the antibody or its antigen-binding fragment may not contain a constant region, such as scFv, which contains the aforementioned HCDR and LCDR. In some embodiments, the antibody or its antigen-binding fragment is selected from Fab, F(ab')2, Fab', Fv, Fd, diabody, or multibody. The diabody and multibody antibodies contain one, two, or more of the aforementioned scFvs and may include other scFvs that specifically bind HBsAg or other antigens. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. Furthermore, as antigen-binding fragments of the antibodies described in this application, various CDRs disclosed in this application, combinations of said CDRs, and antibodies or antibody-binding fragments containing said CDRs (e.g., HCDR3 and / or LCDR3, etc.) and possessing the ability to specifically recognize HBsAg are also covered within the scope of protection of this application.

[0196] In some implementations, the antibody described in this application is a fully human antibody, a humanized antibody, a murine antibody, a chimeric antibody, or a nanobody.

[0197] In some embodiments, the antibody described in this application further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region and / or the light chain constant region are derived from natural immunoglobulins, for example, the heavy chain constant region may be derived from IgG, IgE, IgM, IgD, IgA, and IgY, or subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chain constant region may be selected from the κ chain or the λ chain. In some specific embodiments, the heavy chain constant region is the heavy chain constant region of human IgG1, comprising the amino acid sequence shown in SEQ ID NO: 14, or comprising an amino acid sequence having 70% or more sequence identity with SEQ ID NO: 14, for example, having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14. In some specific embodiments, the light chain constant region is derived from the κ chain and contains an amino acid sequence as shown in SEQ ID NO: 15, or contains an amino acid sequence having 70% or more sequence identity with SEQ ID NO: 15, for example, an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15.

[0198] Nucleic acid, construct, virus, bacteriophage or cell

[0199] On one hand, this application also provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to HBsAg. In some embodiments, the nucleic acid molecule is an RNA or DNA molecule. In some embodiments, the nucleic acid is single-stranded or double-stranded. In some embodiments, the nucleic acid is linear or circular. The nucleic acid can be synthesized by cells or chemically. In some embodiments, the nucleic acid is a chemically modified nucleic acid molecule.

[0200] On one hand, this application also provides a construct comprising the aforementioned nucleic acid molecule, wherein the construct is selected from: plasmids, phage particles, viral vectors, or linear nucleic acids. This application also provides a composition of constructs comprising the aforementioned constructs, wherein each construct in the composition may respectively comprise a portion of a nucleic acid molecule encoding the aforementioned antibody that specifically binds to HBsAg or its antigen-binding fragment. For example, a composition of constructs wherein the construct is a plasmid, and the composition of the construct comprises plasmids respectively encoding the heavy chain and light chain of the aforementioned antibody molecule.

[0201] On one hand, this application also provides a virus, bacteriophage, or cell that expresses or contains the aforementioned antibody specifically binding to HBsAg or its antigen-binding fragment, or a composition containing the aforementioned nucleic acid, the aforementioned construct, or the aforementioned construct. The virus is selected from, for example, AAV, lentivirus, etc. The bacteriophage is selected from, for example, λ phage, T phage, M13 phage, f1 phage, fd phage, etc. The cell can be selected from, for example, mammalian cells, insect cells, bacteria, fungi, etc.

[0202] In addition, this application also includes the use of nucleic acids, constructs, viruses, bacteriophages or cells to produce said specific HBsAg-binding antibodies or antigen-binding fragments thereof.

[0203] Pharmaceutical Compositions and Therapies

[0204] This application also provides a pharmaceutical composition comprising the aforementioned antibody that specifically binds to HBsAg or its antigen-binding fragment, the aforementioned nucleic acid, the aforementioned construct, or the aforementioned virus, bacteriophage, or cell. In some embodiments, the pharmaceutical composition further comprises an appropriate amount of pharmaceutically acceptable excipients. These formulations are included in the formulation to improve delivery and tolerability, etc. A large number of suitable formulations can be found in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA), a pharmacopoeia familiar to all pharmaceutical chemists. In this document, pharmaceutically acceptable excipients refer to non-toxic fillers, stabilizers, diluents, carriers, solvents, or other pharmaceutical excipients. The pharmaceutical composition comprises: diluents and excipients, such as microcrystalline cellulose and mannitol; fillers, such as starch and sucrose; binders, such as starch, cellulose derivatives, alginate, gelatin, and / or polyvinylpyrrolidone; disintegrants, such as calcium carbonate and / or sodium bicarbonate; absorption enhancers, such as quaternary ammonium compounds; surfactants, such as hexadecyl alcohol; carriers and solvents, such as water, physiological saline, kaolin, and soap clay; and lubricants, such as talc, calcium / magnesium stearate, and polyethylene glycol. Furthermore, the pharmaceutical composition of this invention is preferably an injectable formulation.

[0205] In some embodiments of the present invention, the antibody or its antigen-binding fragment in the pharmaceutical composition of the present invention is present at a concentration of 1 mg / ml to 1000 mg / ml, preferably at a concentration of 10 mg / ml to 1000 mg / ml, more preferably at a concentration of 50 mg / ml to 500 mg / ml, and even more preferably at a concentration of 100 mg / ml to 300 mg / ml.

[0206] The pharmaceutical composition of the present invention preferably has a pH of 3.0 to 9.0. It may further comprise a buffer system, preservatives, surface tension agents, chelating agents, stabilizers, and surfactants. In one embodiment of the invention, the pharmaceutical composition is an aqueous formulation. Such a formulation is typically a solution or suspension. In a specific embodiment of the invention, the pharmaceutical composition is a stable aqueous solution. In another specific embodiment of the invention, the pharmaceutical composition is a lyophilized formulation, which is dissolved in a solvent and / or diluent by a physician or patient prior to use.

[0207] On one hand, this application also provides a method for treating HBV infection, comprising administering an appropriate dose of the aforementioned pharmaceutical composition to a patient. The appropriate dose varies depending on the subject's age and body size, target disease, symptoms, route of administration, etc. When the pharmaceutical composition of the present invention is an antibody containing the aforementioned specific HBsAg binding fragment or its antigen-binding fragment used to treat various conditions and diseases related to hepatitis B virus infection in adults, the pharmaceutical composition can be administered intravenously, typically as a single dose of about 0.01 to about 20 mg of antibody per kilogram of body weight, for example, about 0.1 to about 15, about 1 to about 10, about 3 to about 10 mg / kg body weight (mpk), or about 12 mpk body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. For example, the patient may be administered the medication once a week.

[0208] Various drug delivery systems are known to be available for administering the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing variant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. (1987), J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, dural, and oral routes. The pharmaceutical compositions can be administered via any convenient route, such as by perfusion or intravenous bolus, absorption by epithelial and mucosal layers (e.g., oral mucosa, rectal and small intestinal mucosa), and can be administered co-administered with other bioactive agents. Administration can be systemic or local.

[0209] The drug composition can also be delivered via liquid capsules, especially via liposome liquid capsules (see Langer (1990) Science 249: 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327).

[0210] In some cases, the pharmaceutical composition can be delivered via a controlled release system. In one embodiment, a pump may be used (see Langer, as above; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, a polymeric material may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974). For a discussion of other controlled release systems, see Langer (1990) Science 2 49: 1527-1533).

[0211] The pharmaceutical composition can be administered alone or in combination, and improves patient symptoms by reducing the viral load of hepatitis B surface antigen. In some embodiments, the administration is a combination administration, wherein an HBsAg antibody or its antigen-binding fragment is administered in combination with one or more therapeutic agents (or a second therapeutic agent). Co-administration and combination therapy are not limited to simultaneous administration, but also include a treatment regimen in which an anti-HBsAg antibody or its antigen-binding fragment is administered at least once during a course of treatment involving the administration of at least one other therapeutic agent to the patient. The second therapeutic agent may be another HBV treatment, such as another antibody / antibody fragment, or a soluble cytokine receptor (such as interferon, intercytokines, thymosin, etc.), or a nucleoside analog (tenofovir, entecavir, adefovir, etc.).

[0212] The present invention also includes the use of any anti-HBsAg antibody or antigen-binding fragment described herein in the preparation of a medicament for treating a disease or symptom that is improved by reducing the level of HBsAg in the human body.

[0213] Reagents, kits, and detection methods

[0214] On one hand, this application provides a reagent comprising an antibody or antigen-binding fragment thereof that specifically binds to HBsAg, a complementary strand of the aforementioned nucleic acid or a fragment of the complementary strand, or the aforementioned construct, virus, bacteriophage, or cell. In some embodiments, the reagent further comprises a labeling molecule. In some embodiments, the labeling molecule may be selected from enzyme labeling molecules such as horseradish peroxidase and alkaline phosphatase, fluorescent protein molecules, fluorescein molecules, biotin molecules, isotopes, and other contrast agents. In some embodiments, the labeling molecule is coupled with the antibody or antigen-binding fragment thereof that specifically binds to HBsAg, the complementary strand of the aforementioned nucleic acid or a fragment of the complementary strand, or the aforementioned construct, virus, bacteriophage, or cell to form a complex.

[0215] On the other hand, this application also provides a kit. In some embodiments, the kit comprises the aforementioned antibody specifically binding to HBsAg or its antigen-binding fragment. In some embodiments, the kit comprises the aforementioned nucleic acid, the nucleic acid fragment, or a complementary strand of the nucleic acid or a fragment of the complementary strand. In some embodiments, the kit comprises the aforementioned construct, virus, bacteriophage, or cell. In some embodiments, the kit comprises the aforementioned reagent. In some embodiments, the kit is an in vitro detection kit. In some embodiments, the kit is an immunoassay kit, such as an ELISA kit or an immunohistochemical kit, comprising the aforementioned antibody specifically binding to HBsAg or its antigen-binding fragment. In some embodiments, the kit is an in vivo non-invasive diagnostic kit, comprising the aforementioned antibody specifically binding to HBsAg or its antigen-binding fragment, wherein the antibody specifically binding to HBsAg or its antigen-binding fragment forms a complex with a labeling molecule. In some embodiments, the in vivo non-invasive diagnostic kit can be used with an imaging method selected from radioimmunoassay or targeted ultrasound contrast imaging. When used for radioimmunoassay, the labeling molecule is a radionuclide. When used for targeted ultrasound contrast imaging, the labeling molecule is an ultrasound contrast agent.

[0216] In addition, this application also provides a method for detecting HBV, diagnosing HBV infection, or determining the progression of HBV-related diseases using the aforementioned antibody that specifically binds to HBsAg or its antigen-binding fragment, the aforementioned reagents or kits.

[0217] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0218] It should be understood that the above description and the following embodiments are intended to illustrate, not limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention. Example

[0219] Example 1: Construction of HBsAg antibody expression vector

[0220] The HindIII restriction site, Kozak sequence, secretory signal peptide gene, and HBsAg antibody heavy chain coding gene (including the coding gene for the VH amino acid sequence and the coding gene for the constant region of human IgG1), stop codon, and EcoRI coding gene were sequentially fused in tandem, and the gene fragment was obtained by chemical synthesis. The above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1(+) (purchased from Invitrogen Corporation, catalog number V790-20) via the EcoRI and HindIII sites and verified by sequencing to obtain the expression plasmid pCDNA3.1(+)-DH for the HBsAg antibody heavy chain.

[0221] The HindIII restriction site, Kozak sequence, secretion signal peptide gene, HBsAg light chain coding gene (including the coding gene for the VL amino acid sequence and the coding gene for the κ chain constant region), stop codon, and EcoRI coding gene were sequentially fused in tandem, and the gene fragment was obtained through chemical synthesis. The above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1(+) via the EcoRI and HindIII sites and sequenced for verification, yielding the expression plasmid pCDNA3.1(+)-DL for the antibody light chain.

[0222] The amino acid sequence of VH is: Z1Z2Z3LZ4Z5SGAEVKKPGASZ6KVSCKAS X1YX3FX5X6X7Y Z7HWVRQAPGQGZ8EWMGW X 11 NX 13 X 14 X 15 X 16 X 17 X 18 NYAQKFQGRVTZ9TZ 10 DZ 11 SZ 12 STAYMELSZ 13 LRSZ 14 DTAVYYC A RDX 21 WX 23 X 24 X 25 X 26 DX 28 YGMDX 33 WGZ 15 GTZ 16 VTVSS (SEQ ID NO: 47);

[0223] The amino acid sequence of VL is: Z 17Z 18 Z 19 LTQSPZ 20 Z 21 LSZ 22 SZ 23 GZ 24 RZ 25 TZ 26 Z 27 CRAS X 34 X 35 X 36 SX3 8X 39 LZ 28 WYQQKPGZ 29 APZ 30 LLIZ 31 X 40 X 41 X 42 Z 32 Z 33 Z 34 Z 35 GZ 36 PZ 37 RFSGSGSGTZ 38 FTLTIZ 39 SLZ 40 Z 41 Z 42 DZ 43 ATYYC QQSYSTPLX 51 FGZ 44 GTZ 45 Z 46 Z 47 IKR (SEQ ID NO: 48)

[0224] The underlined part is CDR, and the ununderlined part is FR. Xn and Zn (n refers to any integer subscript) can be selected from any amino acid.

[0225] The HBsAg antibodies tested in this application embodiment are numbered 021, 021-M1, 021-M3, 021-M5, 021-M6, 021-M7, 021-M11, 021-M13, 021-M14, 021-M15, 021-M16, 021-M17, 021-M18, 021-M21, 021-M23, and 021-M24, respectively. The Zn and Xn values ​​of the antibodies 021-M25, 021-M26, 021-M28, 021-M33, 021-M34, 021-M35, 021-M36, 021-M38, 021-M39, 021-M40, 021-M41, 021-M42, and 021-M51 are shown in Table 7 (Zn) and Table 3 (Xn) in the invention overview, respectively:

[0226] Table 7:

[0227]

[0228] The antibodies numbered 005, 062, 079, 083, 088, 090, 091, 093, 095, and 096 have Xn and Zn values ​​as shown in Tables 3 (Xn) and 6 (Zn) in the invention overview. Their specific VH and VL sequences are as follows:

[0229] VH sequence:

[0230] 021 and 079: QVQLVESGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLWNDDVDYYGMDVWGKGTLVTVSS (SEQ ID NO: 1)

[0231] 005, 095, 096: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLWNDDVDYYGMDVWGQGTMVTVSS (SEQ IDNO: 2)

[0232] 062: EVQLVESGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLWNDDVDYYGMDVWGQGTLVTVSS (SEQ ID NO: 3)

[0233] 088: QITLKESGAEVKKPGASVKVSCKASGYSFIGYYLHWVRQAPGQGLEWMGWINPYNGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLWNDDVDYYGMDVWGKGTLVTVSS (SEQ ID NO: 4)

[0234] 090, 091, 093: EVQLVQSGAEVKKPGASMKVSCKASGYTFTDYYIHWVRQAPGQGPEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLWNDDVDYYGMDVWGKGTMVTVSS (SEQ IDNO: 5)

[0235] 083: EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDVWQQGGYYYYMDVWGKGTLVTVSS(SEQ ID NO: 6)

[0236] VL sequence:

[0237] 021, 083: DIQLTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKR(SEQ ID NO: 7)

[0238] 005, 062, 079, 088: DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIKR(SEQ ID NO: 8)

[0239] 090: EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPARFSGSGSGTEFTLTISSLQSEDFATYYCQQSYSTPLTFGPGTKVEIKR(SEQ ID NO: 9)

[0240] 091: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSYSTPLTFGGGTKVDIKR(SEQ ID NO: 10)

[0241] 093: DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTEFTLTIRSLQPEDFATYYCQQSYSTPLTFGGGTKLEIKR(SEQ ID NO: 11)

[0242] 095: DIQLTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPGDLATYYCQQSYSTPLTFGGGTKVEIKR (SEQ ID NO: 12)

[0243] 096: ETTLTQSPSTLSASSVGDRVTITCRASQSISSYLNWYQQKPGKAPQLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTRLEIKR (SEQ ID NO: 13)

[0244] The correspondence between the above antibodies VH and VL and the CDR sequence is shown in Table 8 below:

[0245]

[0246]

[0247] Example 2: Expression and purification of anti-HBsAg antibody

[0248] Using the expression plasmids pCDNA3.1(+)-DH and pCDNA3.1(+)-DL described in Example 1, FreeStyle eukaryotic expression cells were used. TM 293-F Cells (Invitrogen Corporation, R790-07) express the target antibody. Following FreeStyle... TM According to the 293 Expression System user manual, adjust the cell density to 1x10⁻¹ one day before plasmid transfection. 6 Cells / mL. On the day of plasmid transfection, the plasmid is combined according to the correspondence between heavy and light chains (i.e., the light chain of the antibody with the same number is combined with the antibody heavy chain), mixed with the transfection reagent, and added to the cell culture medium. After continuous incubation at 37℃ and 8% CO2 for 5-7 days, the cell culture supernatant is collected for antibody purification.

[0249] The expression supernatant was filtered through a 0.22 μM filter membrane, and the expression antibody was captured from the supernatant using a Mabpurix affinity chromatography column (Sepax, 65008). The column was equilibrated with equilibration buffer (120 mM Tris + 100 mM NaCl, pH 7.5), then passed through the affinity chromatography column and eluted with elution buffer (0.15 M glacial acetic acid, pH 2.8). The purified antibody was analyzed by SDS-PAGE and SEC, and the purity was above 95%.

[0250] As can be seen, the amino acids selected for each Xn and each Zn in the antibodies used in the examples are different. Therefore, these antibodies can be used to test the changes in the affinity of the antibody for the antigen when the values ​​of the Xn and / or Zn parameters change.

[0251] Example 3: Antigen Binding Affinity Assay

[0252] HBsAg protein (20 μg / ml, isolated by the applicant, containing the amino acid sequence shown in SEQ ID NO: 16) was coated onto an ELISA plate at 100 μl / well. A sealing film was applied, and the plate was incubated overnight at 4°C. The plate was washed three times with PBST (0.5‰ Tween PBS), and blocked with 10% bovine serum albumin solution (prepared using PBST). 200 μl of this solution was added to each well, and the plate was incubated at 37°C for 2 hours. The plate was then washed three times with PBST (0.5‰ Tween PBS), and 100 μl / well was added with serially diluted test antibodies (20, 4, 0.8, 0.16, 0.032, 0.064, 0.00128, and 0.00256 mg / mL, a total of eight gradients). A system blank control was also included. The plate was incubated at 37°C for 2 hours. Wash the plate three times with PBST (0.5‰ Tween PBS), add 100 μl of secondary antibody (1:10000 dilution, Jackson Immuno Research) to each well, and incubate at 37°C for 1 hour. Finally, wash the plate five times with PBST (0.5‰ Tween PBS), add 100 μl of TMB chromogenic solution to each well, and incubate at room temperature in the dark. After 1 minute, add an equal volume of stop solution to terminate the colorimetric reaction. Use an ELISA reader to measure the absorbance (OD value) at a wavelength of 450 nm. When the OD value of the antigen binding is greater than 0.1, it is generally considered that the antibody can be detected at the corresponding concentration.

[0253] MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTST GPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYIHHHHHH(SEQ ID NO:16)

[0254] The HBsAg antigen binding activity was measured using an ELISA reader at a wavelength of 450 nm, expressed as absorbance (OD value). Eight concentration gradients were used for each antibody: 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μg / mL. Each antibody showed an OD value less than 0.05 (generally considered equivalent to a blank plate, indicating no binding activity). When the OD value of antibody binding to antigen was greater than 0.1, it was generally considered that the antibody was detectable at that concentration. Antibody 021, at a concentration of 0.16 μg / mL, showed an OD value greater than 0.1 for binding to antigen. Using antibody 021 as a base... The activity of the antibody was defined as "++++" level. The ratio of the corresponding OD value of other antibodies at a dilution concentration of 4 μg / mL to the corresponding OD value of the 021 antibody at 4 μg / mL was taken. When the ratio was greater than 1.1, the activity was defined as "++++" level; when the ratio was between 0.8 and 1.1, the activity was defined as "++++" level; when the ratio was between 0.6 and 0.8, the activity was defined as "++" level; when the ratio was between 0.4 and 0.6, the activity was defined as "++" level; when the ratio was between 0.2 and 0.4, the activity was defined as "+" level; and when the ratio was less than 0.2, it was considered that there was no binding activity.

[0255] The affinities of the tested antibodies are shown in Table 9 below:

[0256] Table 9:

[0257]

[0258]

[0259] This result indicates that the antibody in this application has a good ability to specifically bind to HBsAg.

[0260] 021, 021-M1, 021-M3, 021-M5, 021-M6, 021-M7, 021-M11, 021-M13, 021-M14, 021-M15, 021- M16, 021-M17, 021-M18, 021-M21, 021-M23, 021-M24, 021-M25, 021-M26, 021-M28, 021-M33, 021-M34, 021-M35, 021-M36, 021-M38, 021-M39, 021-M40, 021-M41, 021-M42, and 021-M51 have the same FR region, but the amino acids selected at the Xn position in each CDR are different. This indicates that the amino acid substitution at a specific position in the CDR region of the antibody provided in this application can still enable the antibody to maintain its ability to specifically recognize HBsAg.

[0261] The antibodies numbered 005, 062, 079, 083, 088, 090, 091, 093, 095, and 096, possessing the CDR described in this application and different FR regions, can specifically recognize HBsAg, further demonstrating the ability of the CDR to specifically recognize and bind to the HBsAg antigen. Furthermore, we have matched suitable FR regions to the CDR regions; even with different Zn values, the FR regions can still maintain the CDR conformation of the antibodies in this application, enabling the antibodies to specifically recognize HBsAg.

[0262] Example 4: In vivo efficacy test

[0263] In this experiment, the constant region of the antibody was selected from mouse IgG1.

[0264] animal:

[0265] Five-week-old male C57BL / 6 mice, free from specific pathogens, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed in individually ventilated cages. Mice were housed according to the experimental protocol approved by WuXi AppTec IACUC (IACUC#:ID01-013-2020v1.0). After a four-day acclimatization period, mice were injected with AAV / HBV virus (i.e., recombinant AAV virus rAAV8-1.3HBV containing the complete genome of type D HBV; in this article, "AAV / HBV virus" and "rAAV8-1.3HBV" are used interchangeably).

[0266] Solvent: PBS.

[0267] Test compounds: Antibody 005, Antibody 062, Antibody 079, Antibody 083, dose: 12mpk.

[0268] Recombinant rAAV8-1.3HBV: rAAV8-1.3HBV (D type, ayw) was provided by WuXi AppTec, batch number awy1-P4-200102, 1×10 12 Viral genome (vg) / mL. Dilute with sterile PBS to 5×10⁻⁶ before experiment. 11 vg / mL. 200 μL was injected into each mouse, which is equivalent to 1 × 10⁻⁶ vg / mL per mouse. 11 vg.

[0269] Test method:

[0270] Establishment of AAV / HBV mouse model

[0271] AAV / HBV injection. rAAV8-1.3HBV was pre-prepared with sterile PBS to a concentration of 1×10⁻⁶ before injection. 11vg / 200μL solution. 32 mice were injected with 200μL rAAV8-1.3HBV solution via the tail vein.

[0272] Blood was collected before grouping. On days 14, 21, and 35 post-viral injection, approximately 100 μL of blood was collected from the submandibular vein in all infected mice for plasma collection. The collected venous blood was anticoagulated with K2-EDTA, centrifuged at 7000 g / min for 10 minutes at 4°C, and plasma was collected. HBV DNA was detected in the plasma by qPCR, and HBsAg was detected by ELISA. Plasma samples were stored at -80°C until sent to the WuXi AppTec Biotechnology Department's in vitro laboratory for relevant testing.

[0273] The first administration was recorded as day 0, and day 45 post-infection was also designated as day 0. Mice were grouped based on plasma HBV DNA and HBsAg levels collected on days 14, 21, and 35 post-infection. Sixteen mice were selected from 20 mice for the formal experiment and randomly divided into five groups, labeled Group 1 to Group 5. Group 1 consisted of 4 mice, and the remaining groups consisted of 3 mice. All mice were administered PBS or a test antibody at 12 mpk via tail vein injection once. All mice were weighed before administration.

[0274] Blood collection:

[0275] On days -1, 1, 3, 5, 7, 10, 14 and 17 after administration, blood was collected from all mice via the submandibular vein to collect plasma for the detection of HBV DNA and HBsAg.

[0276] Experimental endpoint: Test section: All mice in the test group were euthanized on day 22 after drug administration.

[0277] Weight recording: During the in vivo experiment, the mice's condition was observed daily, and their weight was recorded on the day of infection, the day of drug administration, and the day of blood collection.

[0278] Sample preservation and transfer. All plasma samples were stored at -80°C and transferred to the WuXi AppTec Biotechnology Department's in vitro laboratory on dry ice for relevant testing.

[0279] Quantitative PCR was used to detect the HBV DNA content in mouse plasma. DNA was extracted from the plasma, and the experimental procedures were performed according to the QIAamp96 DNA Blood Kit instructions. The method is briefly described below:

[0280] Prepare the qPCR reaction mixture (Taqman Universal Master Mix (2X)), add the sample and standards, and perform the PCR reaction. Reaction conditions: 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles.

[0281] The HBsAg content in mouse plasma was detected by ELISA. The experimental procedure followed the instructions of the HBsAg ELISA kit (Antu Bio, CL0310). The method is briefly described as follows: Dilute the plasma sample 2, 10, 20 or 600 times, add it to the coated plate, and incubate with the enzyme conjugate (37℃, 60 minutes). Wash the plate 5 times, add the luminescent substrate, and react at room temperature in the dark for 10 minutes. Detect the luminescence intensity using a microplate reader.

[0282] The inhibitory activity of antibodies 005, 072, 079, and 083 (at a dose of 12 mpk) on HBV replication in an AAV / HBV mouse model was evaluated by detecting HBV DNA levels and HBsAg expression in mouse plasma. Results are as follows: Figure 1 and Figure 2 As shown.

[0283] The results are as follows:

[0284] 1) Effects of the test compound on plasma HBV DNA in AAV / HBV mice

[0285] In the solvent group (Group 1, PBS), the plasma HBV DNA level in mice remained relatively stable throughout the experiment, fluctuating between 5.31 and 5.74 log10 copies / μL (with fluctuations not exceeding 0.43 log10 copies / μL). Compared with the solvent group, the plasma HBV DNA level in the treatment group (Group 2, antibody 005, 12 mpk) was significantly reduced on day 1 after administration, with an average reduction of 2.69 log10 copies / μL (p<0.01), and then gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBV DNA level was 5.65 log10 copies / μL. In the treatment group (Group 3, antibody 062, 12 mpk), the plasma HBV DNA level in mice was significantly reduced on day 1 after administration, with an average reduction of 2.81 log10 copies / μL (p<0.01), and then gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBV DNA level was 5.45 log10 copies / μL. In treatment group 4 (antibody 079, 12 mpk), plasma HBV DNA levels in mice significantly decreased on day 1 after drug administration, with an average decrease of 2.68 log10 copies / μL (p<0.01). Subsequently, the levels gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBV DNA level was 5.59 log10 copies / μL. In treatment group 5 (antibody 083, 12 mpk), plasma HBV DNA levels in mice significantly decreased on day 1 after drug administration, with an average decrease of 0.78 log10 copies / μL (p<0.01). By day 3, the levels returned to the solvent group level, at which point the mouse plasma HBV DNA level was 5.36 log10 copies / μL.10 copy / μL.

[0286] 2) Effects of the test compound on plasma HBsAg in AAV / HBV mice

[0287] In the solvent group (group 1), the plasma HBsAg level in mice remained relatively stable throughout the experiment, fluctuating between 4.65 and 5.27 log10 IU / mL (with fluctuations not exceeding 0.62 log10 IU / mL). Compared with the solvent group, the plasma HBsAg level in the treatment group (group 2, antibody 005, 12mpk) mice significantly decreased on day 1 after administration, with an average decrease of 2.73 log10 IU / mL (p<0.01), and then gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBsAg level was 4.87 log10 IU / mL. In the treatment group (group 3, antibody 062, 12mpk) mice, the plasma HBsAg level significantly decreased on day 1 after administration, with an average decrease of 2.95 log10 IU / mL (p<0.01), and then gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBsAg level was 4.78 log10 IU / mL. In treatment group 4 (antibody 079, 12mpk), the plasma HBsAg level in mice decreased significantly on day 1 after administration, with an average decrease of 2.80 log10 IU / mL (p<0.01). Subsequently, it showed a gradual upward trend, reaching the solvent group level by day 10, at which point the mouse plasma HBsAg level was 4.79 log10 IU / mL. In treatment group 5 (antibody 083, 12mpk), the plasma HBsAg level in mice decreased significantly on day 1 after administration, with an average decrease of 1.10 log10 IU / mL (p<0.01). By day 3, it reached the solvent group level, at which point the mouse plasma HBsAg level was 4.93 log10 IU / mL.

[0288] Example 5: In vivo activity assay of antibodies 005, 021, 062, and 088

[0289] The experimental protocol was the same as in Example 4, with 5 groups of 4 mice per group. The IgG1 used was mouse-derived IgG1. The test results were shown in... Figure 3 and Figure 4 The specific results are as follows:

[0290] 1) The content of HBV DNA in the plasma of mice in each group.

[0291] In the solvent group (group 1), the plasma HBV DNA level of mice remained relatively stable throughout the experiment, fluctuating between 5.21 and 6.06 log10 copies / μL (with fluctuations not exceeding 0.85 log10 copies / μL). Compared with the solvent group, the plasma HBV DNA level of mice in the treatment group (group 2, antibody 005, 12mpk) decreased significantly on day 1 after administration, with an average decrease of 2.43 log10 copies / μL (p<0.01), and then showed a gradual upward trend, reaching the solvent group level by day 10, at which point the mouse plasma HBV DNA level was 5.49 log10 copies / μL. In the treatment group (group 3, antibody 021, 12mpk), the plasma HBV DNA level of mice decreased significantly on day 1 after administration, with an average decrease of 2.44 log10 copies / μL (p<0.01), and then showed a gradual upward trend, reaching the solvent group level by day 14, at which point the mouse plasma HBV DNA level was 5.81 log10 copies / μL. In treatment group 4 (antibody 062, 12mpk), the plasma HBV DNA of mice decreased significantly on day 1 after drug administration, with an average decrease of 2.51 log10 copies / μL (p<0.01). Subsequently, the plasma HBV DNA of mice showed a gradual upward trend, reaching the solvent group level on day 10, at which point the plasma HBV DNA of mice was 5.55 log10 copies / μL. In treatment group 5 (antibody 088, 12mpk), the plasma HBV DNA of mice decreased significantly on day 1 after drug administration, with an average decrease of 2.17 log10 copies / μL (p<0.01). Subsequently, the plasma HBV DNA of mice showed a gradual upward trend, reaching the solvent group level on day 7, at which point the plasma HBV DNA of mice was 5.59 log10 copies / μL.

[0292] 2) The content of HBsAg in the plasma of mice in each group.

[0293] In the solvent group (group 1), the plasma HBsAg level in mice remained relatively stable throughout the experiment, fluctuating between 4.74 and 5.11 log10 IU / mL (with fluctuations not exceeding 0.37 log10 IU / mL). Compared with the solvent group, the plasma HBsAg level in the treatment group (group 2, antibody O05m, 12mpk) mice significantly decreased on day 1 after administration, with an average decrease of 2.74 log10 IU / mL (p<0.01), and then gradually increased, reaching the solvent group level by day 10, at which point the mouse plasma HBsAg level was 4.75 log10 IU / mL. In the treatment group (group 3, antibody O21, 12mpk), the plasma HBsAg level in mice significantly decreased on day 1 after administration, with an average decrease of 2.38 log10 IU / mL (p<0.01), and then gradually increased, reaching the solvent group level by day 17, at which point the mouse plasma HBsAg level was 4.98 log10 IU / mL. In treatment group 4 (antibody 062, 12mpk), the plasma HBsAg of mice decreased significantly on day 1 after administration, with an average decrease of 2.41 and 2.60 log10 IU / mL, respectively (p<0.01). Subsequently, the HBsAg levels gradually increased, reaching the solvent group level on day 14, at which point the mouse plasma HBsAg level was 4.95 log10 IU / mL. In treatment group 5 (antibody 088, 12mpk), the plasma HBsAg of mice decreased significantly on day 1 after administration, with an average decrease of 2.19 log10 IU / mL (p<0.01). Subsequently, the HBsAg levels gradually increased, reaching the solvent group level on day 5, at which point the mouse plasma HBsAg level was 5.14 log10 IU / mL.

[0294] Example 6: In vivo experiments with antibody 021 090-097

[0295] The experimental method was the same as in Example 4. The model mice were divided into 8 groups of 4 mice each, and all IgG1 used was human IgG1. The constant regions of the heavy and light chains of the antibody are shown below:

[0296] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:14)

[0297] and

[0298] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 15)

[0299] Except for group 8, which received an intraperitoneal injection of antibody 021 (dose 12 mpk), all other groups of mice were administered PBS or test antibodies (antibody 021, antibody 090, antibody 091, antibody 093, antibody 095, antibody 096, 12 mpk) via tail vein injection. Each group received one dose, while the solvent group received an equal volume of PBS. Test results are as follows: Figure 5 and Figure 6 As shown. Specifically:

[0300] In the solvent group (Group 1), the plasma HBV DNA and HBsAg levels of mice remained relatively stable throughout the experiment, fluctuating between 4.36–4.80 log10 copies / μL and 4.44–4.70 log10 IU / mL, respectively (fluctuations not exceeding 0.44 log10 copies / μL and 0.26 log10 IU / mL). Compared with the solvent group, in the treatment group (Group 2, Antibody 090, 12mpk, IV), the plasma HBV DNA and HBsAg levels of mice were significantly reduced on day 1 after administration, with average reductions of 1.33 log10 copies / μL (p<0.01) and 0.81 log10 IU / mL (p<0.01), respectively. Subsequently, both showed a gradual recovery trend, reaching the solvent group levels on days 11 and 14, respectively, at which point the mouse plasma HBV DNA and HBsAg levels were 4.80 log10 copies / μL and 4.66 log10 IU / mL, respectively. In treatment group 3 (antibody 091, 12mpk, IV), plasma HBV DNA and HBsAg levels in mice were significantly reduced on day 1 after drug administration, with average reductions of 1.12 log10 copies / μL (p<0.01) and 0.93 log10 IU / mL (p<0.01), respectively. Subsequently, both levels gradually increased, reaching the solvent group levels on days 7 and 11, respectively, at which point plasma HBV DNA and HBsAg levels were 4.57 log10 copies / μL and 4.88 log10 IU / mL, respectively. In treatment group 4 (antibody 093, 12mpk, IV), plasma HBV DNA and HBsAg levels in mice were also significantly reduced on day 1 after drug administration, with average reductions of 1.19 log10 copies / μL (p<0.01) and 1.46 log10 IU / mL, respectively. The levels of HBV DNA and HBsAg in mouse plasma were initially low (p<0.01), but gradually increased, reaching the solvent group levels on days 7 and 11, respectively. At this point, the levels were 4.52 log10 copies / μL and 4.90 log10 IU / mL, respectively. In treatment group 5 (antibody 021, 12mpk, IV), both HBV DNA and HBsAg levels significantly decreased on day 1 after drug administration, with average decreases of 1.93 log10 copies / μL (p<0.01) and 1.23 log10 IU / mL (p<0.01), respectively. These levels then gradually increased, reaching the solvent group levels on days 18 and 11, respectively. At this point, the levels were 4.20 log10 copies / μL and 4.59 log10 IU / mL, respectively. In treatment group 6 (antibody 095, 12mpk, IV), the levels of HBV DNA and HBsAg in mouse plasma were also low. Both DNA and HBsAg decreased significantly on day 1 after drug administration, with an average decrease of 2.04 log10 copy / μL (p<0.01) and 1, respectively.The initial levels of HBV DNA and HBsAg in the treatment group (group 7, antibody 096, 12mpk, IV) were 76 log10 IU / mL (p<0.01), and then showed a gradual upward trend, reaching the levels of the solvent group by day 11. At this time, the plasma HBV DNA and HBsAg in mice were 4.66 log10 copy / μL and 4.59 log10 IU / mL, respectively. The plasma HBV DNA and HBsAg in the treatment group (group 7, antibody 096, 12mpk, IV) were significantly reduced on day 1 after administration, with an average reduction of 1.64 log10 copy / μL (p<0.01) and 1.22 log10 IU / mL (p<0.01), respectively. They then showed a gradual upward trend, with the plasma HBV DNA in mice reaching levels close to those of the solvent group by day 7. After that, it fluctuated slightly and remained at the experimental endpoint, which was 3.83 log10 copy / μL on day 18 after administration. By day 11, mouse plasma HBsAg levels returned to those of the solvent group, at which point the mouse plasma HBsAg level was 4.62 log10 IU / mL. Compared to day -3, the plasma HBV DNA levels in treatment group 8 (antibody O21, 12mpk, IP) decreased by 0.60 log10 copy / μL on day 1 after administration, reaching the LLOQ (lower limit of quantification) level by day 5, and subsequently recovered to day -3 levels on day 11, fluctuating between 2.73 and 3.14 log10 copy / μL from day 11 to day 18 (fluctuation not exceeding 0.41 log10 copy / μL). Mouse plasma HBsAg levels decreased significantly on day 1 after administration, with an average decrease of 0.82 log10 IU / mL, and the maximum decrease occurred on day 5, with an average decrease of 1.22 log10 IU / mL, subsequently recovering to day -3 levels on day 11, at which point the mouse plasma HBsAg level was 3.94 log10 IU / mL. .

[0301] In summary, the results of Examples 4-6 show that all the antibodies in this application, including O62 with an affinity of only ++, can be used as neutralizing antibodies against HBV, significantly inhibiting HBV proliferation and reducing HBsAg expression.

[0302] Example 7: Multiple Dosing Experiment

[0303] The AAV / HBV mouse model was established in the same manner as in Example 4.

[0304] The model mice were divided into two groups: a positive control group and an antibody 005 group (using mouse IgG1), with 4 mice in each group. The first administration was counted as day 0, and the drugs were administered once every 7 days thereafter, for a total of 7 administrations. The drugs were administered via tail vein injection at a dose of 12 mpk. The positive control group was given 10 mL / kg of PBS solution.

[0305] On days -25, -18, -11, -4, 1, 3, 5, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 49 and 52 after administration, blood was collected from the submandibular vein of all mice to collect plasma for the detection of HBV DNA and HBsAg.

[0306] The results showed that mouse plasma HBsAg levels decreased significantly on day 1 after administration, with an average decrease of 1.61 log 10 IU / mL (p<0.01), reaching a minimum on day 3, with an average decrease of approximately 1.73 log 10 IU / mL (p<0.01). During continuous administration, from day 0 to day 52, mouse plasma HBsAg levels remained at a low level. These results demonstrate that the antibody described in this application can maintain good resistance to HBV and therapeutic efficacy even with long-term use. Furthermore, it can inhibit viral replication for a long period with injections no more than once a week. sequence list <110> Beijing KAIN Technology Co., Ltd. <120> Antibodies that bind to hepatitis B virus surface antigen and their applications <130> PE01582 <150> 202010659026.2 <151> 2020-07-09 <150> 202010659828.3 <151> 2020-07-10 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial sequence <220> <223> VH of 021 and 079 <400> 1 Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH of 005, 095, 096 <400> 2 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 3 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH of 062 <400> 3 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 123 <212> PRT <213> Artificial sequence <220> VH of 088 <400> 4 Gln Ile Thr Leu Lys Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Gly Tyr 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH of 090, 091, 093 <400> 5 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Lys Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 6 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH of 083 <400> 6 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Val Trp Gln Gln Gly Gly Tyr Tyr Tyr Tyr Met Asp Val 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of 021 and 083 <400> 7 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of 005, 062, 079, 088 <400> 8 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 9 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of 090 <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 10 <211> 108 <212> PRT <213> artificial sequence <220> <223> 091's VL <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 11 <211> 108 <212> PRT <213> artificial sequence <220> <223> 093's VL <400> 11 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 12 <211> 108 <212> PRT <213> artificial sequence <220> <223> 095's VL <400> 12 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Gly Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 13 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of 096 <400> 13 Glu Thr Thr Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45 Tyr Thr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 14 <211> 330 <212> PRT <213> Artificial sequence <220> <223> Human IgG1 heavy chain constant region <400> 14 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 15 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 light chain constant region <400> 15 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 16 <211> 232 <212> PRT <213> artificial sequence <220> <223> HBsAg protein contains sequence <400> 16 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile His His His His His His 225 230 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 17 Gly Tyr Thr Phe Thr Gly Tyr Tyr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR1 <400> 18 Gly Tyr Ser Phe Ile Gly Tyr Tyr 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR1 <400> 19 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR1 <400> 20 Gly Tyr Thr Phe Thr Tyr Tyr Tyr 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR2 <400> twenty one Ile Asn Pro Asn Ser Gly Gly Thr 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR2 <400> twenty two Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> twenty three <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR3 <400> twenty three Ala Arg Asp Leu Trp Asn Asp Asp Val Asp Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR3 <400> twenty four Ala Arg Asp Val Trp Gln Gln Gly Gly Tyr Tyr Tyr Tyr Met Asp Val 1 5 10 15 <210> 25 <211> 6 <212> PRT <213> Artificial sequence <220> <223> LCDR1 <400> 25 Gln Ser Ile Ser Thr Tyr 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial sequence <220> <223> LCDR1 <400> 26 Gln Ser Ile Ser Ser Tyr 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial sequence <220> <223> LCDR1 <400> 27 Gln Ser Val Ser Ser Tyr 1 5 <210> 28 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 28 Ala Ala Ser 1 <210> 29 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 29 Gly Ala Ser 1 <210> 30 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 30 Asp Ala Ser 1 <210> 31 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 31 Thr Ala Ser 1 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 <400> 32 Gln Gln Ser Tyr Ser Thr Pro Leu Thr 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(7) <223> Xaa can be any naturally occurring amino acid <400> 33 Xaa Tyr Xaa Phe Xaa Xaa Xaa Tyr 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (3)..(8) <223> Xaa can be any naturally occurring amino acid <400> 34 Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa 1 5 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <220> <221> misc_feature <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (6)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (16)..(16) <223> Xaa can be any naturally occurring amino acid <400> 35 Ala Arg Asp Xaa Trp Xaa Xaa Xaa Xaa Asp Xaa Tyr Gly Met Asp Xaa 1 5 10 15 <210> 36 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <400> 36 Xaa Xaa Xaa Ser Xaa Xaa 1 5 <210> 37 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <400> 37 Xaa Xaa Xaa 1 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <220> <221> misc_feature <222> (9)..(9) <223> Xaa can be any naturally occurring amino acid <400> 38 Gln Gln Ser Tyr Ser Thr Pro Leu Xaa 1 5 <210> 39 <211> 25 <212> PRT <213> Artificial sequence <220> <223> HFR1 <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18)..(18) <223> Xaa can be any naturally occurring amino acid <400> 39 Xaa Xaa Xaa Leu Xaa Xaa Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Xaa Lys Val Ser Cys Lys Ala Ser 20 25 <210> 40 <211> 17 <212> PRT <213> Artificial sequence <220> <223> HFR2 <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (12)..(12) <223> Xaa can be any naturally occurring amino acid <400> 40 Xaa His Trp Val Arg Gln Ala Pro Gly Gln Gly Xaa Glu Trp Met Gly 1 5 10 15 Trp <210> 41 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> HFR3 <220> <221> misc_feature <222> (12)..(12) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (14)..(14) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (16)..(16) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18)..(18) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (27)..(27) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (31)..(31) <223> Xaa can be any naturally occurring amino acid <400> 41 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Xaa Thr Xaa Asp Xaa 1 5 10 15 Ser Xaa Ser Thr Ala Tyr Met Glu Leu Ser Xaa Leu Arg Ser Xaa Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HFR4 <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid <400> 42 Trp Gly Xaa Gly Thr Xaa Val Thr Val Ser Ser 1 5 10 <210> 43 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> LFR1 <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (9)..(10) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (15)..(15) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (17)..(17) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (19)..(19) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (21)..(22) <223> Xaa can be any naturally occurring amino acid <400> 43 Xaa Xaa Xaa Leu Thr Gln Ser Pro Xaa Xaa Leu Ser Xaa Ser Xaa Gly 1 5 10 15 Xaa Arg Xaa Thr Xaa Xaa Cys Arg Ala Ser 20 25 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> LFR2 <220> <221> misc_feature <222> (2)..(2) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (10)..(10) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (17)..(17) <223> Xaa can be any naturally occurring amino acid <400> 44 Leu Xaa Trp Tyr Gln Gln Lys Pro Gly Xaa Ala Pro Xaa Leu Leu Ile 1 5 10 15 Xaa <210> 45 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> LFR3 <220> <221> misc_feature <222> (1)..(4) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (8)..(8) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18)..(18) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (24)..(24) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (27)..(29) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (31)..(31) <223> Xaa can be any naturally occurring amino acid <400> 45 Xaa Xaa Xaa Xaa Gly Xaa Pro Xaa Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Xaa Phe Thr Leu Thr Ile Xaa Ser Leu Xaa Xaa Xaa Asp Xaa Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LFR4 <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (6)..(8) <223> Xaa can be any naturally occurring amino acid <400> 46 Phe Gly Xaa Gly Thr Xaa Xaa Xaa Ile Lys Arg 1 5 10 <210> 47 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18)..(18) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (28)..(28) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (30)..(32) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (45)..(45) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (51)..(51) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (53)..(58) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (70)..(70) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (72)..(72) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (74)..(74) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (76)..(76) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (85)..(85) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (89)..(89) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (100)..(100) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (102)..(105) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (107)..(107) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (112)..(112) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (115)..(115) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (118)..(118) <223> It can be any naturally occurring amino acid. <400> 47 No No No Leu No No Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser No Lys Val Ser Cys Lys Ala Ser No Tyr No Phe No No 20 25 30 Tyr Haa His Trp Val Arg Gln Ala Pro Gly Gln Gly Haa Glu Trp Met 35 40 45 Gly Trp Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Xaa Thr Xaa Asp Xaa Ser Xaa Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Xaa Leu Arg Ser Xaa Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Xaa Trp Xaa Xaa Xaa Xaa Asp Xaa Tyr Gly Met Asp Xaa 100 105 110 Trp Gly Xaa Gly Thr Xaa Val Thr Val Ser Ser 115 120 <210> 48 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <220> <221> misc_feature <222> (1)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (9)..(10) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (15)..(15) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (17)..(17) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (19)..(19) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (21)..(22) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (27)..(29) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (31)..(32) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (42)..(42) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (45)..(45) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (49)..(56) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (58)..(58) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (60)..(60) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (70)..(70) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (76)..(76) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (79)..(81) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (83)..(83) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (97)..(97) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (100)..(100) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (103)..(105) <223> It can be any naturally occurring amino acid. <400> 48 Thanks Thanks Thanks Leu Thr Gln Ser Pro Thanks Leu Ser Thanks Ser Gly 1 5 10 15 Where Arg Wheel Thr Wheel Cys Arg Ala Ser Wheel Wheel Ser Wheel 20 25 30 Leu No Trp Tyr Gln Gln Lys Pro Gly Not Ala Pro No Leu Ile 35 40 45 Yes Yes Yes Yes Yes Gly Yes Pro Yes Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Xaa Phe Thr Leu Thr Ile Xaa Ser Leu Xaa Xaa 65 70 75 80 No Asp No Ala Thr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Half Phe Gly Half Gly Thr Half Half Isle Lys Arg 100 105

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg), comprising a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein: The heavy chain variable region comprises the complementarity-determining region (CDR) amino acid sequences shown below: HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 21, and HCDR3 as shown in SEQ ID NO: 23; The light chain variable region contains the following CDR amino acid sequences: LCDR1 as shown in SEQ ID NO: 25; LCDR2 as shown in SEQ ID NO: 28; and LCDR3 as shown in SEQ ID NO:

32.

2. The antibody that specifically binds to HBsAg or its antigen-binding fragment of claim 1, comprising the human universal framework region (FR).

3. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, wherein, VH contains the following FR amino acid sequence: HFR1: Z1Z2Z3LZ4Z5SGAEVKKPGASZ6KVSCKAS (SEQ ID NO: 39) HFR2: Z7HWVRQAPGQGZ8EWMGW (SEQ ID NO: 40) HFR3:NYAQKFQGRVTZ9TZ10DZ11SZ12STAYMELSZ13LRSZ14DTAVYYC (SEQ ID NO: 41) HFR4: WGZ15GTZ16VTVSS (SEQ ID NO: 42) VL contains the following FR amino acid sequence: LFR1: Z17Z18Z19LTQSPZ20Z21LSZ22SZ23GZ24RZ25TZ26Z27CRAS (SEQ ID NO: 43) LFR2: LZ28WYQQKPGZ29APZ30LLIZ31 (SEQ ID NO: 44) LFR3: Z32Z33Z34Z35GZ36PZ37RFSGGSGTZ38FTLTIZ39SLZ40Z41Z42DZ43ATYYC (SEQ IDNO: 45) LFR4: FGZ44GTZ45Z46Z47IKR (SEQ ID NO: 46) in, Z1 is selected from Q or E, Z2 from V or I, Z3 from Q or T, Z4 from V or K, Z5 from E or Q, Z6 from V or M, Z7 from M, I or L, Z8 from L or P, Z9 from M or I, Z10 from R or A, Z11 from T or K, Z12 from I or T, Z13 from R or S, Z14 from D or E, Z15 from K or Q, Z16 from L or M, Z17 from D or E, Z18 from I or T, Z19 from Q, T or V, Z20 from S, A or G, Z21 from S or T, Z22 from A or L, Z23 from V or P, Z24 from D or E, Z 25 is selected from V or A, Z26 is selected from I or L, Z27 is selected from T or S, Z28 is selected from N or A, Z29 is selected from K or Q, Z30 is selected from K, Q or R, Z31 is selected from Y or S, Z32 is selected from S or N, Z33 is selected from L or R, Z34 is selected from Q or A, Z35 is selected from S or T, Z36 is selected from V or I, Z37 is selected from S or A, Z38 is selected from D or E, Z39 is S or R, Z40 is selected from Q or E, Z41 is selected from P or S, Z42 is selected from E or G, Z43 is selected from F or L, Z44 is selected from G, Q or P, Z45 is selected from K or R, Z46 is selected from V or L, Z47 is selected from D or E.

4. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, wherein the VH sequence and VL sequence are selected from any one of the following groups of sequences: SEQ ID NO: 1 and SEQ ID NO: 7, and an amino acid sequence having at least 85% identity with the above VH sequence or VL sequence.

5. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, wherein the antibody is selected from: Fab, F(ab')2, Fab', scFv, Fv.

6. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, wherein the antibody is a fully human antibody, a humanized antibody, a murine antibody, a chimeric antibody, or a nanobody.

7. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, wherein it is a monospecific antibody.

8. The antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to claim 1 or 2, further comprising a heavy chain constant region and a light chain constant region, wherein, The heavy chain constant region is selected from the heavy chain constant region of IgG, and the light chain constant region is selected from the κ chain or the λ chain.

9. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of claims 1-8.

10. A construct comprising the nucleic acid molecule of claim 9.

11. The construct of claim 10, wherein the construct is selected from: plasmids, phage particles, viral vectors, or linear nucleic acids.

12. A virus, bacteriophage, or cell expressing an antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of claims 1-8, or comprising the nucleic acid of claim 9 or the construct of claim 10 or 11.

13. A pharmaceutical composition comprising: an antibody that specifically binds to HBsAg according to any one of claims 1-8 or an antigen-binding fragment thereof, a nucleic acid according to claim 9, a construct according to claim 10 or 11, or a virus, bacteriophage, or cell according to claim 12.

14. A kit comprising: an antibody that specifically binds to HBsAg according to any one of claims 1-8 or an antigen-binding fragment thereof, a nucleic acid according to claim 9, a construct according to claim 10 or 11, or a virus, bacteriophage, or cell according to claim 12.

15. Use of the pharmaceutical composition of claim 13 in the preparation of a medicament for treating or preventing hepatitis B virus (HBV) infection, or for relieving symptoms of hepatitis B.

16. Use of the antibody or antigen-binding fragment thereof that specifically binds to HBsAg according to any one of claims 1-8, the nucleic acid according to claim 9, the construct according to claim 10 or 11, or the virus, bacteriophage or cell according to claim 12 in the preparation of a kit for detecting HBV.

Images