KR20220126788A - Antibodies for treatment of hepatitis B infection and related diseases - Google Patents

Abstract

Provided are an antibody against hepatitis B surface antigen, a nucleic acid encoding the same and a method for preparing the antibody, and a pharmaceutical composition comprising the antibody. Also provided are uses of the antibodies and pharmaceutical compositions. Antibodies and pharmaceutical compositions are used to prevent and/or treat HBV infection or diseases associated with HBV infection (eg hepatitis B), neutralize toxicity of HBV in the body of a subject (eg human), and HBV DNA and/or reduce serum levels of HBsAg.

Description

Antibodies for treatment of hepatitis B infection and related diseases

The present invention relates to the field of molecular virology and immunology, in particular the treatment of hepatitis B virus (HBV) infection. Specifically, the present invention relates to an antibody against hepatitis B surface antigen (HBsAg), a nucleic acid molecule encoding the same, a method for preparing the same, and a pharmaceutical composition containing the same. The pharmaceutical composition may be used for preventing and/or treating HBV infection or a disease associated with HBV infection (eg, hepatitis B), neutralizing toxicity of HBV in a subject (eg human), or reducing HBV DNA and/or HBsAg in a subject. It can be used to reduce serum levels. Correspondingly, the present invention also relates to the use of antibodies (in particular humanized antibodies thereof) and variants thereof in the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition comprises HBV infection or a disease associated with HBV infection (eg hepatitis B). for the prevention and/or treatment of, neutralization of toxicity of HBV in a subject (eg human), or reduction of serum levels of HBV DNA and/or HBsAg in a subject.

Hepatitis B virus infection, particularly chronic HBV infection, is one of the most important public health problems in the world (Dienstag JL. Hepatitis B virus infection. N Engl J Med 2008 Oct 2; 359(14): 1486-1500). Chronic HBV infection can lead to a series of liver diseases, such as chronic hepatitis B (CHB), cirrhosis (LC) and hepatocellular carcinoma (HCC) (Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009 Feb 14 ;373(9663): 582-592). According to the report, about 2 billion people worldwide are infected with HBV, about 350 million people are infected with chronic hepatitis B virus, and these infected people eventually 15 due to HBV infection-related liver disease. % to 25%, and more than one million people worldwide die each year from the disease (Dienstag JL., ibid; and Liaw YF et al., ibid.).

Currently, therapeutic drugs for chronic HBV infection can be mainly classified into interferons (INF) and nucleosides or nucleotides (NA) (Dienstag JL., ibid; Kwon H, Lok AS. Hepatitis B therapy. Nat Rev Gastroenterol Hepatol) 2011 May; 8(5): 275-284; and Liaw YF et al., ibid). The former include conventional interferon (IFN) and peg-interferon (Peg-IFN, also known as long-acting interferon), which achieves HBV suppression and CHB therapeutic effects primarily through enhancement of overall immunity in the patient, whereas ; The latter mainly inhibit HBV replication by directly inhibiting the polymerase activity of HBV, such as lamivudine (LMV), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir (tenofovir) mainly include. For HBV-infected patients (eg, CHB patients), the above drugs alone or in combination therapy can effectively inhibit viral replication in vivo and significantly reduce HBV DNA levels; In particular, the response rate of a virological response with HBV DNA levels below the lower limit of detection in patients after 52 weeks or extended period of therapy can reach 40-80% (Kwon H et al., ibid.). However, treatment with the above-mentioned drugs alone or in combination cannot completely eliminate HBV virus in infected patients, which is usually less than 5% of HBsAg negative conversion or HBsAg seroconversion (complete clearance of HBV virus in infected patients). The response rate of (Kwon H et al., ibid.) is derived. Therefore, there is an urgent need to develop innovative therapeutic methods and drugs for HBV-infected patients that can more effectively eliminate HBV virus, particularly HBsAg.

The development of novel drugs for the treatment of chronic HBV infection based on immunological methods is one of the important research directions in this field. Immunotherapy for chronic HBV infection is usually carried out by two methods: active immunotherapy (corresponding to the drug form, such as a vaccine) and passive immunotherapy (corresponding to the drug form, such as an antibody). Active immunotherapy stimulates chronic HBV-infected individuals by administering therapeutic vaccines (including protein vaccines, polypeptide vaccines, and nucleic acid vaccines, etc.) to achieve the purpose of suppression or elimination of HBV, resulting in a cellular immune response (CTL) against HBV. effect, etc.) and/or a method of actively generating a humoral immune response (antibodies, etc.). Currently, there are no drugs or vaccines for active immunotherapy that are significantly effective in the treatment of chronic HBV infection. Passive immunotherapy (eg, with an antibody) administers a therapeutic antibody to an HBV-infected individual, thus preventing naïve hepatocytes from becoming infected with HBV by antibody-mediated viral neutralization, or for antibody-mediated immune clearance. It refers to a method of removing viruses and infected hepatocytes from the body, thereby achieving a therapeutic effect. Currently, anti-HBs polyclonal antibodies, i.e. high-titer hepatitis B immunoglobulin (HBIG), obtained by purification from sera/plasma of either immune responders to prophylactic hepatitis B vaccine or HBV infection survivors, are transferred from mother to infant. It has been widely used to block HBV vertical infection in patients with chronic HBV infection, to prevent HBV re-infection after liver transplantation in chronic HBV-infected patients, and to prevent infection in people who have been accidentally exposed to HBV. However, the therapy of direct administration of HBIG to HBV-infected patients (eg, CHB patients) is not very effective and has a number of limitations such as small sources of high titer plasma, high cost, unstable nature, and potential safety issues.

Therefore, there is an urgent need to develop innovative therapeutic methods and drugs for HBV infection that are more effective for the treatment of HBV infection.

In one aspect, the present invention provides

(a) (i) SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, or said sequence by one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions); VH CDR1 consisting of a sequence selected from a sequence different from;

(ii) SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or a sequence that differs from that sequence by one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR2 consisting of a sequence selected from; and

(iii) SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR3 consisting of a sequence selected from

At least one (eg, 1, 2 or 3) heavy chain variable region (VH) complementarity determining region (CDR) selected from the group consisting of; and/or

(b) (vi) SEQ ID NO: 9, SEQ ID NO: 15, or a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL CDR1 consisting of selected sequences;

(v) SEQ ID NO: 10, SEQ ID NO: 16, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL CDR2 consisting of; and

(vi) SEQ ID NO: 11, SEQ ID NO: 17, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); Consists of VL CDR3

One or more (eg, 1, 2 or 3) light chain variable region (VL) CDRs selected from the group consisting of

It provides an antibody or antigen-binding fragment thereof capable of specifically binding to HBsAg, comprising a.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH CDR1, VH CDR2 and VH CDR3 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VL CDR1, VL CDR2 and VL CDR3 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 as defined above.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from SEQ ID NO: 7, or a sequence different from SEQ ID NO: 7 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR2 consisting of;

(ii) a VH CDR3 consisting of SEQ ID NO: 8, or a sequence selected from a sequence that differs from SEQ ID NO: 8 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR1 consisting of a sequence selected from the sequences of VH CDR1 contained in the heavy chain variable region of any immunoglobulin.

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 9 or a sequence different from SEQ ID NO: 9 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR3 consisting of SEQ ID NO: 11 or a sequence selected from a sequence different from SEQ ID NO: 11 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(vi) a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin.

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH CDR1 is the sequence of the VH CDR1 comprised in the heavy chain variable region of a human immunoglobulin.

In some preferred embodiments, the VH CDR1 is (a) SEQ ID NO: 6 or SEQ ID NO: 6 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VH CDR1 contained in the amino acid sequence encoded by the human heavy chain germline gene. In some exemplary embodiments, the human heavy chain germline gene is selected from the group consisting of IGHV4-4 ^* 08 and IGHV4-61 ^* 01.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention is a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and SEQ ID NO: 6, SEQ ID NO: 137 or sequence and a VH CDR1 having the amino acid sequence of number 138.

In some preferred embodiments, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of a human immunoglobulin.

In some preferred embodiments, the VL CDR2 is (a) SEQ ID NO: 10 or SEQ ID NO: 10 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene. In some exemplary embodiments, the human light chain germline gene is selected from the group consisting of IGKV1-39 ^* 01 and IGKV1-5 ^* 03.

In some exemplary embodiments, the VL of an antibody or antigen-binding fragment thereof of the present invention is a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and SEQ ID NO: 10, SEQ ID NO: 139 or sequence and a VL CDR2 having the amino acid sequence of number 140.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and an amino acid of SEQ ID NO: 6, 137 or 138 a VH CDR1 having the sequence, wherein the VL of the antibody or antigen-binding fragment thereof is a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and an amino acid sequence of SEQ ID NO: 10, 139 or 140 VL CDR2 with

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from among SEQ ID NO: 12, or a sequence different from SEQ ID NO: 12 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR1 consisting of;

(ii) a VH CDR3 consisting of SEQ ID NO: 14 or a sequence selected from a sequence that differs from SEQ ID NO: 14 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR2 consisting of a sequence selected from the sequences of the VH CDR2 contained in the heavy chain variable region of any immunoglobulin.

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 15, or a sequence different from SEQ ID NO: 15 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR3 consisting of SEQ ID NO: 17 or a sequence selected from a sequence that differs from SEQ ID NO: 17 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(vi) a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin.

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH CDR2 is the sequence of the VH CDR2 contained in the heavy chain variable region of a human immunoglobulin.

In some preferred embodiments, the VH CDR2 is (a) SEQ ID NO: 13, or SEQ ID NO: 13 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VH CDR2 contained in the amino acid sequence encoded by the human heavy chain germline gene. In some exemplary embodiments, the human heavy chain germline gene is selected from the group consisting of IGHV4-30-4 ^* 07 and IGHV4-4 ^* 01.

In some exemplary embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises (a) a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: 141 and a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 142.

In some preferred embodiments, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of a human immunoglobulin.

In some preferred embodiments, the VL CDR2 comprises (a) SEQ ID NO: 16 or SEQ ID NO: 16 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions). different sequences; or (b) the sequence of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene. In some exemplary embodiments, the human light chain germline gene is selected from the group consisting of IGKV2-28 ^* 01 and IGKV3-15 ^* 01.

In some exemplary embodiments, the VLs of an antibody or antigen-binding fragment thereof of the present invention are VL CDR1 as set forth in SEQ ID NO: 15, VL CDR3 as set forth in SEQ ID NO: 17, and SEQ ID NO: 16, SEQ ID NO: 143 and SEQ ID NO: and a VL CDR2 having an amino acid sequence selected from the group consisting of number 144.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention is a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: 141 and the sequence and a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 142, wherein the VL of the antibody or antigen-binding fragment thereof is a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR3 as set forth in SEQ ID NO: 17, and SEQ ID NO: 16, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 143 and SEQ ID NO: 144.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from SEQ ID NO: 18, or a sequence different from SEQ ID NO: 18 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH CDR1 consisting of;

(ii) a VH CDR2 consisting of SEQ ID NO: 19 or a sequence selected from a sequence different from SEQ ID NO: 19 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR3 consisting of SEQ ID NO: 20 or a sequence selected from a sequence different from SEQ ID NO: 20 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions)

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 15, or a sequence different from SEQ ID NO: 15 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR2 consisting of SEQ ID NO: 16 or a sequence selected from a sequence that differs from SEQ ID NO: 16 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) , and

(vi) a VL CDR3 consisting of SEQ ID NO: 17 or a sequence selected from a sequence that differs from SEQ ID NO: 17 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions)

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a VH CDR1 as set forth in SEQ ID NO: 18, a VH CDR2 as set forth in SEQ ID NO: 19, and a VH CDR3 as set forth in SEQ ID NO: 20 wherein the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 16, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is humanized. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least It has a degree of humanization of 97%, or at least 98%. In some preferred embodiments, the non-CDR region of the antibody or antigen-binding fragment thereof of the invention is 19 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less. no more than 6, no more than 5, no more than 4, or no more than 3 amino acid residues from a non-human source (eg, a cynomolgus monkey source).

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention further comprises a framework region (FR).

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(a) (i) SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, or one or multiple amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions; VH FR1 consisting of a sequence selected from sequences different from the above sequences by deletion or addition);

(ii) SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 51, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH FR2 consisting of a sequence selected from a sequence different from the sequence;

*(iii) SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 52, or one or multiple amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or VH FR3 consisting of a sequence selected from a sequence different from the above sequence by addition); and

(iv) SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 53, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH FR4 consisting of a sequence selected from a sequence different from the sequence

at least one (eg 1, 2, 3 or 4) heavy chain variable region (VH) framework region (FR) selected from; and/or

(b) (v) SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 54, or one or multiple amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) VL FR1 consisting of a sequence selected from a sequence different from the above sequence by;

(vi) SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 55, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR2 consisting of a sequence selected from a sequence different from the sequence;

(vii) SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 56, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR3 consisting of a sequence selected from a sequence different from the sequence; and

(viii) SEQ ID NO: 28, SEQ ID NO: 36, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR4 made

One or more (eg 1, 2, 3 or 4) light chain variable region (VL) framework regions (FR) selected from

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3 and VH FR4 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL FR2, VL FR3 and VL FR4 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3 and VL FR4 as defined above.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(a) at least 80%, at least 85%, compared to a heavy chain framework region contained in a heavy chain variable region selected from among heavy chain variable regions as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164 , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology. heavy chain framework region; and/or

(b) at least 80%, at least 85%, at least a light chain framework region contained in a light chain variable region selected from among the light chain variable regions as set forth in SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 A light chain frame having 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology. work area

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(a) four FRs contained in a heavy chain variable region selected from among the heavy chain variable regions as set forth in any one of SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164; and/or

(b) four FRs contained in a light chain variable region selected from among the light chain variable regions as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166

includes

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof is

(a) VH FR1 as set forth in SEQ ID NO: 21, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 23, and VH FR4 as set forth in SEQ ID NO: 24;

(b) VH FR1 as set forth in SEQ ID NO: 29, VH FR2 as set forth in SEQ ID NO: 30, VH FR3 as set forth in SEQ ID NO: 31, and VH FR4 as set forth in SEQ ID NO: 32;

(c) VH FR1 as set forth in SEQ ID NO: 37, VH FR2 as set forth in SEQ ID NO: 38, VH FR3 as set forth in SEQ ID NO: 39, and VH FR4 as set forth in SEQ ID NO: 32;

(d) VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 45, and VH FR4 as set forth in SEQ ID NO: 46; or

(e) VH FR1 as set forth in SEQ ID NO: 50, VH FR2 as set forth in SEQ ID NO: 51, VH FR3 as set forth in SEQ ID NO: 52, and VH FR4 as set forth in SEQ ID NO: 53

and/or the VL of the antibody or antigen-binding fragment thereof is

(a) VL FR1 as set forth in SEQ ID NO: 25, VL FR2 as set forth in SEQ ID NO: 26, VL FR3 as set forth in SEQ ID NO: 27, and VL FR4 as set forth in SEQ ID NO: 28;

(b) VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36;

(c) VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, VL FR3 as set forth in SEQ ID NO: 49, and VL FR4 as set forth in SEQ ID NO: 28; or

(d) VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, VL FR3 as set forth in SEQ ID NO: 56, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a framework region of a human immunoglobulin (eg, a framework region contained in the amino acid sequence encoded by a human germline antibody gene), the framework region optionally comprises one or more back mutations from human residues to cynomolgus monkey residues.

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

In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in the amino acid sequence encoded by IGHV 4-4 ^* 08, and contained in the amino acid sequence encoded by IGKV 1-39 ^* 01 light chain framework regions, wherein the heavy chain framework regions and/or light chain framework regions optionally comprise one or more reverse mutations from human residues to cynomolgus monkey residues.

In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in the amino acid sequence encoded by IGHV 4-4 ^* 02, and contained in the amino acid sequence encoded by IGKV 4-1 ^* 01. light chain framework regions, wherein the heavy chain framework regions and/or light chain framework regions optionally comprise one or more reverse mutations from human residues to cynomolgus monkey residues.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) VH FR1 as set forth in SEQ ID NO: 21, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 23, and VH FR4 as set forth in SEQ ID NO: 24; and VL FR1 as set forth in SEQ ID NO: 25, VL FR2 as set forth in SEQ ID NO: 26, VL FR3 as set forth in SEQ ID NO: 27, and VL FR4 as set forth in SEQ ID NO: 28;

(b) VH FR1 as set forth in SEQ ID NO: 29, VH FR2 as set forth in SEQ ID NO: 30, VH FR3 as set forth in SEQ ID NO: 31, and VH FR4 as set forth in SEQ ID NO: 32; and VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36;

(c) VH FR1 as set forth in SEQ ID NO: 37, VH FR2 as set forth in SEQ ID NO: 38, VH FR3 as set forth in SEQ ID NO: 39, and VH FR4 as set forth in SEQ ID NO: 32; and VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36;

(d) VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 45, and VH FR4 as set forth in SEQ ID NO: 46; and VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, VL FR3 as set forth in SEQ ID NO: 49, and VL FR4 as set forth in SEQ ID NO: 28; or

(e) VH FR1 as set forth in SEQ ID NO:50, VH FR2 as set forth in SEQ ID NO:51, VH FR3 as set forth in SEQ ID NO:52, and VH FR4 as set forth in SEQ ID NO:53; and VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, VL FR3 as set forth in SEQ ID NO: 56, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164 At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least compared to the amino acid sequence of a heavy chain variable region selected from the group consisting of 97%, at least 98%, at least 99%, or 100% sequence homology. In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof is a heavy chain variable region as set forth in any one of SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164.

In some preferred embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a light chain variable as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology. In some preferred embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof is a light chain variable region as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region as defined above and a light chain variable region as defined above.

In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in any one of SEQ ID NOs: 1, 57, 157 and 158; the light chain variable region of the antibody or antigen-binding fragment thereof is a light chain variable region as set forth in any one of SEQ ID NOs: 2, 58, 159 and 160; or

The heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in any one of SEQ ID NOs: 3, 59, 163 and 164; The light chain variable region of the antibody or antigen-binding fragment thereof is a light chain variable region as set forth in any one of SEQ ID NOs: 4, 60, 165 and 166.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(1) VH as set forth in SEQ ID NO: 1 and VL as set forth in SEQ ID NO: 2;

(2) VH as set forth in SEQ ID NO:3 and VL as set forth in SEQ ID NO:4;

(3) VH as set forth in SEQ ID NO:5 and VL as set forth in SEQ ID NO:4;

(4) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 58;

(5) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 60;

(6) VH as set forth in SEQ ID NO: 157 and VL as set forth in SEQ ID NO: 58;

(7) VH as set forth in SEQ ID NO: 158 and VL as set forth in SEQ ID NO: 58;

(8) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 159;

(9) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 160;

(10) VH as set forth in SEQ ID NO:163 and VL as set forth in SEQ ID NO:60;

(11) VH as set forth in SEQ ID NO: 164 and VL as set forth in SEQ ID NO: 60;

(12) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 165; or

(13) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 166

includes

In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention are scFv, Fab, Fab', (Fab') ₂ , Fv fragments, diabodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, and humanized antibodies. selected from the group consisting of antibodies. In some preferred embodiments, the antibody is a chimeric antibody or a humanized antibody.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is of the class IgG (eg class IgG1, IgG2, IgG3 or IgG4).

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is capable of specifically binding to HBsAg, neutralizing the toxicity of HBV, and/or of HBV DNA and/or serum levels of HBsAg in a subject. can reduce

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is labeled. In some preferred embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, eg, a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme (eg horseradish peroxidase).

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the invention, or a heavy chain variable region and/or a light chain variable region thereof.

In another aspect, the invention provides a vector (eg, a cloning vector or expression vector) comprising an isolated nucleic acid molecule according to the invention.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule according to the invention or a vector according to the invention.

In another aspect, the present invention comprises culturing a host cell according to the invention under conditions permissive for expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell. A method for producing an antibody or antigen-binding fragment thereof according to the present invention is provided.

In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof according to the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In a preferred embodiment, the kit further comprises a secondary antibody that specifically recognizes the antibody according to the invention or an antigen-binding fragment thereof. Preferably, the secondary antibody further comprises a detectable label. Such detectable labels well known to those skilled in the art include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, chromogenic substances and enzymes (eg horseradish peroxidase), and the like.

In another aspect, the present invention provides a method for detecting the presence or level of HBsAg protein in a sample comprising using an antibody or antigen-binding fragment thereof according to the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In another preferred embodiment, the method further comprises using a secondary antibody having a detectable label for detecting the antibody or antigen-binding fragment thereof according to the invention. The method can be used diagnostically or non-diagnostic (eg, the sample is a cell sample that is not a sample from a patient).

In another aspect, the present invention provides a method for diagnosing whether a subject is infected with HBV, comprising detecting the presence of HBsAg protein in a sample from the subject using an antibody or antigen-binding fragment thereof according to the present invention . In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In another preferred embodiment, the method further comprises using a secondary antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof according to the invention.

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

The antibody or antigen-binding fragment thereof according to the present invention is used to prevent or treat HBV infection or a disease associated with HBV infection (eg hepatitis B) in a subject (eg human), in vitro or in a subject (eg, It can be used to neutralize HBV toxicity in humans) and to reduce serum levels of HBV DNA and/or HBsAg in subjects (eg humans).

Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier and/or excipient. In a preferred embodiment, the pharmaceutical composition according to the present invention may further comprise an additional pharmaceutically active agent. In a preferred embodiment, the additional pharmaceutically active agent is an agent for the prophylaxis or treatment of HBV infection or a disease associated with HBV infection (eg hepatitis B), for example an interferon-type agent, for example interferon or pegylated interferon.

In another embodiment, preventing or treating HBV infection or a disease associated with HBV infection (eg, hepatitis B) in a subject (eg, a human) and reducing HBV toxicity in vitro or in a subject (eg, a human) Use of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for neutralizing and/or reducing the serum level of HBV DNA and/or HBsAg in a subject (eg human) provides

In another aspect, the present invention prevents or treats HBV infection or a disease associated with HBV infection (eg, hepatitis B) in a subject (eg, a human) and neutralizes HBV toxicity in a subject (eg, a human) and/or provides a method for reducing the serum level of HBV DNA and/or HBsAg in a subject (eg human), said method comprising administering to said subject an effective amount of an antibody according to the invention or an antigen-binding fragment thereof or It comprises administering the pharmaceutical composition according to the present invention.

The medicaments and pharmaceutical compositions provided herein can be used alone or in combination with an additional pharmaceutically active agent (e.g., other antiviral agents, e.g., interferon-type agents, e.g., interferon or pegylated interferon) can be used with

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are for illustrative purposes only and do not limit the scope of the present invention. will know Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments and the accompanying drawings.

1 shows the results of an ELISA assay for determining the binding activity of three cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 and M3-13 to the antigen HBsAg in Example 2, The abscissa indicates the antibody concentration (Log ₁₀ ng/ml) and the ordinate indicates the OD value. The results showed that all three cynomolgus monkey-human chimeric monoclonal antibodies all had good antigen-binding activity.
2 is a competitive ELISA assay for cross-blocking of binding of three cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 and M3-13 to the antigen HBsAg by the 6D11 mouse antibody in Example 2. shows the results of The results showed that the binding of M1-23, M3-23 and M3-13 to HBsAg could be significantly inhibited by 6D11.
3A and 3B show the evaluation results of in vivo therapeutic effects of cynomolgus monkey-human chimeric monoclonal antibodies M1-23 and M3-23 in HBV transgenic mice in Example 2. FIG. Figure 3a shows the change in HBsAg level in the serum of mice, where the abscissa represents the number of days after injection of the chimeric antibody, and the ordinate represents the relative reduction level of HBsAg in the serum of mice (Log ₁₀ IU/ml). indicate; Figure 3b shows the change in HBV DNA level in the serum of mice, where the abscissa represents the number of days after injection of the chimeric antibody, and the ordinate represents the relative reduction level of HBV DNA in the serum of mice (Log ₁₀ IU/ml ) is indicated. The results showed that M1-23 and M3-23 can clearly clear HBsAg and HBV DNA in animals.
Figure 4 shows the results of ELISA analysis of the binding activity of humanized antibodies M1D, M3D to antigen HBsAg in Example 4, wherein the abscissa represents the antibody concentration (Log ₁₀ ng/ml) and the ordinate represents the OD value. indicates The results showed that both humanized antibodies had good antigen binding activity and their affinity for HBsAg was superior to that of reference antibody 162.
5 depicts the results of a competitive ELISA assay for cross-blocking of binding of humanized antibodies M1D, M3D to antigen HBsAg by 6D11 mouse antibody in Example 4. The results showed that the binding of humanized antibodies M1D, M3D to antigen HBsAg could be significantly inhibited by 6D11.
6A and 6B show the measurement results of the neutralizing activity of the humanized antibody M1D against HBV virus in Example 4, wherein FIG. 6A shows a standard curve obtained using HBIG as a standard, and FIG. 6B shows The measurement result of the neutralizing activity in the sample is shown, wherein the ordinate indicates the number of standard units (mIU) of HBIG having a neutralizing activity equivalent to the neutralizing activity per mg of antibody. The results showed that the neutralizing activity of M1D against HBV was comparable to that of the reference antibody 162.
7 depicts the evaluation results of in vivo therapeutic effects of humanized antibodies M1D and M3D and chimeric antibody M3-13 in HBV transgenic mice in Example 5. 7A depicts changes in HBsAg levels in the serum of mice, where the abscissa represents days after injection of humanized antibody, and the ordinate represents the relative reduction level of HBsAg in the serum of mice (Log ₁₀ IU/ml). indicate; Figure 7b shows the change of HBV DNA level in the serum of mice, where the abscissa represents the number of days after injection of the chimeric antibody, and the ordinate represents the relative reduction level of HBV DNA in the serum of mice (Log ₁₀ IU/ml ) is indicated. The results showed that all three antibodies were able to clear HBsAg and HBV DNA in animals and their effect was superior to that of the reference antibody 162.
8A-8E show the results of an ELISA analysis of the binding activity of antibodies containing substitutions of CDR regions as compared to humanized antibody M1D against the antigen HBsAg in Example 6, wherein the abscissa is the antibody concentration ( Log ₁₀ ng/ml) and the ordinate indicates OD values. The results showed that the substitution of HCDR1 and LCDR2 of antibody M1D did not affect the binding activity to antigen HBsAg, indicating that the HCDR1 and LCDR2 regions of antibody M1D are not important CDR regions for binding to antigen HBsAg.
9A-9E show the results of an ELISA analysis of the binding activity of antibodies containing substitutions of CDR regions as compared to humanized antibody M3D against the antigen HBsAg in Example 6, wherein the abscissa is the antibody concentration ( Log ₁₀ ng/ml) and the ordinate indicates OD values. The results showed that the substitution of heavy chain CDR2 (HCDR2) and light chain CDR2 (LCDR2) did not affect the binding activity to antigen HBsAg, indicating that the HCDR2 and LCDR2 regions of antibody M3D are not important CDR regions for binding to antigen HBsAg. implies
Figure 10 depicts the results of Western blot analysis of the binding of antibodies M1D, M3D, 162 to various single-site mutated polypeptide fragments of HBsAg aa 113-135 in Example 8. The results show that antibody 162 loses binding activity to HBsAg when sites in aa 120, aa 122 and aa 123 are mutated to alanine and its core epitope is CKTC (aa 121-124); M1D has binding activity to HBsAg when the site in aa 118-124 is mutated to alanine and its core epitope is TGPCKTCT (aa 118-124) longer than that of reference antibody 162 (aa 118-124) and is in the forward position compared to that of reference antibody 162 loses; It was shown that M3D loses binding activity to HBsAg when a site in aa 121-125 is mutated to alanine and its core epitope is CKTCT (aa 121-125) in a reverse position compared to that of 162.
11A and 11B show the results of ELISA analysis of the binding activity of humanized antibodies M1D, M3D to different subtypes of HBsAg-aa 113-135 in Example 9, where the abscissa is the antibody concentration (Log ₁₀ ng/ml) and the ordinate represents the OD value. The results showed that M1D and M3D could bind to most HBV subtypes.
12A and 12B show the blood of humanized antibody M1D in the serum of each cynomolgus monkey (Group 1) as a function of time after a single intravenous injection of 20 mg/kg humanized antibody M1D, M3D in Example 11; A graph of curve versus concentration is shown, where the abscissa represents time (h) and the ordinate represents blood concentration (ng/ml).
13A and 13B depict the Tm values of humanized antibodies M1D, M3D in 3 different buffers in Example 12. The abscissa indicates temperature (°C) and the ordinate indicates CP (cal/°C). The results showed that the humanized antibody M1D had a Tm onset of greater than 61.7°C, and that of M3D both had a Tm onset value of greater than 61.6°C, indicating that M1D and M3D had good thermal stability.

sequence information

Information of some sequences related to the present invention is provided in Table 1 below.

description of the sequence SEQ ID NO: sequence information One QVQLQESGPGLVKPSEILSVTCSVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLSRVTLSVDTSKNQLSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGLRVTVSS 2 EIVMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPQLLIYYANRLESGVPSRFRGSGSGTEFTLTISSLQPEDFTTYYCQQYHSLPLTFGGGTKVEIK 3 QLQLQESGPGLVKPSETLSLTCAVSGVSISSPYDWTWIRQPPGKGLEWIGRIHGNGGSTSYNPSLKNRVTISKDTSKNQFSLILSSVTAADTAVYYCVRDETWGQGVLVTVSS 4 DIQMSQSPDSLAVSLGERVTINCKSSQSLLYSSNNKNYLAWYQQKPGQAPKLLFYWASTRESGVPNRFSGSGSGTDFTLTIRGLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 5 QVQLQESGPRLVKSSETLPLTCAVSGASNSSNYWSWIRQPPGKGLEWIGRIYGNSGSTDYNPSLKSRVSISTDTSKNQFSLKLSSVTAADTAVYFCARGSVYTYSWGQGVLVTVSS 6 GGSITSNF 7 ISGSGTYT 8 ARSHDYGSNDYAFDF 9 QDISSS 10 YAN 11 QQYHSLPLT 12 GVSISSPYDW 13 IHGNGST 14 VRDET 15 QSLLYSSNNKNY 16 WAS 17 QQHYTTPLT 18 GASNSSNY 19 IYGNSGST 20 ARGSVYTYS 21 QVQLQESGPGLVKPSEILSVTCSVS 22 WSWIRQPPGKGLEWIGY 23 DYNPSLRSRVTLSVDTSKNQLSLKLSSVTAADTAVYYC 24 WGQGLRVTVSS 25 EIVMTQSPSSLSASVGDRVTITCRAT 26 LNWYQQKPGKAPQLLIY 27 RLESGVPSRFRGSGSGTEFTLTISSLQPEDFTTYYC 28 FGGGTKVEIK 29 QLQLQESGPGLVKPSETLSLTCAVS 30 TWIRQPPGKGLEWIGR 31 SYNPSLKNRVTISKDTSKNQFSLILSSVTAADTAVYYC 32 WGQGVLVTVSS 33 DIQMSQSPDSLAVSLGERVTINCKSS 34 LAWYQQKPGQAPKLLFY 35 TRESGVPNRFSGSGSGTDFTLTIRGLQAEDVAVYYC 36 FGQGTKVEIK 37 QVQLQESGPRLVKSSETLPLTCAVS 38 WSWIRQPPGKGLEWIGR 39 DYNPSLKSRVSISTDTSKNQFSLKLSSVTAADTAVYFC 40 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYTSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 41 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP 42 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCAR 43 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP 44 QVQLQESGPGLVKPSETLSLTCTVS 45 DYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC 46 WGQGTTVTVSS 47 DIQMTQSPSSSLSASVGDRVTITCRAT 48 LNWYQQKPGKAPKLLIY 49 RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 50 QVQLQESGPGLVKPSGTLSLTCAVS 51 TWVRQPPGKGLEWIGR 52 SYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYC 53 GQGTLVTVSS 54 DIVMTQSPDSLAVSLGERATINCKSS 55 LAWYQQKPGQPPKLLFY 56 TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 57 QVQLQESGPGLVKPSETLSLTCTVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS 58 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPKLLIYYANRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSLPLTFGGGTKVEIK 59 QVQLQESGPGLVKPSGTLSLTCAVSGVSISSPYDWTWVRQPPGKGLEWIGRIHGNGGSTSYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCVRDETWGQGTLVTVSS 60 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLFYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 61 GGACAGCCKGGAAGGTGTGC 62 GAGGCAGTTCCAGATTTCAA 63 CCGCGTACTTGTTGTTGCTCTGT 64 5'-AGTAGCAACTGCAACCGGTGTACATTCTcaggtgcagctgcaggagtcaggcccag 65 5'-caggagtcaggcccaggactggtgaagccttcggagaCCCTGTCCCTCACCTGCAC 66 5'-aagccttcggagaCCCTGTCCCTCACCTGCACtgtctctggtggctccatc 67 5'- GTTCTTGGACGTGTCTACGGATATGGTGACTCGACTCTTGAGGGAGGGGTTGTAGT 68 5'-TCCGTAGACACGTCCAAGAACCAGTtctccctgaaactgagctc 69 5'-GATGGCCCTTGGTCGACGCtgaagagacggtgacGGTGGTcccttggccccagaaatc 70 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTCTCCATCC 71 5'-CAGCAAAAACCGGGGAAAGCCCCTAAGCTCCTGATCTATTATGC 72 5'-GAACCTTGATGGGACCCCACTTTGCAAACGATTGCATAATAGATCAGGAGC 73 5'-CTGTCCCAGATCCACTGCCACTGAACCTTGATGGGACCCC 74 5'- GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAGG 75 5'-GCAGCCTGCAACCTGAGGATTTTGCAACTTATTACTGTCAAC 76 5'-GACCAAGGTGGAAATCAACGTACGGTGGCTGCACCAT 77 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC 78 5'-ATTCCCGGGTagatgcccaGTAAAAGAGCAGCTTAGGAGGCTGTCCTGGTTTCTGCTG 79 5'-ggtgtctccatcagcagtccttatgacTGGACCTGGGTCCGCCAGCCCCCAGGGAAG 80 5'-AACTGGTTCTTGGACTTGTCTACTGAAATGGTGACTCGACTCTTGAGGGAGGGGTTG 81 5'- AGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGC 82 5'-gaaaccTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC 83 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTCCAGACTCC 84 5'-CCCAGTCTCCAGACTCCCTGGCTTGTGTCTCTGGGAGAGAGGGCCACCATCAACTGCAAGTCCAG 85 5'-CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCTTTTACtgggcatctACCCGGGAAT 86 5'- GTGAAATCTGTCCCAGATCCACTGCCACTGAATCGGTCAGGGACCCCGGATTCCCGGGTagatgcc 87 5'-GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATG 88 5'-ATGGTGCAGCCACCGTACGTTGATTTCCACCTTGGTCC 89 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCAGGC 90 5'-GATGGCCCTTGGTCGACGCTGAAGAGACGGTGAC 91 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTC 92 5'-ATGGTGCAGCCACCGTACTTTGATTTCCACCTTGGTCCC 93 5'-GTAGTAACTACTGATGGAGCCACCAGAGACAGTGCAGGTGAG 94 5'-GgtggctccatcagtagttactacTGGAGCTGGATCCGCCAG 95 5'-ATAGTAACTACCACTGCTGACGGAGCCACCAGAGACAGTGCAGGTGAG 96 5'-GgtggctccgtcagcagtggtagttactATTGGAGCTGGATCCGCCAG 97 5'-GGTGCTCCCACTGGTATAGATATACCCAATCCACTCCAG 98 5'-AtctataccagtgggagcaccGACTACAACCCCTCCCTC 99 5'-TGTGTAACTACTACTACTACTAATATACCCAATCCACTCCAG 100 5'-AttagtagtagtagtagttacacaGACTACAACCCCTCCCTC 101 5'-GTAGCTGCTGCTAACACTCTGAGTTGCCCGGCAAGTGATGG 102 5'-CagagtgttagcagcagctacTTAAATTGGTATCAGC 103 5'-AGTATTACTTCCGATGTTGGAGCTAGTTGCCCGGCAAGTGATGG 104 5'-AgctccaacatcggaagtaatactTTAAATTGGTATCAGC 105 5'-GACCCCACTTTGCAAACGGGATGCAGCATAGATCAGGAGCTTAGG 106 5'-CCTAAGCTCCTGATCTATGctgcatccCGTTTGCAAAGTGGGGTC 107 5'-GACCCCACTTTGCAAACGAGACGCCTTATAGATCAGGAGCTTAGG 108 5'-CCTAAGCTCCTGATCTATAaggcgtctCGTTTGCAAAGTGGGGTC 109 5'-AGTGAGAGGGTAATTGTAATCTTGTAGACAGTAATAAGTTGCAAAATC 110 5'-CtacaagattacaattaccctCTCACTTTCGGCGGAGGGACCAAG 111 5'-AGTGAGAGGTAAACTACTACTCTGATGACAGTAATAAGTTGCAAAATC 112 5'-catcagagtagtagtttacctCTCACTTTCGGCGGAGGGACCAAG 113 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCG 114 5'-GATGGCCCTTGGTCGACGCTGAGGAGACGGTGACCAG 115 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTC 116 5'-ATGGTGCAGCCACCGTACGTTGATTTCCACCTTGGTC 117 5'-GGAGTAACCACCACTGCTGATGGAGCCACCAGAGACAGCGCAGGTGAGGGACAG 118 5'-ggtggctccatcagcagtggtggttactccACCTGGGTCCGCCAGCCC 119 5'-CCAGTTACTACTGCTGATGGAGCCACCAGAGACAGCGCAGGTGAGGGACAG 120 5'-GgtggctccatcagcagtagtaactggACCTGGGTCCGCCAGCCC 121 5'-GGTGCTCCCACTATGATAGATTCGTCCAATCCACTCCAG 122 5'-AtctatcatagtgggagcaccAGCTACAACCCCTCCCTCAAG 123 5'-TGTGCTACCACCACTACCACTAATTCGTCCAATCCACTCCAG 124 5'- attagtggtagtggtggtagcacaAGCTACAACCCCTCCCTCAAG 125 5'-ATAGGTCTTTCCATCACTATGCAGGAGGCTCTGGCTGGACTTGCAGTTGATGGTGGC 126 5'- cagagcctcctgcatagtgatggaaagacctatTTAGCCTGGTACCAGCAGAAAC 127 5'-GTAGCTGCTGCTAACACTCTGGCTGGACTTGCAGTTGATGGTGGC 128 5'-CagagtgttagcagcagctacTTAGCCTGGTACCAGCAGAAAC 129 5'- GGGACCCCGGATTCCCGGGTAGAACCCAAGTAAAAGAGCAGCTTAGGAGG 130 5'-CCTCCTAAGCTGCTCTTTTACTtgggttctACCCGGGAATCCGGGGTCCC 131 5'- GGGACCCCGGATTCCCGGGTGGATGCACCGTAAAAGAGCAGCTTAGGAGG 132 5'-CCTCCTAAGCTGCTCTTTTACGgtgcatccACCCGGGAATCCGGGGTCCC 133 5'-CGTCCAAGGAGTACTATAATATTGCTGACAGTAATACACTGCCACATC 134 5'-CagcaatattatagtactccttggacgTTCGGCCAAGGGACCAAG 135 5'-CGTCCAAGGAGTTTGTAGAGCTTGCATACAGTAATACACTGCCACATC 136 5'-AtgcaagctctacaaactccttggacgTTCGGCCAAGGGACCAAG 137 GGSISSYY 138 GGSVSSGSYY 139 AAS 140 KAS 141 IYHSGST 142 ISGSGGST 143 LGS 144 GAS 145 IYTSGST 146 ISSSSSYT 147 QSVSSSY 148 SSNIGSNT 149 LQDYNYPLT 150 HQSSSLPLT 151 GGSISSGGYS 152 GGSISSSNW 153 QSLLHSDGKTY 154 QSVSSSY 155 QQYYSTPWT 156 MQALQTPWT 157 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS 158 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS 159 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSLPLTFGGGTKVEIK 160 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSLPLTFGGGTKVEIK 161 QVQLQESGPGLVKPSETLSLTCTVSGGSITSNFWSWIRQPPGKGLEWIGYISSSSSYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS 162 DIQMTQSPSSLSASVGDRVTITCRATSSNIGSNTLNWYQQKPGKAPKLLIYYANRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSLPLTFGGGTKVEIK 163 QVQLQESGPGLVKPSGTLSLTCAVSGVSISSPYDWTWVRQPPGKGLEWIGRIYTSGSTSYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCVRDETWGQGTLVTVSS 164 QVQLQESGPGLVKPSGTLSLTCAVSGVSISSPYDWTWVRQPPGKGLEWIGRISGSGGSTSYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCVRDETWGQGTLVTVSS 165 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLFYLGSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 166 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLFYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 167 DIVMTQSPDSLAVSLGERATINCKSSQSLLHSDGKTYLAWYQQKPGQPPKLLFYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 168 SSTTSTGPCKTCTTPAQGTSMFP 169 SSTTATGPCKTCTTPAQGTSMFP 170 SSTTSAGPCKTCTTPAQGTSMFP 171 SSTTSTAPCKTCTTPAQGTSMFP 172 SSTTSTGACKTCTTPAQGTSMFP 173 SSTTSTGPSKTCTTPAQGTSMFP 174 SSTTSTGPCATCTTPAQGTSMFP 175 SSTTSTGPCKACTTPAQGTSMFP 176 SSTTSTGPCKTSTTPAQGTSMFP 177 SSTTSTGPCKTCATPAQGTSMFP 178 SSTTSTGPCKTCTAPAQGTSMFP 179 SSTTSTGPCKTCTTAAQGTSMFP 180 SSTTSTGPCKTCTTPAAGTSMFP 181 SSTTSTGPCKTCTTPAQGASMFP 182 SSTTSTGPCKTCTTPAQGTAMFP 183 ATCAACTACCGCCACGGGACCATGCAAGACCTGCAC 184 GGTCCCGTGGCGGTAGTTGATGTTCCTGGAAGTAGA 185 AACTACCAGCGCGGGACCATGCAAGACCTGCACGAT 186 CATGGTCCCGCGCTGGTAGTTGATGTTCCTGGAAGT 187 ACCAGCACGGCACCATGCAAGACCTGCACGATTCCT 188 CTTGCATGGTGCCGTGCTGGTAGTTGATGTTTCCTGG 189 CAGCACGGGAGCATGCAAGACCTGCACGATTCCTGC 190 GTCTTGCATGCTCCCGTGCTGGTAGTTGATGTTTCCT 191 ACGGGACCATCCAAGACCTGCACGATTCCTGCTCAA 192 GCAGGTCTTGGATGGTCCCGTGCTGGTAGTTGATGTGT 193 GGGACCATGCCGGACCTGCACGATTCCTGCTCAAGG 194 GTGCAGGTCCGGCATGGTCCCGTGCTGGTAGTTGAT 195 GGGACCATGCGCGACCTGCACGATTCCTGCTCAAGG 196 GTGCAGGTCGCGCATGGTCCCGTGCTGGTAGTTGAT 197 ACCATGCAAGGCCTGCACGATTCCTGCTCAAGGAAC 198 ATCGTGCAGGCCTTGCATGGTCCCGTGCTGGTAGTT 199 TGCAAGACCTCCACGATTCCTGCTCAAGGAACCTCT 200 AGGAATCGTGGAGGTCTTGCATGGTCCCGTGCTGGT 201 CAAGACCTGCGCGATTCCTGCTCAAGGAACCTCTAT 202 GCAGGAATCGCGCAGGTCTTGCATGGTCCCGTGCTG 203 GACCTGCACGGCTCCTGCTCAAGGAACCTCTATGTT 204 TGAGCAGGAGCCGTGCAGGTCTTGCATGGTCCCGTG 205 CTGCACGATTGCTGCTCAAGGAACCTCTATGTTTCC 206 CCTTGAGCAGCAATCGTGCAGGTCTTGCATGGTCCC 207 GATTCCTGCTGCAGGAACCTCTATGTTTCCCTCTTG 208 GAGGTTCCTGCAGCAGGAATCGTGCAGGTCTTGCAT 209 TGCTCAAGGAGCCTCTATGTTTCCCTCTTGTTGCTG 210 AACATAGAGGCTCCTTGAGCAGGAATCGTGCAGGTC 211 TCAAGGAACCGCTATGTTTCCCTCTTGTTGCTGTAC 212 GGAAACATAGCGGTTCCTTGAGCAGGAATCGTGCAG 213 STTTSTGPCKTCTTPAQGNSMFP 214 TSTTSTGPCKTCTIPAQGTSMFP 215 SSTTSTGPCRTCTTPAQGTSMYP 216 SSTTSTGPCRTCTTLAQGTSMFP 217 STTTSTGPCKTCTTLAQGTSMFP 218 STTTSTGPCKTCTALAQGTSMFP 219 STTTSTGPCRTCTTLAQGTSMLP 220 SSTTSTGPCKTCTTPAQGNSMYP

Definition of Terms

In the present invention, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art unless otherwise stated. Moreover, as used herein, the execution steps of cell culture, biochemistry, nucleic acid chemistry, immunology experiments, etc. are conventional steps widely used in the corresponding fields. In addition, for a better understanding of the present invention, relevant definitions and explanations are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. refers to Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of at least about 12 amino acids, and the heavy chain further comprises a “D” region of at least about 3 amino acids. Each heavy chain consists of a heavy chain variable region (V _H ) and a heavy chain constant region ( _CH ). The heavy chain constant region consists of three domains ( _CH 1 , C _H 2 and C _H 3 ). Each light chain consists of a light chain variable region (V _L ) and a light chain constant region ( _CL ). The light chain constant region consists of one domain, C _L . The constant regions of an antibody are capable of mediating binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg effector cells) and the first component of the classical complement system (C1q). The V _H and V _L regions can also be subdivided into relatively conserved regions, termed framework regions (FR), and spaced hypervariable regions (referred to as complementarity determining regions (CDRs)). Each V _H and V _L consists of 3 CDRs and 4 FRs arranged in the following order: from amino terminus to carboxy terminus FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V _H and V _L ) of each heavy/light chain pair each form an antibody binding site. The distribution of amino acids in the various regions or domains is described in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or in Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883].

As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding, generally residues 24-34 {LCDR1} in the light chain variable region; 50-56{LCDR2}, 89-97{LCDR3}, and residues 31-35{HCDR1}, 50-65{HCDR2}, 95-102{HCDR3} in the heavy chain variable region (see, e.g., Kabat et al, Sequences of Proteins of lmmunological lnterest, 5 ^th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)), or residues 26-32 {Ll}, 50-52 {L2} in the light chain variable region; 91-96{L3}, and residues 26-32{H1}, 53-55{H2}, 96-101{H3} in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917) (1987)]).

As used herein, the term "framework region" or "FR" refers to amino acid residues other than CDR residues as defined above in the variable region of an antibody.

The term "antibody" is not limited by any particular method by which the antibody is generated. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be an antibody of a different isotype, for example an IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a full-length antibody that specifically binds to and/or retains the ability to specifically bind to the same antigen to which the full-length antibody binds. refers to a polypeptide comprising a fragment of a full-length antibody that competes with and is also referred to as an “antigen-binding portion”. See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)], which is incorporated herein by reference in its entirety for all purposes. Can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibody In some cases, antigen-binding fragments are Fab, Fab', F(ab') ₂ , Fd, Fv, dAb and complementarity. fragments of a determining region (CDR), single chain antibodies (eg scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.

As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the V _H and C _H 1 domains; The term "dAb fragment" refers to an antibody fragment consisting of the V _H domain (Ward et al, Nature 341:544 546 (1989)); The term “Fab fragment” refers to an antibody fragment consisting of the V _L , V _H , C _L and C _H 1 domains; The term “F(ab′) ₂ fragment” refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

As used herein, the term “Fv fragment” refers to an antibody fragment consisting of the single branched V _L and V _H domains of an antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (eg an Fd fragment containing only three CDRs specific for an antigen) is capable of recognizing and binding antigen, albeit with an affinity less than that of the entire binding site.

In some cases, the antigen-binding fragment of the antibody is a single chain antibody (eg scFv), wherein the V _L and V _H domains are monovalent via a linker that pairs, allowing them to generate a single polypeptide chain. form molecules (eg, Bird et al., Science 242: 423-426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). ; and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994). Such scFv molecules may have a general structure: NH ₂ -V _L -Linker-V _H -COOH or NH ₂ -V _H -Linker-V _L -COOH. A linker suitable in the prior art may consist of a repeating amino acid sequence of GGGGS or a variant thereof. For example, linkers having the amino acid sequence (GGGGS) ₄ can be used, and variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

As used herein, the term "single chain antibody-Fc" or "scFv-Fc" refers to an engineered antibody formed by linking an scFv to an Fc fragment of an antibody. As used herein, the term "Fc fragment" refers to an antibody fragment formed by linking the second and third constant regions of a first heavy chain of an antibody to the second and third constant regions of a second heavy chain via disulfide bonds. refers to Fc fragments of antibodies have a variety of different functions, but are not involved in antigen binding.

In some cases, antigen-binding fragments of antibodies are diabodies, i.e., the V _L and V _H domains are expressed on a single polypeptide chain, but the linker used is too short to pair between the two domains of the same chain, such that one domain is a bivalent antibody that forces pairing with the complementary domain of another chain to generate two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad.Sci.USA 90 : 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

Antigen-binding fragments of an antibody (e.g. the spheroid fragments described above) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) to a given antibody (e.g., monoclonal antibodies provided herein), and can be screened for specificity in the same manner as complete antibodies are screened.

As used herein, unless expressly specified, when referring to the term “antibody”, antibody includes both intact antibodies as well as antigen-binding fragments of antibodies.

As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody from a population of highly homologous antibody molecules, i.e., a population of antibody molecules that are exactly the same except for natural mutations that may occur spontaneously. refers to fragments. Monoclonal antibodies have high specificity for a single epitope of an antigen. In comparison to monoclonal antibodies, polyclonal antibodies generally comprise at least two or more different antibodies that generally recognize different epitopes on the antigen. Monoclonal antibodies are generally obtained by the hybridoma technique first reported by Kohler et al. (Nature, 256:495, 1975), and may also be obtained by recombinant DNA techniques (e.g., U.S. Pat. 4,816,567).

For example, monoclonal antibodies can be prepared as follows. First, a non-human primate (eg cynomolgus monkey) or other suitable host animal is immunized by injection of an immunogen (adjuvanted if necessary). The means of injection of the immunogen or adjuvant is generally subcutaneous multi-point injection or intraperitoneal injection. Pre-conjugation of an immunogen to some known protein (eg serum albumin or soybean trypsin inhibitor) can promote the immunogenicity of the antigen in the host. The adjuvant may be Freund's adjuvant or MPL-TDM. After immunization of the animal, lymphocytes are produced in the animal that secrete antibodies that specifically bind to the immunogen. Lymphocytes can also be obtained by in vitro immunization. The lymphocytes of interest are collected and fused to myeloma cells using a suitable fusing agent (eg PEG) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). The hybridoma cells prepared above are seeded in a suitable culture medium and grown in the medium, and the culture medium preferably contains one or more substances capable of inhibiting proliferation of unfused, parental myeloma cells. For example, in the case of parental myeloma cells deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), substances such as hypoxanthine, aminopterin, and thymidine (HAT culture medium) are added to the culture medium. Inhibits proliferation of HGPRT-deficient cells. Preferred myeloma cells should have a high confluent rate, a stable ability to secrete antibodies, be sensitive to HAT culture medium, and the like. A culture medium for proliferation of hybridoma cells is used to detect the production of monoclonal antibodies to specific antigens. The following methods, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) can be used to determine the binding specificity of monoclonal antibodies produced by hybridoma cells. can See, eg, Munson et al., Anal. Biochem. 107: 220 (1980) can be used to determine the affinity of monoclonal antibodies. After determination of the specificity, affinity and reactivity of the antibodies produced by the hybridomas, the cell lines of interest are analyzed in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996]. A suitable culture medium may be DMEM or RPMI-1640 or the like. In addition, hybridoma cells can be proliferated in the form of ascites tumors in an animal body. Cell culture of monoclonal antibodies secreted by subclonal cells by using traditional methods for the purification of immunoglobulins, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. It can be isolated from water, ascites or serum.

Monoclonal antibodies can also be obtained by genetically engineered recombinant techniques. DNA molecules encoding MAb heavy and light chains are obtained from hybridoma cells by PCR amplification of nucleic acid primers that specifically bind to genes of MAb heavy and light chains. The obtained DNA molecule is inserted into an expression vector, and host cells (eg E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) are transfected with the vector, and by recombinant expression. Incubate under conditions suitable for obtaining the antibody of interest.

As used herein, the terms "antigenic epitope" and "epitope" refer to a portion on an antigen to which an immunoglobulin or antibody specifically binds. An “epitope” is also known as an “antigenic determinant”. Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and usually have a specific three-dimensional structure and specific charge characteristics. For example, an epitope generally has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or contains 15 contiguous or non-contiguous amino acids. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)]. In a linear epitope, all interaction sites between a protein and an interacting molecule (eg an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, the interaction sites span amino acid residues that separate the interaction sites from each other in the protein. Antibodies can be selected according to their competitiveness for binding to the same epitope by conventional techniques known by those skilled in the art. For example, competition or cross-competition studies can be conducted to obtain antibodies that compete or cross-compete with each other for binding to antigen. A high-throughput method for obtaining antibody binding to the same epitope, based on cross-competition, is described in International Patent Application WO 03/48731.

As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to those that have not undergone a maturation process capable of inducing gene rearrangements and mutations for expression of a particular immunoglobulin. , refers to a sequence present in the genome of a gene encoding an immunoglobulin. In the present invention, the expression "heavy chain germline gene" refers to a germline antibody gene encoding an immunoglobulin heavy chain comprising a V gene (variable), a D gene (diversity), a J gene (binding) and a C gene (constant) or refers to a gene fragment; Similarly, the expression "light chain germline gene" refers to a germline antibody gene or gene fragment encoding an immunoglobulin light chain comprising a V gene (variable), a J gene (binding) and a C gene (constant). In the present invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also referred to as "germline sequence". Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (eg IMGT, unswag, NCBI or VBASE2).

As used herein, the terms "specifically binds" and "specifically binds" refer to the binding of two molecules in a non-random manner, eg, a reaction between an antibody and an antigen against it. do. In some embodiments, an antibody that specifically binds an antigen (or an antibody specific for an antigen) is less than about 10 ^-5 M, such as about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ⁻ Refers to an antibody that binds to an antigen with an affinity (K _D ) of less than or less than ⁹ M or 10 ^-10 M.

As used herein, the term "K _D " refers to the dissociation constant of a particular antibody-antigen interaction used to describe the binding affinity of an antibody for an antigen. The smaller the dissociation constant, the tighter the binding to the antibody and the higher the affinity between the antibody and the antigen. In general, an antibody (eg an antibody according to the invention) is less than about 10 ⁻⁵ M, eg about 10 ⁻⁶ M, 10 as measured by surface plasmon resonance (SPR), eg on a BIACORE device. Binds to antigen (eg HBsAg) with a K _D of less than or less than ^-7 M, 10 ^-8 M, 10 ^-9 M or 10 ^-10 M.

As used herein, the term “immunogenicity” refers to the ability to stimulate the formation of specific antibodies or sensitizing lymphocytes in an organism. It refers not only to the property of an antigen to activate, proliferate and differentiate specific immune cells to ultimately produce immunological effector substances, such as antibodies and sensitizing lymphocytes, but also to stimulate the antigen to produce an antibody or sensitizing T Lymphocytes refer to a specific immune response that can be formed in an organism's immune system. When a heterologous antibody is applied to a subject, immunogenicity of the heterologous antibody in the subject is unnecessary. Such immunogenicity may lead to rejection of the heterologous antibody by the subject's immune system/immune cells, which may result in reduced efficacy of the heterologous antibody in the subject or unwanted side effects in the subject. Thus, prior to administration to a human subject, a heterologous antibody (eg, a cynomolgus monkey-derived antibody) generally needs to be engineered to reduce its immunogenicity as much as possible.

As used herein, the term "chimeric antibody" means that a portion of its light and/or heavy chain is derived from an antibody (either from a particular species or from a particular antibody type) so long as the antibody still retains the activity of binding to the antigen of interest. or may belong to a subtype) and different portions of its light and/or heavy chains are derived from another antibody (which may originate from the same or different species or belong to the same or different antibody type or subtype). (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, the term "chimeric antibody" may include such antibodies (eg, human-monkey chimeric antibodies), wherein the heavy and light chain variable regions of the antibody comprise a primary antibody (eg, cynomolgus monkey-derived antibodies), whereas the heavy and light chain constant regions of the antibody are derived from secondary antibodies (eg human antibodies).

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody in which the amino acid sequence has been modified to increase its homology to that of a human antibody. Generally, some or all of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and some or all of the non-CDR regions (eg, variable region FRs and/or constant regions) are derived from human immunity. It is derived from globulin (recipient antibody). Humanized antibodies typically retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to neutralize and/or clear viruses, and the like. The donor antibody may be a mouse, rat, rabbit or non-human primate (eg cynomolgus monkey) having the expected properties (eg antigen specificity, affinity, reactivity, virus neutralizing ability, and/or virus clearance ability). may be an antibody of

A humanized antibody can retain the expected properties of a non-human donor antibody (eg, a cynomolgus monkey-derived antibody) and in a human subject a non-human donor antibody (eg, a cynomolgus monkey-derived antibody). ) is particularly advantageous because it is effective in reducing the immunogenicity of However, due to congruence issues between the CDRs of the donor antibody and the FRs of the recipient antibody, the expected properties of the humanized antibody (eg antigen specificity, affinity, reactivity, ability to neutralize virus, and/or ability to clear virus) are non- generally lower than for human donor antibodies (eg cynomolgus monkey-derived antibodies).

Thus, although researchers in the art have conducted intensive studies on the humanization of antibodies and made some advances (eg, Jones et al., Nature, 321:522 525 (1986); Reichmann et al. , Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000) ), the prior art provides detailed guidance on how to humanize several donor antibodies sufficiently so that the resulting humanized antibodies can have as high a degree of humanization as possible while retaining as much as possible the expected properties of the donor antibody. it's not happening Those skilled in the art should investigate, explore and engineer specific donor antibodies, and have a high degree of humanization (e.g., at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% degree of humanization), as well as retaining the expected properties of the donor antibody. should be done

In the present invention, in order to prepare a humanized antibody that retains as much as possible the properties of the donor antibody (including, for example, specificity, affinity, reactivity, virus neutralizing ability and/or virus clearance ability), the humanized antibody of the present invention The framework region (FR) of a may comprise both amino acid residues of an acceptor antibody of human origin and amino acid residues of a corresponding donor antibody of non-human origin.

In addition, in order to further increase the degree of humanization in order to reduce as much as possible the immunogenicity caused by non-human amino acid residues, in the humanized antibody of the present invention some of the amino acid residues derived from the CDR regions of the donor antibody, for example For example, the corresponding amino acid residues or other amino acid residues of the CDR regions of human immunoglobulin may be substituted.

In addition, in order to further improve or optimize the performance of the humanized antibody of the invention, some of the amino acid residues in the heavy and light chain variable regions of the humanized antibody of the invention, for example, are neither derived from the recipient antibody nor from the donor antibody. It may be further substituted with an amino acid residue not shown.

Specifically, in the present application, the inventors first described three cynomolgus monkey-human chimeric monoclonal antibodies with excellent properties, named M1-23, M3-23 and M3-13, respectively (their heavy and light chain variable regions). are shown in SEQ ID NOs: 1-5, respectively): the three antibodies can specifically recognize/bind HBsAg and neutralize HBV toxicity, as well as serum levels of HBV DNA and/or HBsAg in the subject. , and can effectively eliminate HBV and HBV-infected cells from the body. Accordingly, the M1-23, M3-23 and M3-13 antibodies have potential for use in the prevention and treatment of HBV infection and diseases associated with HBV infection, such as hepatitis B.

Based on this, the inventors made extensive creative efforts to diligently study and modify the two chimeric antibodies M1-23 and M3-23, and thus developed humanized antibodies of M1-23 and M3-23: Humanized antibodies not only have a very high degree of humanization (the degree of humanization can be as high as 98%), but also have substantially the same (or even superior) expected properties (including but not limited to HBsAg binding activity, HBV neutralizing activity, biologic abatement activity of HBV DNA or HBsAg in vivo, or removal activity of HBV and HBV-infected cells in vivo, etc.).

Therefore, the antibody (especially humanized antibody) of the present invention is very advantageous because it can retain the functions and properties of the parent antibody, and thus the possibility of preventing and treating HBV infection and diseases associated with HBV infection (eg hepatitis B). have; Moreover, antibodies can have a very high degree of humanization (up to 98% degree of humanization) and thus can be safely administered to human subjects without elevating the immunogenic response. Antibodies according to the invention (especially humanized antibodies) may have significant clinical value.

In the present application, the expected properties of the antibody of the present invention are the activity of specifically binding to HBsAg, the neutralizing activity of HBV, the removal activity of HBV DNA or HBsAg in vivo, and/or the removal activity of HBV and HBV-infected cells in vivo. includes A humanized antibody according to the invention retains one or more of the above-mentioned expected properties of its parent antibody (cynomolgus monkey-human chimeric antibody), preferably its parent antibody (cynomolgus monkey-human chimeric antibody). antibody) retains all of the above-mentioned expected properties.

In the present application, the humanized antibody of the present invention is obtained by modifying the parent antibody of the present invention (cynomolgus monkey-human chimeric antibody). For example, some amino acid residues in the FRs of the antibody are substituted (eg, conservative substitutions). Such substitutions may, for example, (1) decrease the sensitivity of the antibody to proteolysis; (2) reduce the sensitivity of the antibody to oxidation; (3) alter (eg, enhance) the antigen binding affinity of the antibody; (4) alter (eg, enhance) the HBV-neutralizing activity of the antibody; (5) alter (eg, enhance) the HBV-clearing activity of the antibody; (6) further enhance the degree of humanization of the antibody to reduce the immunogenicity of the antibody; or (7) alter other biochemical or functional properties of the antibody; The expected properties of the antibody can still be maintained. Such substitutions may be in the CDRs and/or FRs, and may be single amino acid substitutions or multiple amino acid substitutions.

As used herein, the term “degree of humanization” is an index indicating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be calculated, for example, by the formula: degree of humanization = (number of amino acids in FR - number of non-human amino acids in FR)/number of amino acids in FR x 100%.

As used herein, the term "neutralizing antibody" refers to an antibody or antigen-binding fragment thereof that is capable of significantly reducing or completely inhibiting the toxicity (eg, ability to infect cells) of a target virus. . In general, neutralizing antibodies are capable of recognizing and binding the target virus and preventing the target virus from entering/infecting cells in a subject. The antibody according to the invention is a neutralizing antibody.

However, in the present application, it goes without saying that the virus-neutralizing ability of the antibody is not directly equivalent to the virus-removing ability of the antibody. As used herein, “viral neutralization” refers to neutralizing the toxicity of a target virus by inhibiting the target virus from entering/infecting cells of a subject (ie, the toxicity of the target virus is significantly reduced or completely reduced). inhibited). As used herein, "viral clearance" means removing a target virus (whether infecting or not infecting cells) from an organism, thus returning the organism to a state prior to infection by the virus (e.g., For example, the serological test result of the virus turns negative). Thus, in general, neutralizing antibodies do not necessarily have virus-clearing ability. However, in the present application, the inventors surprisingly found that the antibody according to the present invention can not only neutralize HBV, but also eliminate viruses (ie can remove HBV DNA and/or HBsAg in vivo, and HBV and HBV-infected cells can be eliminated), and thus have important clinical value.

As used herein, the term “isolated” refers to a condition obtained from a natural state by artificial means. When some “isolated” substance or component exists in nature, it is possible because its natural environment has changed, or the substance has been isolated from its natural environment, or both. For example, some non-isolated polynucleotides or polypeptides exist naturally in some living animals, and the same polynucleotide or polypeptide isolated with high purity from such a natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor other impure substances that do not affect the activity of the isolated substance.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector may have transferred genetic material expressed in a host cell by transformation, transduction or transfection into the host cell. vectors such as, but not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); Phages, such as λ phages or M13 phages and animal viruses, are well known to those skilled in the art. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (eg herpes simplex virus), varicella viruses, baculoviruses, papillomaviruses, papova. viruses (eg SV40). A vector can include a number of elements for regulating expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. A vector may also contain an origin of replication.

As used herein, the term "host cell" refers to cells into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, and fungal cells, such as For example yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 refers to cells or human cells.

As used herein, the term "homology" refers to the degree of agreement between two polypeptides or between two nucleic acids. When two sequences for comparison have the same monomeric subunit of a base or amino acid at some site (e.g., two respective DNA molecules have an adenine at some site, or each of the two polypeptides has an adenine at some site) lysine), the two molecules are identical at this site. The percent homology between two sequences is a function of the number of identical sites shared by the two sequences over the total number of sites for comparison×100. For example, if 6 out of 10 sites of two sequences are congruent, then these two sequences have 60% homology. For example, the DNA sequences: CTGACT and CAGGTT share 50% homology (3 out of 6 sites are congruent). In general, comparison of two sequences is performed in a manner that produces maximum homology. Such alignment is performed using a computer program, for example, the Align program (DNAstar, Inc.) (based on the method of Needleman, et al., J. Mol. Biol. 48:443-453, 1970). It can be done using The percent homology between the two amino acid sequences was also calculated using the PAM120 weighted residue table, a break length penalty of 12 and a break penalty of 4, incorporated into the ALIGN program (version 2.0) by E. Meyers and W. Miller (W). Miller) (Comput. Appl. Biosci., 4:11-17 (1988)). In addition, the percent homology between the two amino acid sequences is determined by a Blossum 62 matrix or a PAM250 matrix and a break weight of 16, 14, 12, 10, 8, 6, or 4, and 1, 2, 3, 4, 5, or 6 Using length weights, Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)).

As used herein, the terms "conservative substitution" and "conservative amino acid substitution" refer to amino acid substitutions that do not adversely affect or change the expected properties of a protein/polypeptide comprising an amino acid sequence. refers to For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is another amino acid residue having a similar side chain to the corresponding amino acid residue, e.g., physically or functionally similar (e.g., similar in size, shape, charge, covalent bond, or hydrogen bond). (having chemical properties, including the ability, etc.) Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine and histidine), amino acids with acidic side chains (eg aspartic acid and glutamic acid), amino acids with uncharged polar side chains (eg glycine, aspa) Ragine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains ( threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Accordingly, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for the identification of amino acid conservative substitutions are well known in the art (eg, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10) ): 879-884 (1999), and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997) (incorporated herein by reference).

Twenty conventional amino acids relevant to this specification are shown according to conventional methods. See, e.g., Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), herein incorporated by reference. In the present invention, "polypeptide The terms " and "protein" have the same meaning and can be used interchangeably. Furthermore, in the present invention, amino acids are generally referred to as one-letter codes and three-letter codes. For example, alanine as A or Also, as used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" has the same meaning and may be used interchangeably.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, and these are those in the art (See, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic-pressure-regulating agents, absorption delaying agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, and sorbic acid. Osmolality modifiers include, but are not limited to, sugars, NaCl and analogs thereof. Absorption delaying agents include, but are not limited to, monostearate and gelatin.

As used herein, an "adjuvant" is an antigen that, when delivered to an organism together with an antigen or when pre-delivered to an organism, is capable of enhancing an immune response to an antigen in an organism or changing the type of immune response. Refers to non-specific immunopotentiators. Various adjuvants such as, but not limited to, aluminum adjuvant (eg aluminum hydroxide), Freund's adjuvant (eg Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parbium, lipo Polysaccharides, cytokines, and the like are present. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

As used herein, the term "preventing/preventing" refers to a method performed to inhibit or delay the development of a disease, condition or condition (eg, HBV infection or a disease associated with HBV infection) in a subject. do. As used herein, the term "treating/treating" refers to a method performed to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical outcomes, whether detectable or undetectable, include, but are not limited to, alleviation of symptoms, reduction of the scope of disease, stabilization (ie, not worsening) of the disease state; delaying or slowing disease progression, and alleviation (partial or complete) of symptoms. "Treatment" also refers to prolonged survival as compared to expected survival (if treatment is not received). In the present application, the antibody according to the present invention has the ability to neutralize HBV, and thus can be used to prevent/protect an intact subject or its cells from infection by HBV. In addition, the antibody according to the invention has the ability to eliminate HBV (i.e. can remove HBV DNA and/or HBsAg in vivo, and can eliminate HBV and HBV-infected cells in vivo), thus It can be used to treat HBV infection or diseases associated with HBV infection in an infected subject.

As used herein, the term "subject" refers to a mammal, eg, a primate mammal, eg, a human.

As used herein, the term “effective amount” refers to an amount sufficient to achieve or at least partially achieve the expected effect. For example, an amount effective for preventing a disease (eg, HBV infection or a disease associated with HBV infection) is an amount effective for preventing, suppressing, or delaying the onset of a disease (eg, HBV infection or disease associated with HBV infection). refers to An amount effective for the treatment of a disease refers to an amount effective for curing or at least partially blocking the disease and its complications in a patient with the disease. Determination of such effective amounts is within the skill of one of ordinary skill in the art. For example, the amount effective for therapeutic use depends on the severity of the disease to be treated, the general state of the immune system in the patient, the general condition of the patient, such as age, weight and sex, the means of administration of the drug, and additional therapies used concurrently, and the like. change

antibodies of the invention

In the present application, the inventors first described three cynomolgus monkey-human chimeric monoclonal antibodies with excellent properties, designated M1-23, M3-23 and M3-13, respectively, whose heavy and light chain variable regions are each sequenced 1-5) were developed: three antibodies were able to specifically recognize/bind HBsAg and neutralize the toxicity of HBV, as well as reduce serum levels of HBV DNA and/or HBsAg in a subject. and can effectively remove HBV and HBV-infected cells from the body. Accordingly, the M1-23, M3-23 and M3-13 antibodies have potential for use in the prevention and treatment of HBV infection and diseases associated with HBV infection, such as hepatitis B.

Based on this, the inventors have carried out intensive studies and modifications of the two chimeric antibodies M1-23 and M3-23, and have developed humanized antibodies of the M1-23 and M3-23 antibodies: the humanized antibodies of the present invention are Not only has a very high degree of humanization (the degree of humanization can be as high as 98%), but also has substantially the same (or even superior) expected properties (including but not limited to HBsAg binding activity, HBV neutralizing activity, in vivo HBV DNA or HBsAg clearance, or HBV and HBV-infected cells in vivo).

Thus, the antibodies of the present invention (especially humanized antibodies) can retain the functions and properties of the parent antibody, and thus have potential for use in the prevention and treatment of HBV infection and diseases associated with HBV infection (eg hepatitis B). As well as; It can have a very high degree of humanization (up to 98%) and is therefore very advantageous in that it can be safely administered to human subjects without inducing an immunogenic response. The antibodies of the present invention (particularly humanized antibodies) have significant clinical value.

Accordingly, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to HBsAg, wherein the antibody or antigen-binding fragment thereof comprises:

(a) (i) SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, or said sequence by one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions); VH CDR1 consisting of a sequence selected from a sequence different from;

(ii) SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or a sequence that differs from that sequence by one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR2 consisting of a sequence selected from; and

(iii) SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR3 consisting of a sequence selected from

At least one (eg, 1, 2 or 3) heavy chain variable region (VH) complementarity determining region (CDR) selected from the group consisting of; and/or

(b) (vi) SEQ ID NO: 9, SEQ ID NO: 15, or a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL CDR1 consisting of selected sequences;

(v) SEQ ID NO: 10, SEQ ID NO: 16, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL CDR2 consisting of; and

(vi) SEQ ID NO: 11, SEQ ID NO: 17, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); Consists of VL CDR3

One or more (eg, 1, 2 or 3) light chain variable region (VL) CDRs selected from the group consisting of

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH CDR1, VH CDR2 and VH CDR3 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VL CDR1, VL CDR2 and VL CDR3 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 as defined above.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from SEQ ID NO: 7, or a sequence different from SEQ ID NO: 7 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR2 consisting of;

(ii) a VH CDR3 consisting of SEQ ID NO: 8, or a sequence selected from a sequence that differs from SEQ ID NO: 8 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR1 consisting of a sequence selected from the sequences of VH CDR1 contained in the heavy chain variable region of any immunoglobulin.

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 9 or a sequence different from SEQ ID NO: 9 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR3 consisting of SEQ ID NO: 11 or a sequence selected from a sequence different from SEQ ID NO: 11 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(vi) a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin.

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH CDR1 is the sequence of the VH CDR1 comprised in the heavy chain variable region of a human immunoglobulin.

In some preferred embodiments, the VH CDR1 is (a) SEQ ID NO: 6 or SEQ ID NO: 6 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VH CDR1 contained in the amino acid sequence encoded by the human heavy chain germline gene. In some exemplary embodiments, the human heavy chain germline gene is selected from the group consisting of IGHV4-4 ^* 08 and IGHV4-61 ^* 01.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention is a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and SEQ ID NO: 6, SEQ ID NO: 137 or sequence and a VH CDR1 having the amino acid sequence of number 138.

In some preferred embodiments, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of a human immunoglobulin.

In some preferred embodiments, the VL CDR2 is (a) SEQ ID NO: 10 or SEQ ID NO: 10 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene. In some exemplary embodiments, the human light chain germline gene is selected from the group consisting of IGKV1-39 ^* 01 and IGKV1-5 ^* 03.

In some exemplary embodiments, the VL of an antibody or antigen-binding fragment thereof of the present invention is a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and SEQ ID NO: 10, SEQ ID NO: 139 or sequence and a VL CDR2 having the amino acid sequence of number 140.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is VH CDR2 as set forth in SEQ ID NO:7; VH CDR3 as set forth in SEQ ID NO:8; and a VH CDR1 consisting of a sequence selected from the sequence of a VH CDR1 contained in the heavy chain variable region of any immunoglobulin (eg SEQ ID NO: 6, 137 or 138);

The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and a VL CDR2 having the amino acid sequence of SEQ ID NO: 10.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is VH CDR2 as set forth in SEQ ID NO:7; VH CDR3 as set forth in SEQ ID NO:8; and a VH CDR1 as set forth in SEQ ID NO:6;

The VL of the antibody or antigen-binding fragment thereof is contained in a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and a light chain variable region (eg, a κ light chain variable region) of any immunoglobulin. and a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 (eg, SEQ ID NO: 10, 139 or 140).

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is the VH CDR2 as set forth in SEQ ID NO: 7, the VH CDR3 as set forth in SEQ ID NO: 8, and the amino acid sequence of SEQ ID NO: 6, 137 or 138 comprises a VH CDR1 having; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and a VL CDR2 having the amino acid sequence of SEQ ID NO: 10, 139 or 140.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR2 as set forth in SEQ ID NO: 10, and a VL CDR3 as set forth in SEQ ID NO: 11.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 137, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR2 as set forth in SEQ ID NO: 10, and a VL CDR3 as set forth in SEQ ID NO: 11.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 138, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR2 as set forth in SEQ ID NO: 10, and a VL CDR3 as set forth in SEQ ID NO: 11.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR2 as set forth in SEQ ID NO: 139, and a VL CDR3 as set forth in SEQ ID NO: 11.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR2 as set forth in SEQ ID NO: 140, and a VL CDR3 as set forth in SEQ ID NO: 11.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from among SEQ ID NO: 12, or a sequence different from SEQ ID NO: 12 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VH CDR1 consisting of;

(ii) a VH CDR3 consisting of SEQ ID NO: 14 or a sequence selected from a sequence that differs from SEQ ID NO: 14 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR2 consisting of a sequence selected from the sequences of the VH CDR2 contained in the heavy chain variable region of any immunoglobulin.

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 15, or a sequence different from SEQ ID NO: 15 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR3 consisting of SEQ ID NO: 17 or a sequence selected from a sequence that differs from SEQ ID NO: 17 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(vi) a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin.

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH CDR2 is the sequence of the VH CDR2 contained in the heavy chain variable region of a human immunoglobulin.

In some preferred embodiments, the VH CDR2 is (a) SEQ ID NO: 13, or SEQ ID NO: 13 by one or multiple amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) different sequences; or (b) the sequence of VH CDR2 contained in the amino acid sequence encoded by the human heavy chain germline gene. In some exemplary embodiments, the human heavy chain germline gene is selected from the group consisting of IGHV4-30-4 ^* 07 and IGHV4-4 ^* 01.

In some exemplary embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises (a) a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: 141 and a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 142.

In some preferred embodiments, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of a human immunoglobulin.

In some preferred embodiments, the VL CDR2 comprises (a) SEQ ID NO: 16 or SEQ ID NO: 16 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions). different sequences; or (b) the sequence of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene. In some exemplary embodiments, the human light chain germline gene is selected from the group consisting of IGKV2-28 ^* 01 and IGKV3-15 ^* 01.

In some exemplary embodiments, the VLs of an antibody or antigen-binding fragment thereof of the present invention are VL CDR1 as set forth in SEQ ID NO: 15, VL CDR3 as set forth in SEQ ID NO: 17, and SEQ ID NO: 16, SEQ ID NO: 143 and SEQ ID NO: and a VL CDR2 having an amino acid sequence selected from the group consisting of number 144.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is comprised in the VH CDR1 as set forth in SEQ ID NO: 12, the VH CDR3 as set forth in SEQ ID NO: 14, and the heavy chain variable region of any immunoglobulin. a VH CDR2 comprising a sequence selected from the sequences of the VH CDR2 (eg, SEQ ID NO: 13, SEQ ID NO: 141 or SEQ ID NO: 142);

The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR3 as set forth in SEQ ID NO: 17, and a VL CDR2 as set forth in SEQ ID NO: 16.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is VH CDR1 as set forth in SEQ ID NO: 12; VH CDR3 as set forth in SEQ ID NO: 14; and a VH CDR2 as set forth in SEQ ID NO: 13;

The VL of the antibody or antigen-binding fragment thereof is contained in the VL CDR1 as set forth in SEQ ID NO: 15, the VL CDR3 as shown in SEQ ID NO: 17, and the light chain variable region (eg kappa light chain variable region) of any immunoglobulin. and a VL CDR2 consisting of a sequence selected from the sequences of the VL CDR2 (eg, SEQ ID NO: 16, SEQ ID NO: 143, or SEQ ID NO: 144).

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is the group consisting of VH CDR1 as set forth in SEQ ID NO: 12, VH CDR3 as set forth in SEQ ID NO: 14, and SEQ ID NO: 13, 141 or 142 a VH CDR2 having a sequence selected from; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR3 as set forth in SEQ ID NO: 17, and a VL CDR2 having a sequence selected from the group consisting of SEQ ID NO: 16, 143 or 144 do.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and a VH CDR3 as set forth in SEQ ID NO: 14. including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 16, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 141, and a VH CDR3 as set forth in SEQ ID NO: 14 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 16, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 142, and a VH CDR3 as set forth in SEQ ID NO: 14 including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 16, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and a VH CDR3 as set forth in SEQ ID NO: 14. including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 143, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and a VH CDR3 as set forth in SEQ ID NO: 14. including; The VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 144, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) (i) to a sequence selected from SEQ ID NO: 18, or a sequence different from SEQ ID NO: 18 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH CDR1 consisting of;

(ii) a VH CDR2 consisting of SEQ ID NO: 19 or a sequence selected from a sequence different from SEQ ID NO: 19 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) ; and

(iii) a VH CDR3 consisting of SEQ ID NO: 20 or a sequence selected from a sequence different from SEQ ID NO: 20 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions)

a heavy chain variable region (VH) comprising; and/or

(b) (iv) to a sequence selected from SEQ ID NO: 15, or a sequence different from SEQ ID NO: 15 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) VL CDR1 consisting of;

(v) a VL CDR2 consisting of SEQ ID NO: 16 or a sequence selected from a sequence that differs from SEQ ID NO: 16 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) , and

(vi) a VL CDR3 consisting of SEQ ID NO: 17 or a sequence selected from a sequence that differs from SEQ ID NO: 17 by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions)

A light chain variable region (VL) comprising

includes

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a VH CDR1 as set forth in SEQ ID NO: 18, a VH CDR2 as set forth in SEQ ID NO: 19, and a VH CDR3 as set forth in SEQ ID NO: 20 wherein the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 15, a VL CDR2 as set forth in SEQ ID NO: 16, and a VL CDR3 as set forth in SEQ ID NO: 17.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention further comprises a framework region.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(a) (i) SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, or one or multiple amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions; VH FR1 consisting of a sequence selected from sequences different from the above sequences by deletion or addition);

(ii) SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 51, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH FR2 consisting of a sequence selected from a sequence different from the sequence;

(iii) SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 52, or one or multiple amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) VH FR3 consisting of a sequence selected from a sequence different from the above sequence by );

(iv) SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 53, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VH FR4 consisting of a sequence selected from a sequence different from the sequence

at least one (eg 1, 2, 3 or 4) heavy chain variable region (VH) framework region (FR) selected from; and/or

(b) (v) SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 54, or one or multiple amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) VL FR1 consisting of a sequence selected from a sequence different from the above sequence by;

(vi) SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 55, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR2 consisting of a sequence selected from a sequence different from the sequence;

(vii) SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 56, or one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR3 consisting of a sequence selected from a sequence different from the sequence; and

(viii) SEQ ID NO: 28, SEQ ID NO: 36, or a sequence selected from a sequence that differs from said sequence by one or more amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); VL FR4 made

One or more (eg 1, 2, 3 or 4) light chain variable region (VL) framework regions (FR) selected from

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3 and VH FR4 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL FR2, VL FR3 and VL FR4 as defined above. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3 and VL FR4 as defined above.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof is

(a) VH FR1 as set forth in SEQ ID NO: 21, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 23, and VH FR4 as set forth in SEQ ID NO: 24;

(b) VH FR1 as set forth in SEQ ID NO: 29, VH FR2 as set forth in SEQ ID NO: 30, VH FR3 as set forth in SEQ ID NO: 31, and VH FR4 as set forth in SEQ ID NO: 32; or

(c) VH FR1 as set forth in SEQ ID NO: 37, VH FR2 as set forth in SEQ ID NO: 38, VH FR3 as set forth in SEQ ID NO: 39, and VH FR4 as set forth in SEQ ID NO: 32

includes

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof is

(a) VL FR1 as set forth in SEQ ID NO: 25, VL FR2 as set forth in SEQ ID NO: 26, VL FR3 as set forth in SEQ ID NO: 27, and VL FR4 as set forth in SEQ ID NO: 28; or

(b) VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) VH FR1 as set forth in SEQ ID NO: 21, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 23, and VH FR4 as set forth in SEQ ID NO: 24; and VL FR1 as set forth in SEQ ID NO: 25, VL FR2 as set forth in SEQ ID NO: 26, VL FR3 as set forth in SEQ ID NO: 27, and VL FR4 as set forth in SEQ ID NO: 28;

(b) VH FR1 as set forth in SEQ ID NO: 29, VH FR2 as set forth in SEQ ID NO: 30, VH FR3 as set forth in SEQ ID NO: 31, and VH FR4 as set forth in SEQ ID NO: 32; and VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36; or

(c) VH FR1 as set forth in SEQ ID NO: 37, VH FR2 as set forth in SEQ ID NO: 38, VH FR3 as set forth in SEQ ID NO: 39, and VH FR4 as set forth in SEQ ID NO: 32; and VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is humanized. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, a degree of humanization of at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some preferred embodiments, the non-CDR region of the antibody or antigen-binding fragment thereof of the invention is 19 or less, 15 or less, 14 or less, 13 or less from a non-human (eg cynomolgus monkey) source. No more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 or the non-CDR region does not contain amino acid residues from a non-human (eg, cynomolgus monkey) source. In some preferred embodiments, the FR region of the antibody or antigen-binding fragment thereof of the invention is 19 or less, 15 or less, 14 or less, 13 or less, of non-human (eg cynomolgus monkey) origin; 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acids residues, or the FR region does not contain amino acid residues of non-human (eg cynomolgus monkey) origin.

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

In such embodiments, the heavy chain framework region and/or light chain framework region of the antibody or antigen-binding fragment thereof of the invention comprises one or more amino acid residues of non-human (eg cynomolgus monkey) origin. can do. In some preferred embodiments, the heavy chain framework region and/or the light chain framework region comprises conservative substitutions of one or more amino acid residues or corresponding residues back-mutated to the corresponding residues of cynomolgus monkey (such mutations as above) is called a reverse mutation).

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain framework region comprised in the amino acid sequence encoded by the human heavy chain germline gene IGHV 4-4 ^* 08, wherein the heavy chain framework region is a human - optionally comprising one or more back mutations from a derived residue to a cynomolgus monkey-derived residue. In some preferred embodiments, IGHV 4-4 ^* 08 encodes an amino acid sequence as set forth in SEQ ID NO:40.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof is

(i) SEQ ID NO: 21, or

(01) T at H17;

(02) L at H20; and

(03) H23 to T

VH FR1 different from SEQ ID NO: 21 by one or more amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from the group consisting of;

(ii) VH FR2 of SEQ ID NO: 22;

(iii) SEQ ID NO: 23, or

(01) K at H64;

(02) I at H69; and

(03) H78 to F

VH FR3 different from SEQ ID NO: 23 by one or more amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from the group consisting of;

(iv) SEQ ID NO: 24, or

(01) T at H107; and

(02) T in H108

VH FR4 different from SEQ ID NO: 24 by one or multiple amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from the group consisting of

wherein all amino acid positions mentioned above are numbered according to the Kabat numbering system.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof is VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 45, and SEQ ID NO: 46 as shown in VH FR4.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain framework region comprised in the amino acid sequence encoded by the human heavy chain germline gene IGHV 4-4 ^* 02, wherein the heavy chain framework region is a human - optionally comprising one or more back mutations from a derived residue to a cynomolgus monkey-derived residue. In some preferred embodiments, the amino acid sequence encoded by IGHV 4-4 ^* 02 is set forth in SEQ ID NO:42.

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof is

(i) SEQ ID NO: 29, or

(01) H2 to V; and

(02) H16 to G

VH FR1 different from SEQ ID NO: 29 by one or more amino acid substitutions (eg, 1 or 2) selected from the group consisting of;

(ii) is SEQ ID NO: 30, or a VH FR2 that differs from SEQ ID NO: 30 by an amino acid substitution of: H37 to V;

(iii) SEQ ID NO: 31, or

(01) S at H65;

(02) V at H71;

(03) K at H73; and

(04) K in H81

VH FR3 different from SEQ ID NO: 31 by one or more amino acid substitutions (eg, 1, 2, 3 or 4 amino acid substitutions) selected from the group consisting of;

(iv) SEQ ID NO: 32, or a VH FR4 that differs from SEQ ID NO: 32 by an amino acid substitution of: H107 to T

wherein all amino acid positions mentioned above are numbered according to the Kabat numbering system.

In some exemplary embodiments, the VH of the antibody or antigen-binding fragment thereof is VH FR1 as set forth in SEQ ID NO: 50, VH FR2 as set forth in SEQ ID NO: 51, VH FR3 as set forth in SEQ ID NO: 52, and SEQ ID NO: 53 as shown in VH FR4.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof comprises a light chain framework region contained in the amino acid sequence encoded by the human light chain germline gene IGKV1-39 ^* 01, wherein the light chain framework region comprises optionally comprising one or more back mutations from human-derived residues to cynomolgus monkey-derived residues. In some preferred embodiments, the amino acid sequence encoded by IGKV1-39 ^* 01 is set forth in SEQ ID NO: 41.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof is

(i) SEQ ID NO: 25, or

(01) L1 to D; and

(02) L3 to Q

VL FR1 different from SEQ ID NO: 25 by one or more amino acid substitutions (eg, one or two amino acid substitutions) selected from the group consisting of;

(ii) is SEQ ID NO: 26, or a VL FR2 that differs from SEQ ID NO: 26 by K at L45 by an amino acid substitution of:

(iii) SEQ ID NO: 27, or

(01) Q at L55;

(02) S at L63;

(03) D at L70; and

(04) A at L84

VL FR3 different from SEQ ID NO: 27 by one or multiple amino acid substitutions (eg, 1, 2, 3 or 4 amino acid substitutions) selected from the group consisting of

(iv) VL FR4 of SEQ ID NO:28

wherein all amino acid positions mentioned above are numbered according to the Kabat numbering system.

In some exemplary embodiments, the VL of the antibody or antigen-binding fragment thereof is VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, VL FR3 as set forth in SEQ ID NO: 49, and SEQ ID NO: 28 as shown in VL FR4.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof comprises a light chain framework region contained in the amino acid sequence encoded by the human light chain germline gene IGKV4-1 ^* 01, wherein the light chain framework region comprises optionally comprising one or more back mutations from human-derived residues to cynomolgus monkey-derived residues. In some preferred embodiments, the amino acid sequence encoded by IGKV4-1 ^* 01 is set forth in SEQ ID NO:43.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof is

(i) SEQ ID NO: 33, or

(01) L3 to V;

(02) L5 to T; and

(03) A at L19

VL FR1 different from SEQ ID NO: 33 by one or more amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from the group consisting of;

(ii) is SEQ ID NO: 34, or a VL FR2 that differs from SEQ ID NO: 34 by an amino acid substitution of: L43 to V;

(iii) SEQ ID NO: 35, or

(01) D at L60;

(02) S at L76; and

(03) L77 to S

VL FR3 different from SEQ ID NO: 35 by one or more amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from the group consisting of

(iv) VL FR4 of SEQ ID NO:36

wherein the aforementioned amino acid positions are numbered according to the Kabat numbering system.

In some exemplary embodiments, the VL of the antibody or antigen-binding fragment thereof is VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, VL FR3 as set forth in SEQ ID NO: 56, and SEQ ID NO: VL FR4 as shown in 36.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is

(a) a heavy chain framework region contained in the amino acid sequence encoded by IGHV4-4 ^* 08, and a light chain framework region contained in the amino acid sequence encoded by IGKV1-39 ^* 01; or

(b) a heavy chain framework region contained in the amino acid sequence encoded by IGHV4-4 ^* 02, and a light chain framework region contained in the amino acid sequence encoded by IGKV4-1 ^* 01

includes

In some preferred embodiments, the antibody or antigen-binding fragment thereof is

(a) VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 45, and VH FR4 as set forth in SEQ ID NO: 46; and VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, VL FR3 as set forth in SEQ ID NO: 49, and VL FR4 as set forth in SEQ ID NO: 28; or

(b) VH FR1 as set forth in SEQ ID NO: 50, VH FR2 as set forth in SEQ ID NO: 51, VH FR3 as set forth in SEQ ID NO: 52, and VH FR4 as set forth in SEQ ID NO: 53; and VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, VL FR3 as set forth in SEQ ID NO: 56, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention is

(a) VH FR1 as set forth in SEQ ID NO: 21, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 23, and VH FR4 as set forth in SEQ ID NO: 24;

(b) VH FR1 as set forth in SEQ ID NO: 29, VH FR2 as set forth in SEQ ID NO: 30, VH FR3 as set forth in SEQ ID NO: 31, and VH FR4 as set forth in SEQ ID NO: 32;

(c) VH FR1 as set forth in SEQ ID NO: 37, VH FR2 as set forth in SEQ ID NO: 38, VH FR3 as set forth in SEQ ID NO: 39, and VH FR4 as set forth in SEQ ID NO: 32;

(d) VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, VH FR3 as set forth in SEQ ID NO: 45, and VH FR4 as set forth in SEQ ID NO: 46; or

(e) VH FR1 as set forth in SEQ ID NO: 50, VH FR2 as set forth in SEQ ID NO: 51, VH FR3 as set forth in SEQ ID NO: 52, and VH FR4 as set forth in SEQ ID NO: 53

includes

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof of the invention is

(a) VL FR1 as set forth in SEQ ID NO: 25, VL FR2 as set forth in SEQ ID NO: 26, VL FR3 as set forth in SEQ ID NO: 27, and VL FR4 as set forth in SEQ ID NO: 28;

(b) VL FR1 as set forth in SEQ ID NO: 33, VL FR2 as set forth in SEQ ID NO: 34, VL FR3 as set forth in SEQ ID NO: 35, and VL FR4 as set forth in SEQ ID NO: 36;

(c) VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, VL FR3 as set forth in SEQ ID NO: 49, and VL FR4 as set forth in SEQ ID NO: 28; or

(d) VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, VL FR3 as set forth in SEQ ID NO: 56, and VL FR4 as set forth in SEQ ID NO: 36

includes

In some preferred embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 as defined above. . In some preferred embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4 as defined above. . In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 as defined above, ; The light chain variable region comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4 as defined above.

In some preferred embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164 At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 in the amino acid sequence of a heavy chain variable region selected from the group consisting of %, at least 98%, at least 99%, or 100% sequence homology.

In some preferred embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention is a heavy chain variable region as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164.

In some preferred embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention consists of a light chain variable region as set forth in SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology.

In some preferred embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof of the invention is a light chain variable region as set forth in SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166.

In some preferred embodiments, an antibody of the invention comprises a heavy chain variable region as defined above and a light chain variable region as defined above.

In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in any one of SEQ ID NOs: 1, 57, 157 and 158; The light chain variable region of the antibody or antigen-binding fragment thereof is a light chain variable region as set forth in any one of SEQ ID NOs: 2, 58, 159 and 160.

In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is a heavy chain variable region as set forth in any one of SEQ ID NOs: 3, 59, 163 and 164; The light chain variable region of the antibody or antigen-binding fragment thereof is a light chain variable region as set forth in any one of SEQ ID NOs: 4, 60, 165 and 166.

In some preferred embodiments, the antibody of the invention is

(1) VH as set forth in SEQ ID NO: 1 and VL as set forth in SEQ ID NO: 2;

(2) VH as set forth in SEQ ID NO:3 and VL as set forth in SEQ ID NO:4;

(3) VH as set forth in SEQ ID NO:5 and VL as set forth in SEQ ID NO:4;

(4) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 58;

(5) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 60;

(6) VH as set forth in SEQ ID NO: 157 and VL as set forth in SEQ ID NO: 58;

(7) VH as set forth in SEQ ID NO: 158 and VL as set forth in SEQ ID NO: 58;

(8) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 159;

(9) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 160;

(10) VH as set forth in SEQ ID NO:163 and VL as set forth in SEQ ID NO:60;

(11) VH as set forth in SEQ ID NO: 164 and VL as set forth in SEQ ID NO: 60;

(12) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 165; or

(13) VH as set forth in SEQ ID NO: 59 and VL as set forth in SEQ ID NO: 166

includes

Antibodies of the present invention can be obtained by genetically engineered recombinant techniques. For example, DNA molecules of genes encoding the heavy and light chains of the antibody of the present invention can be obtained by chemical synthesis or PCR. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells such as E. coli cells, simian COS, CHO cells, or other myeloma cells that do not produce immunoglobulins. The transfected host cells are then cultured under specific conditions and the antibodies of the present invention are expressed.

The antibody of the present invention has high specificity and high affinity for the HBsAg protein. For example, an antibody of the invention is capable of binding HBsAg with a KD of less than 1 x 10 ^-5 M; Preferably the KD value is less than 1 x 10 ^-6 M; more preferably the KD value is less than 1 x 10 ^-7 M; Most preferably the KD value is less than 1 x 10 ^-8 M.

An antibody of the invention may be an antibody comprising two heavy and two light chains and having a conventional "Y" type structure. In addition, the antibody of the present invention maintains affinity for the HBsAg protein and is capable of binding to the HBsAg protein with higher or lower affinity than an antibody having a conventional "Y" type structure, Fab fragments, Fab', F (ab) ₂ , Fv, or other types of fragments of antibodies with the conventional “Y” type structure.

Antigen-binding fragments of the invention can be obtained by hydrolysis of intact antibody molecules (Morimoto et al., J. biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al. ., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can be generated directly from recombinant host cells (Hudson, curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from E. coli cells; Fab' fragments can be chemically linked to form F(ab') ₂ fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)). Furthermore, Fv, Fab or F(ab′) ₂ fragments can also be isolated directly from the culture of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in some preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is combined with scFv, Fab, Fab', (Fab') ₂ , Fv fragment, diabody, bispecific antibody, multispecific antibody, chimeric antibody, or a humanized antibody. Particularly preferably, the antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or a humanized antibody.

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is capable of specifically binding to HBsAg, neutralizing the toxicity of HBV, and/or serum levels of HBV DNA and/or HBsAg in the subject. can reduce

In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is of the IgG class. For example, the antibody or antigen-binding fragment thereof of the invention may be of class IgG1 or IgG2 or IgG3 or IgG4.

fusion antibody

In another embodiment, a fusion antibody or immunoadhesin may be prepared, for example an antibody according to the invention or antigen-binding fragment thereof may be linked to another polypeptide. In some preferred embodiments, the fusion antibody comprises a heavy chain variable region and a light chain variable region of an antibody according to the invention. In some preferred embodiments, the fusion antibody comprises the VH domain and the VL domain of an antibody according to the invention; wherein the VH domain is linked to the first polypeptide; The VL domain is linked to a second polypeptide.

Derivatized Antibodies

An antibody or antigen-binding fragment thereof according to the invention may be derivatized, for example linked to another molecule (eg another polypeptide or protein). In general, derivatization (eg labeling) of the antibody or antigen-binding fragment thereof will not adversely affect its binding to HBsAg. Accordingly, the antibody or antigen-binding fragment thereof according to the present invention is also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof according to the invention may be combined with one or more other groups of molecules, for example another antibody (eg forming a bispecific antibody), a detection agent, a drug, and/or an antibody or its functionally (by chemical coupling, gene fusion, non-covalent association or other means) to a protein or polypeptide capable of mediating the association of an antigen-binding fragment with another molecule (eg, avidin or polyhistidine-tag) can do it

One type of derivatized antibody (eg a bispecific antibody) is generated by crosslinking two or more antibodies (either belonging to or not belonging to the same type). Suitable crosslinking agents include, for example, heterobifunctional crosslinking agents comprising two different reactive groups separated by a suitable spacer (eg m-maleimidobenzoyl-N-hydroxylsuccinimide ester); and a homobifunctional crosslinking agent (disuccinimidyl suberate). Such crosslinking agents may be purchased from Pierce Chemical Company (Rockford, II).

Another type of derivatized antibody is a labeled antibody. For example, an antibody or antigen-binding fragment thereof according to the invention can be linked to a useful detection agent. Such detection agents include, for example, fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, lanthanide phosphor, and the like. In addition, the antibody may also be labeled by enzymes such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody is labeled with an enzyme, a reagent that can be used by the enzyme to produce an identifiable signal or reaction product can be added to detect the labeled antibody. For example, when horseradish peroxidase is used to label an antibody, hydrogen peroxide and diaminobenzidine can be added to determine the presence or amount of the labeled antibody to generate a detectable chromogenic reaction product. Antibodies can also be labeled with biotin. In this case, the presence or amount of the labeled antibody can be measured by indirectly measuring the binding of avidin. In addition, the antibody can be labeled with a tag that can be recognized by a second reporter molecule (eg, a leucine zipper pair sequence, a metal-binding domain, an epitope tag, etc.). In some specific embodiments, the label can be linked to the antibody through spacer branches of different lengths to reduce potential steric hindrance.

In addition, the antibodies or antigen-binding fragments thereof according to the invention may also be derivatized with chemical groups, for example polyethylene glycol (PEG), methyl or ethyl, or saccharide groups. These groups can be used to improve the biological properties of the antibody, for example to increase the serum half-life.

Nucleic Acid Molecules, Vectors and Host Cells

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

In another aspect, the invention provides a vector (eg, a cloning vector or expression vector) comprising an isolated nucleic acid molecule according to the invention. In some preferred embodiments, the vector according to the invention is, for example, a plasmid, a cosmid, a phage and the like. In some preferred embodiments, the vector is capable of expressing an antibody according to the invention or an antigen-binding fragment thereof in a subject (eg mammal, eg human).

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule according to the invention or a vector according to the invention. Such host cells include, but are not limited to, prokaryotic cells, such as E. coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (eg mammalian cells, such as for example, mouse cells and human cells). The cell according to the present invention may be a cell line, for example a 293T cell.

In another embodiment, the method comprises culturing a host cell according to the invention under conditions permissive for expression of the antibody or antigen-binding fragment thereof according to the invention, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell. It provides a method for producing an antibody or antigen-binding fragment thereof according to the present invention, comprising.

Diagnostic methods and kits

The antibody or antigen-binding fragment thereof according to the present invention can specifically bind to HBsAg, and thus can be used to detect the presence or level of HBsAg protein in a sample, and can be used to diagnose whether a subject is infected with HBV. .

Accordingly, in another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof according to the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In a preferred embodiment, the kit further comprises a secondary antibody that specifically recognizes the antibody according to the invention or an antigen-binding fragment thereof. Preferably, the secondary antibody further comprises a detectable label.

According to the above-described method, the antibody or antigen-binding fragment thereof or secondary antibody according to the present invention may be labeled. For example, the antibody or antigen-binding fragment thereof according to the present invention may be labeled with a detectable label. Such detectable labels as are well known by those skilled in the art include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, chromogenic substances and enzymes (eg horseradish peroxidase), and the like. In addition, such a detectable label may be, for example, a radioisotope, such as ¹²⁵ I; fluorescent substances such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors; enzymes capable of producing an identifiable signal or reaction product, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase; tags that can be recognized by the second reporter molecule, such as biotin, avidin, leucine zipper pair sequences, metal-binding domains, and epitope tags. In some specific embodiments, a detection agent (eg, a tag) can be linked to the antibody via linkers of different lengths to reduce potential steric hindrance.

In another aspect, the invention provides a method for detecting the presence or level of HBsAg protein in a sample comprising using an antibody or antigen-binding fragment thereof according to the invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In another preferred embodiment, the method further comprises using a secondary antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof according to the invention. The method may be used for diagnostic purposes or for non-diagnostic purposes (eg the sample is a cell sample that is not a sample from a patient).

In another aspect, the present invention provides a method for diagnosing whether a subject is infected with HBV, comprising using an antibody or antigen-binding fragment thereof according to the present invention to detect the presence of HBsAg protein in a sample from the subject. to provide. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention further comprises a detectable label. In another preferred embodiment, the method further comprises using a secondary antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof according to the invention.

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

Methods of treatment and pharmaceutical compositions

The antibody or antigen-binding fragment thereof according to the present invention is used to prevent or treat HBV infection or a disease associated with HBV infection (eg hepatitis B) in a subject (eg human), in vitro or in a subject (eg, It can be used to neutralize HBV toxicity in humans) and to reduce serum levels of HBV DNA and/or HBsAg in subjects (eg humans).

Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier and/or excipient. In a preferred embodiment, the pharmaceutical composition according to the present invention may further comprise an additional pharmaceutically active agent. In a preferred embodiment, the additional pharmaceutically active agent is an agent for the prophylaxis or treatment of HBV infection or diseases associated with HBV infection (eg hepatitis B), eg other antiviral agents, eg interferon-type an agent such as interferon or pegylated interferon.

In another embodiment, preventing or treating HBV infection or a disease associated with HBV infection (eg, hepatitis B) in a subject (eg, a human) and reducing HBV toxicity in vitro or in a subject (eg, a human) Use of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for neutralizing and/or reducing the serum level of HBV DNA and/or HBsAg in a subject (eg human) provides

In another aspect, the present invention provides for preventing or treating HBV infection or a disease associated with HBV infection (eg hepatitis B) in a subject, neutralizing HBV toxicity in a subject (eg human), and/or a subject There is provided a method for reducing serum levels of HBV DNA and/or HBsAg in (eg human), said method comprising administering to said subject an effective amount of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention including administering

An antibody or antigen-binding fragment thereof according to the present invention or a pharmaceutical composition according to the present invention can be administered by conventional routes, including but not limited to oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intravaginal, intracisternal, intragroin, Administration may be by intravesical, topical (eg, powder, ointment or drops), or nasal routes. The antibody or antigen-binding fragment thereof according to the present invention may be administered by multiple routes known in the art. However, for many therapeutic applications, the preferred route/mode of administration is parenteral administration (eg, intravenous injection, subcutaneous injection, intraperitoneal injection, and intramuscular injection). It is understood by those skilled in the art that the route/mode of administration may vary depending on the desired purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention is administered by intravenous infusion or injection.

The antibody or antigen-binding fragment thereof according to the present invention or the pharmaceutical composition according to the present invention may be prepared in multiple dosage forms, for example liquid, semi-solid, and solid forms, for example solutions (eg injections), dispersions or suspensions, tablets, Powders, granules, emulsions, pills, syrups, powders, liposomes, capsules and suppositories can be prepared. Preferred dosage forms vary depending on the anticipated route of administration and therapeutic use.

For example, one preferred dosage form is injection. Such injection may be a sterile injectable solution. For example, sterile injectable solutions can be prepared by the following method: the required dose of the antibody or antigen-binding fragment thereof according to the invention is incorporated in a suitable solvent, and optionally other contemplated ingredients, including but not limited to pH adjusting agents. , surfactant, adjuvant, ionic strength enhancing agent, isotonicity agent, preservative, diluent, or any combination thereof), followed by filtration sterilization. In addition, sterile injectable solutions may be prepared as a sterile powder for convenience of storage and use (eg, by vacuum drying or freeze drying). Such sterile powders may be dispersed prior to use in a suitable vehicle, for example, sterile, pyrogen-free water.

Another preferred dosage form is a dispersion. Dispersions can be prepared by the following methods: The antibody or antigen-binding fragment thereof according to the invention is prepared in a basic dispersion medium and optionally other contemplated components including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonic agents, preservatives, diluents, or any combination thereof). In addition, absorption delaying agents, such as monostearate salts and gelatin, may also be incorporated into the dispersion to obtain the expected pharmacokinetic properties.

Another preferred dosage form is an oral solid dosage form including capsules, tablets, powders, granules, and the like. Such solid dosage forms generally contain (a) an inert drug excipient (or vehicle) such as sodium citrate and calcium phosphate; (b) fillers such as starch, lactose, sucrose, mannose and silicic acid; (c) binders such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (d) wetting agents, such as glycerol; (e) disintegrants such as agar, calcium carbonate, potato or tapioca starch; (f) retarders such as olefins; (g) absorption enhancers such as quaternary ammonium compounds; (h) moisturizing agents such as cetyl alcohol and glyceryl monostearate; (i) adsorbents such as kaolin and bentonite; (j) a lubricant such as at least one of talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or any combination thereof. In the case of tablet and capsule dosage forms, buffers may also be included.

In addition, release rate modifying agents (ie agents capable of altering the drug release rate) may also be added to the oral solid dosage form to obtain a modified release or pulsed release dosage form. Such release rate modifiers include, but are not limited to, carboxypropyl methylcellulose, methylcellulose, carboxymethyl cellulose sodium, ethyl cellulose, cellulose acetate, polyethylene oxide, xanthan gum, isoacryl amino copolymer, hydrogenated flavor oil, carnauba wax, paraffin, cellulose acetate phthalate, carboxypropyl methylcellulose phthalate, methacrylic acid copolymer, or any combination thereof. The modified release or pulsed release dosage form may include one or a group of release rate modifying agents.

Another preferred dosage form is an oral liquid dosage form including emulsions, solutions, suspensions, syrups and the like. In addition to the active ingredient, such oral liquid dosage forms may contain inert solvents commonly used in the art, such as water or other solvents such as ethyl alcohol, isopropanol, propylene glycol, 1,3-butylene glycol, oil (eg cottonseed oil, peanut oil, corn oil, olive oil, flavor oil and sesame oil), glycerol, polyethylene glycol and sorbitan fatty acid esters, and any combination thereof. In addition to these inert solvents, the oral liquid dosage form as described above may further contain a moisturizing agent, an emulsifying agent, a suspending agent, a sweetening agent, a flavoring agent, a fragrance agent, and the like.

In addition, the antibody or antigen-binding fragment thereof according to the present invention may be present in a unit dosage form in the pharmaceutical composition for convenience of administration. Pharmaceutical compositions according to the present invention must be sterile and stable under the conditions of manufacture and storage.

The medicaments and pharmaceutical compositions provided herein, used alone or in combination, or additional pharmaceutically active agents (e.g., other antiviral agents, e.g., interferon-type agents, e.g., interferon or pegylated interferon) can be used with In some preferred embodiments, the antibody or antigen-binding fragment thereof according to the present invention is used in combination with other antiviral agent(s) to prevent and/or treat diseases associated with HBV infection. The antibody or antigen-binding fragment thereof according to the present invention and the above antiviral agent(s) may be administered simultaneously, separately or sequentially. Such antiviral agent(s) include, but are not limited to, interferon-type agents, ribavirin, adamantane, hydroxyurea, IL-2, L-12 and pentacarboxy cytosolic acid, and the like.

The pharmaceutical composition according to the present invention may contain a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof according to the present invention. A “prophylactically effective amount” refers to an amount sufficient to prevent, inhibit, or delay the development of a disease (eg, HBV infection or disease associated with HBV infection). A “therapeutically effective amount” refers to an amount sufficient to cure or at least partially inhibit a disease and its complications in a patient with the disease. A therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the present invention may vary depending on the following factors: the severity of the disease to be treated, the general state of the immune system in the patient, the general conditions of the patient, such as age, weight and Gender, mode of administration of the drug, and any additional treatments used at the same time.

Dosage regimens may be adjusted to provide the optimal desired effect (eg, a therapeutic or prophylactic effect). For example, a single dose may be administered, or multiple doses may be administered within a period of time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation.

For an antibody or antigen-binding fragment thereof according to the invention, an exemplary, non-limiting range for a therapeutically or prophylactically effective amount is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more preferably 0.1 to 25 mg/kg, 0.1 to 10 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the disease being treated. In addition, those skilled in the art should, for any particular patient, adjust a particular dosage regimen over time according to the patient's needs and professional evaluation performed by the physician; It is understood that the dosage ranges provided herein are provided for illustrative purposes only, rather than limiting the use or scope of the pharmaceutical composition according to the present invention.

beneficial effect of the invention

Compared with the prior art, the technical solution of the present invention has the following advantageous effects:

(1) the antibody of the present invention can specifically recognize and / bind HBsAg and neutralize the toxicity of HBV, as well as reduce the serum level of HBV DNA and / or HBsAg in a subject, and HBV in vivo and cells infected by HBV. Accordingly, the antibody of the present invention can be used for the prevention and treatment of HBV infection and diseases associated with HBV infection, for example, hepatitis B.

(2) the antibody (especially humanized antibody) of the present invention retains the function and properties of the parent monkey antibody, and thus can be used for the prevention and treatment of HBV infection and diseases associated with HBV infection (eg hepatitis B) not; It has a very high degree of humanization (up to 98%) that can be safely administered to human subjects without inducing an immunogenic response. Thus, the antibodies of the present invention (especially humanized antibodies) have significant clinical value.

A specific model for carrying out the invention

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

Unless otherwise specified, molecular biology laboratory methods and immunoassays used in the present invention are essentially those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989 and FM Ausubel et al., Guide to Editing Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995 ]; Restriction endonucleases are used according to the manufacturer's recommended conditions. Those skilled in the art will appreciate that the examples illustrate the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: Preparation of human-cynomolgus monkey chimeric monoclonal antibody that specifically binds to HBsAg

1.1 Immunization of Cynomolgus Monkeys

Cynomolgus monkeys over 6 months old and weighing 4±1 kg were immunized by intramuscular injection using a recombinant HBV vaccine previously developed in the laboratory (recombinant vaccine is detailed in Chinese patent application 201710085194.3). , the injection dose was 20 μg/monkey/hour. Immunization time points were 0, 2, 6, 10, 14, 18, 22, and 26 weeks, respectively. After the serum titers of cynomolgus monkeys reached a plateau at the 4th immunization injection (10 weeks), we calculated that at 13, 17, 25 and 29 weeks, respectively (i.e. the 3rd week and multiple memory after each immunization) 10 ml/monkey/hour of peripheral blood of cynomolgus monkeys were sampled (at the time B cells were generated).

1.2 Isolation of Cynomolgus Monkey Peripheral Blood Mononuclear Cells

Isolation of cynomolgus monkey peripheral blood mononuclear cells was performed with reference to the description of SepMate ^™ . Mononuclear cells (MNCs) were isolated by density gradient centrifugation from human peripheral whole blood and cord blood samples using SepMate ^™ tubes. Prior to separation, samples, PBS buffer containing 2% fetal bovine serum (FBS) (PBS + 2% FBS), density gradient medium, and centrifuge were stored at room temperature (15° C. to 25° C.). The specific steps were as follows:

1) Density gradient medium was carefully added to the SepMate ^™ tube through the center hole of the SepMate ^™ insert using a pipette. The top of the density gradient medium should be above the insert. Note: Density gradient media may generate small air bubbles after pipetting use, which will not affect performance.

2) Dilute the sample with an equal volume of PBS + 2% FBS and mix slowly and thoroughly.

3) The SepMate ^™ tube was held upright and the diluted sample was pipetted down the side of the tube. The sample will be mixed with the density gradient medium over the insert. Note: The sample could be poured directly down the side of the tube. Note: Diluted samples should not be poured directly through the center hole.

4) Centrifugation was performed at 1200 × g for 10 minutes at room temperature without stopping the centrifuge brake. Note: For samples stored longer than 24 hours, a centrifugation time of 20 minutes is recommended.

5) Pour the top layer containing concentrated MNC into a new tube. The SepMate ^TM tube must not be placed in an inverted position for more than 2 seconds. Note: After centrifugation, some red blood cells (RBCs) may appear on the surface of the SepMate ^TM insert, but this did not affect the performance.

6) MNCs were washed with PBS + 2% FBS. Washing was repeated once. NOTE: Washing of cells should be performed under centrifugation at 1200 × g for 8 min at room temperature without the need to stop the centrifuge brake.

1.3 Isolation of Memory B Cells

Since there are no specific reagents on the market for sorting the memory B cells of cynomolgus monkeys, their isolation was performed using the EasySep ^™ Human Pan-B Cell Enrichment Kit.

1) PBMC cells isolated by the method described in 1.2 above were centrifuged at 300 g for 10 minutes and then washed once with 10 ml of serum-free 1640 culture medium.

2) Washing was repeated once with 10 ml of filtered MACS buffer.

3) Cells were resuspended in 1 ml MACS buffer and then transferred to 5 ml polystyrene round bottom tube.

4) 50 μl/ml of ^{EasySep™ Human} Pan-B Cell Enrichment Cocktail was added to the cells.

5) After incubation at room temperature for 10 minutes, 75 μl/ml of ^{EasySep TM D} Magnetic Particles was added to the cells.

6) MACS buffer was added to reach 2.5 ml and slowly mixed up and down.

7) Place the tube on a magnetic separator for 5 minutes, then transfer the desired positive cells to a new tube, while unwanted negative cells remain in the original tube.

1.4 In vitro stimulation and culture

1.4.1 HEK293-CD154 investigation

The irradiation dose was 40 Gy, and the specific implementation steps were described in the instructions of the RS 2000 biological X-ray irradiator.

1.4.2 Cell smear

Cells resuspended at 5 cells/well in RPMI 1640 medium were mixed with 50 ng/ml of IL21 and 10 ⁴ cells/well of irradiated HEK293-CD154, cultured and plated at 250 μl/well in 96-well plates. Cells were incubated at 37° C. in a CO ₂ incubator and cultured for 14 days for stimulation.

1.4.3 Cell Culture Positive Well Assay

A commercial reaction plate for HBsAg (purchased from Beijing Wantai) was used, and 100 μl of the supernatant to be tested was added to each well and incubated at 37° C. for 1 hour. Subsequently, the ELISA plate was washed 5 times with PBST, and 100 μl of GAH-HRP diluted at 1:5000 was added and incubated at 37° C. for 30 minutes. Then, the ELISA plate was washed 5 times with PBST, and the substrate TMB solution was added. After 15 minutes of color development, the color reaction was stopped with H ₂ SO ₄ and the reading was measured at OD450/620. RPMI 1640 culture medium was used as a negative control.

1.5 Acquisition of antibody variable region genes from B cells

1.5.1 Cell Treatment

Plates with culture wells for positive B cells were centrifuged at 300 x g for 5 minutes, and the cell supernatant was aspirated. 150 μl of lysis buffer (purchased from Beijing GenMagBio) was added, pipetted up and down several times and transferred to RNase-free 1.5 mL Eppendorf tubes. Samples can be stored in the refrigerator at -80°C for 1 year.

1.5.2 RNA Extraction

RNA extraction methods are described in the instructions of the viral DNA/RNA co-extraction kit (GenMagBio).

1) 1.5 of 10 μl protease K, 4 μl acrylic carrier, 300 μl lysis buffer and 20 μl magnetic beads (mix evenly by inverting magnetic beads up-down or pipetting up and down before use) into ml nuclease-free Eppendorf tubes; An additional 200 μl of sample was added, and each centrifuge tube was shaken up and down at room temperature for 10 minutes to mix uniformly and to allow sufficient binding between magnetic beads and nucleic acids.

2) The tube was placed on a magnetic shelf for 1 minute to absorb the magnetic beads in the centrifuge tube, the liquid in the tube was removed with a pipette, and the centrifuge tube was taken out.

3) The magnetic beads were resuspended by adding 500 μl of wash buffer I, and adsorbed by a magnetic shelf, the liquid in the tube was removed after 1 minute, and the centrifuge tube was taken out.

4) The magnetic beads were resuspended by adding 500 μl of Wash Buffer II and adsorbed by a magnetic shelf, the liquid in the tube was removed after 1 minute, and the centrifuge tube was kept on the magnetic shelf at the same time, so that the magnetic beads were continuously adsorbed.

5) 550 μl of Wash Buffer III was added in such a way as to avoid washing as many magnetic beads as possible; The liquid in the tube was removed after 1 minute and the centrifuge tube was removed.

6) adding 50 μl of elution buffer to resuspend the magnetic beads; It was carried out in a water bath at 55° C. for 5 minutes, and light shaking was performed twice while completely eluting the nucleic acid in the water bath.

7) Place the centrifuge tube on a magnetic shelf for 1 minute to allow the magnetic beads to adsorb, and transfer the liquid to a new 1.5 ml nuclease-free centrifuge tube.

1.5.3 RT-PCR for Antibody Variable Regions in B Cells

Primers used for reverse transcription (Meng W, Li L, Xiong W, et al. Efficient generation of monoclonal antibodies from single rhesus macaque antibody securing cells [C] // mAbs. Taylor & Francis, 2015, 7 (4): 707-718] are shown in Table 2.

Reverse transcription primers for cynomolgus monkey antibody variable region genes Primer name SEQ ID NO: primer sequence Rh Gamma-PCR1 61 5'GGACAGCCKGGAAGGTGTGC Rh kappa-PCR1 62 5'GAGGCAGTTCCAGATTTCAA Rh lambda-PCR1 63 5'CCGCGTACTTGTTGTTGCTCTGT

The specific steps are as follows: (1) First, solution I was prepared according to the following recipe:

(2) It was maintained at 75° C. for 5 minutes in a water bath.

(3) Put it in an ice bath quickly and keep it for 5 minutes.

(4) Thereafter, solution II was prepared according to the following preparation method.

(5) Mix evenly, and add 5.2 μl to each tube.

(6) Mix uniformly, and reverse transcription was performed in a 42°C water bath for 40 minutes.

(7) Packed and stored, stored at -20℃ for short-term use or -80℃ for short-term use.

1.5.4 Amplification of Antibody Variable Region Genes

B cell antibody H chain, κ light chain and λ light chain variable region genes were amplified by nested PCR, and amplification primers were described in references (Meng W, Li L, Xiong W, et al. Efficient generation). of monoclonal antibodies from single rhesus macaque Antibody secreting cells [C]//mAbs. Taylor & Francis, 2015, 7(4): 707-718), wherein among the primers for the second round of nested PCR homologous to the vector The contained sequences were replaced with fragments of our laboratory's eukaryotic expression vector (see Example 1.5.5.1) as homologous sequences. PCR reaction systems were prepared according to Table 3, each system being 25 μl. Reverse transcribed cDNA was used as template in the first round of nested reaction, reaction conditions were 95°C, 5 min; 95°C, 30 seconds; 55° C., 60 seconds; 72° C., 90 seconds; 72° C., 7 minutes; The number of amplification cycles was 35; The amplification product of the first round was used as a template in the second round of the nested reaction, and the reaction conditions were 95° C., 5 min; 95°C, 30 seconds; 58° C., 60 seconds; 72° C., 90 seconds; 72° C., 7 minutes; The number of amplification cycles was 35. Then, the amplification product was analyzed by agarose gel electrophoresis, and the amplification product was recovered using a DNA purification recovery kit (TianGen, DP214-03).

Nested PCR reaction system buffer Volume (μl) of first round PCR Volume of second round PCR (μl) number of DEPCs 12.5 μl 15.5 μl 10ХPCR buffer 2.5 μl 2.5 μl 2.5 mmol/L dNTPs 2.5 μl 2.5 μl HS Taq DNA Polymerase 0.5 μl 0.5 μl F _mix 1 μl 1 μl R _mix 1 μl 1 μl template 5 μl 2 μl

1.5.5 Expression and Purification of Antibody Genes 1.5.5.1 Construction of Recombinant Vectors for Eukaryotic Expression

In the present invention, a large amount of antibody recombination is required, and therefore it was necessary to construct a set of light and heavy chain vectors capable of efficiently recombination of the antibody. In the present invention, the existing eukaryotic expression vector pTT5 in our laboratory was specifically modified to construct a set of light and heavy chain recombinant vectors for co-transfection of double plasmids. The signal peptide for the light and heavy chains is MGWSCIILFLVATATGVHS. A set of pTT5-CH, pTT5-Cκ and pTT5-Cλ eukaryotic expression vectors was ligated downstream of the signal peptide with sequences encoding the light or heavy chain constant regions of human antibodies to facilitate antibody recombination. The constructed eukaryotic expression vector was subjected to PCR products (primers having sequences homologous to the vector) of the antibody variable region genes recovered in 1.5.4 by using a laboratory-made Gibson assembly solution and Recombinant vectors VH+pTT5-CH, VH+pTT5-Cκ and VH+pTT5-Cλ were obtained by ligation. Recombinant vectors were transformed into E. coli DH5α strains, plated on LB plates, and incubated overnight at 37° C. in an incubator. Monoclonal colonies were collected from the plate and sequenced, and the sequencing results were aligned using Igblast to confirm the feasibility of the gene and to exclude a pseudogene.

1.5.5.2 Small- and large-scale expression of antibody genes

The constructed recombinant vectors VH+pTT5-CH and VH+pTT5-Cκ and VH+pTT5-Cλ were co-transfected into HEK293-cells, and the double plasmids for small expression were co-transfected at 500 μl/well in 24-well plates. let; If the small expressing cell supernatant had antigenic activity, the transfection system was scaled up to 100 ml (depending on the amount of antibody) CHO-S suspension cells (cell density: about 2 x 10 ⁶ cells/ml). Transfected cells were cultured in shake flasks at 32° C. in a 5% CO ₂ incubator, and the supernatant was collected after 7 days of expression.

1.5.5.3 Antibody Purification

Cell expression supernatants were collected and purified using a Protein A chromatography column according to the manufacturer's instructions. Its specific steps were as follows: the collected cell culture supernatant was centrifuged at 8000 rpm for 10 minutes to maintain the resulting supernatant, and its pH was adjusted to 8.4 with Na ₂ HPO ₄ in dry powder form, and then to 0.22 μm. Filtered through a filter membrane having a pore size. The column was charged with 10 ml of Sepharose 4B medium conjugated with protein A, and the column was connected to an AKTA Explorer 100 system. Pump A was connected to a 0.2 M disodium hydrogen phosphate solution, and pump B was connected to a 0.2 M citric acid solution. The detection wavelength was UV 280 nm, the flow rate was 5 ml/min, and the sample injection rate of pump A/B was adjusted. The column was first washed with 100% B (pH 2.3) to remove protein impurities, the pH was balanced with 10% B (pH 8.0), and the signal at the detection wavelength returned to zero after stabilization, followed by loading the sample did. After the pass peak passed, 10% B was used for balancing until the signal of the detection wavelength decreased to zero and stabilized, and elution was performed using 70% B (pH 4.0), and the elution peak was collected. The elution peak sample was dialyzed into PBS buffer and concentration analysis and SDS-PAGE analysis were performed to determine the purity of the IgG antibody.

Example 2: Analysis of properties of cynomolgus monkey-human chimeric monoclonal antibody that specifically binds to HBsAg

Three cynomolgus monkey-human chimeric monoclonal antibodies specifically binding to HBsAg (designated as M1-23, M3-23 and M3-13) were prepared according to the method of Example 1. The VH and VL amino acid sequences of the three strains are shown in the table below (SEQ ID NOs: 1 to 5). In addition, the CDR sequences of the three antibody strains were measured using IMGT, and the amino acid sequences of the CDRs of the heavy chain variable region and light chain variable region of these antibodies are shown in Table 5 (SEQ ID NOs: 6 to 20).

Amino acid sequences of light and heavy chain variable regions of M1-23/M3-23/M3-13 sequence name SEQ ID NO: amino acid sequence M1-23 VH One QVQLQESGPGLVKPSEILSVTCSVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLSRVTLSVDTSKNQLSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGLRVTVSS M1-23 VK 2 EIVMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPQLLIYYANRLESGVPSRFRGSGSGTEFTLTISSLQPEDFTTYYCQQYHSLPLTFGGGTKVEIK M3-23 VH 3 QLQLQESGPGLVKPSETLSLTCAVSGVSISSPYDWTWIRQPPGKGLEWIGRIHGNGGSTSYNPSLKNRVTISKDTSKNQFSLILSSVTAADTAVYYCVRDETWGQGVLVTVSS M3-23 VK 4 DIQMSQSPDSLAVSLGERVTINCKSSQSLLYSSNNKNYLAWYQQKPGQAPKLLFYWASTRESGVPNRFSGSGSGTDFTLTIRGLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK M3-13 VH 5 QVQLQESGPRLVKSSETLPLTCAVSGASNSSNYWSWIRQPPGKGLEWIGRIYGNSGSTDYNPSLKSRVSISTDTSKNQFSLKLSSVTAADTAVYFCARGSVYTYSWGQGVLVTVSS M3-13 VK 4 DIQMSQSPDSLAVSLGERVTINCKSSQSLLYSSNNKNYLAWYQQKPGQAPKLLFYWASTRESGVPNRFSGSGSGTDFTLTIRGLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK

CDR sequences of light and heavy chains of M1-23/M3-23/M3-13 M1-23 VH CDR1 GGSITSNF SEQ ID NO: 6 VH CDR2 YISGSGTYT SEQ ID NO: 7 VH CDR3 ARSHDYGSNDYAFDF SEQ ID NO: 8 VL CDR1 QDISSS SEQ ID NO: 9 VL CDR2 YAN SEQ ID NO: 10 VL CDR3 QQYHSLPLT SEQ ID NO: 11 M3-23 VH CDR1 GVSISSPYDW SEQ ID NO: 12 VH CDR2 IHGNGST SEQ ID NO: 13 VH CDR3 VRDET SEQ ID NO: 14 VL CDR1 QSLLYSSNNKNY SEQ ID NO: 15 VL CDR2 WAS SEQ ID NO: 16 VL CDR3 QQHYTTPLT SEQ ID NO: 17 M3-13 VH CDR1 GASNSSNY SEQ ID NO: 18 VH CDR2 IYGNSGST SEQ ID NO: 19 VH CDR3 ARGSVYTYS SEQ ID NO: 20 VL CDR1 QSLLYSSNNKNY SEQ ID NO: 15 VL CDR2 WAS SEQ ID NO: 16 VL CDR3 QQHYTTPLT SEQ ID NO: 17

We performed a series of characterizations on three purified monoclonal antibodies. First, the binding ability of M1-23, M3-23 and M3-13 to HBsAg was detected by ELISA. A 3-fold gradient dilution was performed starting from 10 μg/ml to obtain 12 gradients. The diluted antibody was then applied to a commercial reaction plate for HBsAg (purchased from Beijing Wantai), incubated at 37° C. for 1 hour, washed 5 times with PBST and dried. Then, a GAH-HRP enzyme-labeled secondary antibody was added, incubated for 30 minutes, washed 5 times with PBST, and dried. Substrate TMB solution was added. After 15 minutes of color development, the color reaction was stopped with H ₂ SO ₄ and the reading was taken at OD450/630. The results are shown in Fig. 1, wherein M1-23, M3-23 and M3-13 all have good antigen-binding activity. The blockade of binding of M1-23, M3-23 and M3-13 to HBsAg by HBsAg (described in 201610879693.5) was studied by a competitive ELISA method. See Example 4.2 for specific steps of the competitive ELISA method. As shown in FIG. 2 , the results showed that mouse mAb 6D11 significantly blocked the binding of M1-23, M3-23 and M3-13 to HBsAg, which resulted in M1-23, M3-23 and M3- 13 and mouse monoclonal antibody 6D11 recognize the same HBsAg SA linear epitope.

The virus clearance ability of cynomolgus-human chimeric antibodies in animals was evaluated in HBV transgenic mice. Cynomolgus monkey-human chimeric antibody was administered as a single dose to HBV transgenic mice (6 HBV transgenic mice per group) at a dose of 10 mg/kg by tail vein injection. Then, the blood of the mice was collected from the posterior orbital venous plexus, and the changes in the HBsAg level and HBV DNA level in the serum of the mice were measured. Methods for measuring HBsAg levels and HBV DNA levels in mouse serum can be seen in Examples 5.1 and 5.2. The results were M1-23, M3-23 and M3-13 (the results of M1-23 and M3-23 are shown in FIGS. 3A and 3B; the results of M3-13 removal for HBsAg are shown in FIGS. 7A and 7B. ) has been shown to have potential in the treatment of HBV infection or diseases associated with HBV infection, such as hepatitis B.

Example 3: Humanization of Antibodies M1-23 and M3-23

To reduce the immunogenicity caused by heterologous antibodies administered to human subjects, we humanized two antibodies, M1-23 and M3-23. Due to the high homology of amino acid sequences between cynomolgus monkey and human immunoglobulins, in the case of the two antibodies M1-23 and M3-23, only a few variable region FR amino acids are needed to make the two antibodies suitable for human therapy. to the corresponding amino acid in the human variable region FR sequence. However, while an antibody recognizes an antigen by contacting it primarily through its CDRs, some of the residues in the FR region of an antibody may also be involved in antigen-antibody interactions, thereby affecting the conformational configuration of the CDRs. For example, after replacing the FR region of a mouse antibody with that of a human antibody, the conformational configuration of the CDRs of a mouse antibody can often change, which significantly reduces the affinity of the humanized antibody to recognize/bind antigen and , may even lose the ability of humanized antibodies to bind antigen (Ge Yan, Development Strategy Analysis and Application Research of Humanized Antibodies [J]. Foreign Medical Sciences, Immunology, 2004, 27(5): 271). Therefore, in the process of humanization of M1-23 and M3-23, the location of the point mutation in the variable region FR sequence and the selection of amino acids that can be replaced with the original are very important.

Through the use of the IMGT Information System (http://www.imgt.org) together with experimental experience, and based on numerous analyzes and experiments, the inventors unexpectedly found that the human germline gene sequence IGHV4-4*08 (SEQ ID NO: 40) ) and IGKV1-39*01 (SEQ ID NO: 41) are particularly advantageous as templates for humanization of M1-23, and IGHV4-4*02 (SEQ ID NO: 42) and IGKV4-1*01 (SEQ ID NO: 43) are M3- 23 has been found to be particularly advantageous as a template for humanization, and the relevant germline gene sequences are listed in Table 6 below.

Amino acid sequence of human germline genes germ line
gene corresponding
name of sequence SEQ ID NO: amino acid sequence IGHV4-4*08 M1-23 VH 40 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYTSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR IGKV1-39*01 M1-23 VK 41 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP IGHV4-4*02 M3-23 VH 42 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCAR IGKV4-1*01 M3-23 VK 43 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

Corresponding to germline gene sequences, and considering that changes in amino acids in the FR regions proximal to the CDRs may affect the affinity of the humanized antibody to recognize/bind antigen, the present inventors have determined that the FR regions of the two antibodies To be modified, the following single point mutations were designed while retaining the amino acids near the CDR regions. Humanized M1-23 and M3-23 were named M1D and M3D, respectively.

Single point mutation of an amino acid residue in the FR region of antibody M1-23 No. ^※ usually
amino acid after humanization
amino acid No. ^※ usually
amino acid amino acids after humanization HFR1 LFR1 H17 I T L1 E D H20 V L L3 V Q H23 S T LFR2 HFR3 L45 Q K H64 R K LFR3 H69 L I L55 E Q H78 L F L63 R S HFR4 L70 E D H107 L T L84 T A H108 R T

※: The amino acid positions mentioned in the table above are numbered according to the Kabat numbering system.

Single point mutation of an amino acid residue in the FR region of antibody M3-23 No. ^※ usually
amino acid after humanization
amino acid No. ^※ usually
amino acid after humanization
amino acid HFR1 LFR1 H2 L V L3 Q V H16 E G L5 S T HFR2 L19 V A H37 I V LFR2 HFR3 L43 A P H65 N S LFR3 H71 K V L60 N D H73 T K L76 R S H81 I K L77 G S HFR4 H107 V T

※: The amino acid positions mentioned in the table above are numbered according to the Kabat numbering system.

Primers for humanization of FR regions of M1-23, M3-23 Primer name order
number primer sequence PTT5-1-23H-F 64 5'-AGTAGCAACTGCAACCGGTGTACATTCTcaggtgcagctgcaggagtcaggcccag 1-23H-F1 65 5'-caggagtcaggcccaggactggtgaagccttcggagaCCCTGTCCCTCACCTGCAC 1-23H-F2 66 5'-aagccttcggagaCCCTGTCCCTCACCTGCACtgtctctggtggctccatc 1-23H-R1 67 5'-GTTCTTGGACGTGTCTACGGATATGGTGACTCGACTCTTGAGGGAGGGGTTGTAGT 1-23H-F3 68 5'-TCCGTAGACACGTCCAAGAACCAGTtctccctgaaactgagctc PTT5-1-23H-R 69 5'-GATGGGCCCTTGGTCGACGCtgaagagacggtgacGGTGGTcccttggccccagaaatc PTT5-1-23K2-F 70 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTCTCCATCC 1-23K-R2 71 5'-CAGCAAAAACCGGGGAAAGCCCCTAAGCTCCTGATCTATTATGC 1-23K2-R1 72 5'-GAACCTTGATGGGACCCCACTTTGCAAACGATTGCATAATAGATCAGGAGC 1-23K2-R2 73 5'-CTGTCCCAGATCCACTGCCACTGAACCTTGATGGGACCCC 1-23K-F2 74 5'- GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAGG 1-23K-F3 75 5'-GCAGCCTGCAACCTGAGGATTTTGCAACTTATTACTGTCAAC PTT5-1-23K-R 76 5'-GACCAAGGTGGAAATCAACGTACGGTGGCTGCACCAT PTT5-3-23HV2-F 77 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC 3-23HV2-R1 78 5'-ATTCCCGGGTagatgcccaGTAAAAGAGCAGCTTAGGAGGCTGTCCTGGTTTCTGCTG 3-23HV2-F1 79 5'-ggtgtctccatcagcagtccttatgacTGGACCTGGGTCCGCCAGCCCCCAGGGAAG 3-23HV2-R2 80 5'-AACTGGTTCTTGGACTTGTCTACTGAAATGGTGACTCGACTCTTGAGGGAGGGGTTG 3-23HV2-F2 81 5'- AGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGC PTT5-3-23HV1-R 82 5'-gaaaccTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC PTT5-3-23KV1-F 83 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTCCAGACTCC 3-23KV1-F1 84 5'-CCCAGTCTCCAGACTCCCTGGCTTGTGTCTCTGGGAGAGAGGGCCACCATCAACTGCAAGTCCAG 3-23KV2-R1 85 5'-CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCTTTTACtgggcatctACCCGGGAAT 3-23KV1-R2 86 5'-GTGAAATCTGTCCCAGATCCACTGCCACTGAATCGGTCAGGGACCCCGGATTCCCGGGTagatgcc 3-23KV1-F2 87 5'-GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATG PTT5-3-23KV1-R 88 5'-ATGGTGCAGCCACCGTACGTTGATTTCCACCTTGGTCC

According to the single point mutations listed in Tables 7 and 8, the relevant primers were designed (see Table 9). Mutations of all original amino acids at the corresponding positions were performed by PCR, where M1-23-H, M1-23-L, M3-23-H and M3-23-L were used separately as templates. Considering humanization of the M1-23 heavy chain variable region (M1-23-H) as an example, oligonucleotide primers PTT5-1-23H-F, 1-23H-F1, 1-23H-F2, 1-23H-R1, 1-23H-F3 and PTT5-1-23H-R (their sequences are shown in the table above) were designed. The specific protocol of PCR was as follows: the gene encoding M1-23-H was used as a template, and primer pairs 1-23H-F1/1-23H-R1 and 1-23H-F3/PTT5-1-23H- R was used for amplification to obtain fragment 1 and fragment 2, respectively; Using fragment 1 as a template, primer pair 1-23H-F2/1-23H-R1 was used for amplification to obtain fragment 3; Using fragment 3 as a template, primer pair PTT5-1-23H-F/1-23H-R1 was used for amplification to obtain fragment 4; Fragment 4 and fragment 2 were ligated together by overlap extension PCR and then PTT5-1-23H-F/PTT5-1-23H-R was used as upstream and downstream primers for amplification to obtain humanized gene fragment M1D-H .According to the above method, humanized gene fragment M1D-L of M1-23-L, humanized gene fragment M3D-H of M3-23-H, and humanized gene fragment M3D-L of M3-23-L were obtained. did.

The humanized gene fragments M1D-H and M3D-H as obtained were ligated with the eukaryotic expression vector pTT5-CH, respectively, using a laboratory-made Gibson assembly liquid, and M1D-L and M3D-L were respectively transferred to eukaryotes. The recombinant vector was obtained by ligation with the expression vector pTT5-Cκ. The recombinant vector was transformed into Escherichia coli DH5α strain, plated on LB plates, and incubated overnight at 37°C in an incubator. Monoclonal colonies were harvested from the plates and sequenced. A plasmid having the correct sequence was selected, and expression and purification of the antibody were performed according to the method of Example 1.

The amino acid sequences of humanized antibodies M1D and M3D are shown in Table 11. In addition, the degree of humanization of M1D and M3D was calculated according to the following recipe. As shown in Table 10 below, after humanization of the two antibodies, the degree of humanization increased from about 90% to about 98%.

Sequencing of Humanized Antibodies before humanization after humanization Antibody name degree of humanization Antibody name degree of humanization M1-23 90% M1D 98.33% M3-23 91.67% M3D 97.78%

Degree of humanization = (number of amino acids in the FR region - the number of monkey-derived amino acids retained in the FR region)/number of amino acids in the FR region × 100%

Amino acid sequences of the light and heavy chain variable regions of antibodies M1D, M3D corresponding
name of sequence SEQ ID NO: amino acid sequence M1D VH 57 QVQLQESGPGLVKPSETLSLTCTVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGTTVTVSS M1D VK 58 DIQMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPKLLIYYANRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSLPLTFGGGTKVEIK M3D VH 59 QVQLQESGPGLVKPSGTLSLTCAVSGVSISSPYDWTWVRQPPGKGLEWIGRIHGNGGSTSYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCVRDETWGQGTLVTVSS M3D VK 60 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLFYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK

Example 4: Evaluation of in vitro binding activity of humanized antibodies M1D, M3D

4.1 Determination of Antigen-Binding Activity of Antibodies

The binding activity of humanized antibodies M1D, M3D to the antigen HBsAg was investigated by ELISA method (enzyme-linked immunosorbent assay). A 3-fold gradient dilution was performed starting from 10 μg/ml to obtain 12 gradients. Humanized antibody 162 (described in detail in Chinese patent application CN201610879693.5) was used as a reference antibody. Subsequently, the diluted antibody was applied to a commercial reaction plate for HBsAg (purchased from Beijing Wantai), incubated at 37° C. for 1 hour, washed 5 times with PBST and dried. Thereafter, a GAH-HRP enzyme-labeled secondary antibody was added, incubated for 30 minutes, washed 5 times with PBST, and dried. Substrate TMB solution was then added. After 15 minutes of color development, the color reaction was stopped with H ₂ SO ₄ and the reading was taken at OD450/630. The results are shown in Figure 4, wherein both humanized antibodies M1D and M3D had excellent antigen-binding activity, and their affinity for antigen HBsAg was superior to that of reference antibody 162. The results indicated that the two humanized antibodies not only had a very high degree of humanization (>97%), but also substantially retained the antigen-binding activity of their parent antibody, and even showed better antigen-binding activity than their parent antibody. .

4.2 Identification of Preliminary Epitopes of Antibodies

We also performed a preliminary identification of the epitope on the antibody to determine whether the selected human-monkey chimeric and humanized antibodies recognized the SA epitope on HBsAg. 6D11, which recognizes a high concentration of the SA type epitope, was used to cross-block the binding of the antibody to be tested to HBsAg, wherein if there was a significant blocking (competition) effect, the antibody to be tested had the same epitope as 6D11 recognized. The specific steps were as follows:

(1) After the measurement of the antibody concentration, the antibody was added to a commercial ELISA plate for HBsAg at 10 μg/well, then the volume was made up to 50 μl with 20% NBS, and incubation was performed at 37° C. for 30 minutes.

(2) 6D11-HRP diluted 1:20000 with ED-11 was added to an ELISA plate at 50 μl/well, and incubated at 37° C. for 30 minutes.

(3) The plate was washed 5 times with PBST and dried.

(4) Substrate TMB solution was added, and after 15 minutes of color development, the color reaction was terminated with H ₂ SO ₄ .

(5) Readings were taken at OD450/630.

The results are shown in Figure 5, where 162 was used as a positive control in the above experiment. The binding of M1D, M3D to HBsAg could be competed by mouse antibody 6D11, and the competitive effect was comparable to that of positive control 162 and parental antibodies M1-23, M3-23. The results indicated that humanized antibodies M1D, M3D also recognized SA epitopes.

4.3 Determination of neutralizing activity of antibodies

We used the laboratory-made cell model HepaG2-NTCP to evaluate the ability of the antibody to neutralize HBV infection (with an HBV infection titer of 100 MOI). An amount of HBV and gradient-diluted antibody were co-cultured in HepaG2 cells, and HBV was able to replicate in the cells by infiltrating the differentiated HepaG2 cells. With the addition of different antibodies and changes in the amount of antibody added, invasion and replication of HBV was attenuated or inhibited to different degrees, and thus different levels of HBV antigen (HBeAg) would have been present in the cell supernatant. Thus, by measuring the level of HBV antigen (HBeAg), it was possible to determine the ability of different humanized antibodies to neutralize HBV.

Humanized antibody was diluted in a 2-fold gradient and antibody 162 was used as a positive control. From 10 μg/ml, a total of 18 dilution gradients were made. The viral infection system was mixed with the antibody separately and pre-incubated for 1 hour. This was then applied to cell plates pre-plated with HepaG2, incubated at 37° C. for 24 hours, and the medium was replaced with fresh every 2 days. On day 7 post-infection, the cell culture supernatant was harvested and the level of HBeAg was measured. The measurement method included the following steps:

(1) Preparation of reaction plate: Anti-HBeAg monoclonal antibody was diluted to 2 μg/ml with 20 mM PB buffer (Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer, pH 7.4). Diluted anti-HBeAg monoclonal antibody (100 μl/well) was added to 96-well chemiluminescent plates and incubated at 37° C. for 2 hours and then at 4° C. overnight. The raw material used was purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd. 96-well chemiluminescent plates were washed once with PBST and dried. A blocking solution was added to a 96-well chemiluminescence plate at 200 μl/well and blocked at 37° C. for 2 hours. The blocking solution was then discarded and the plates were placed in a dry room to dry and stored at 2-8° C. until use.

(2) Culture: 100 μl/well of the sample to be tested was added to the prepared reaction plate, and incubated at 37° C. for 60 minutes.

(3) Washing: The 96-well chemiluminescent plate was washed 5 times with PBST and dried.

(4) Culture: Horseradish peroxidase-labeled anti-HBeAg antibody (100 μl/well) was added and incubated at 37° C. for 30 minutes. Raw materials were purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.

(5) Washing: The 96-well chemiluminescent plate was washed 5 times with PBST and dried.

(6) Color development: Luminol chemiluminescence reagent (100 μl/well) was added.

(7) Reading Plate: Values were measured using a chemiluminescent microplate reader.

The experimental results are shown in FIGS. 6A and 6B, indicating that the neutralizing activity of M1D against HBV is comparable to that of the reference antibody 162.

Example 5: Determination of the therapeutic effect of humanized antibodies M1D, M3D

The five antibodies were administered separately to HBV transgenic mice at a single dose of 10 mg/kg, each group containing 6 HBV transgenic mice. Then, blood was collected from the retro-orbital venous plexus of mice, and changes in HBsAg levels and HBV DNA levels in the mice's serum were measured.

5.1 Quantitative determination of HBsAg

(1) Preparation of reaction plate: mouse monoclonal antibody HBs-45E9 was diluted to 2 μg/ml with 20 mM PB buffer (Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer, pH 7.4), and coating buffer was diluted to 100 μl/well applied to a chemiluminescent plate. Plates were coated at 6°C to 8°C for 16-24 hours and then at 37°C for 2 hours, washed once with PBST wash and dried. After washing, 200 μl of blocking solution was added to each well and blocking was performed at 37° C. for 2 hours. The blocking solution was then discarded and the plates were placed in a dry room to dry and stored at 2°C to 8°C until use.

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

(3) Denaturing treatment of samples: 15 μl of the diluted serum sample was thoroughly mixed with 7.5 μl of denaturation buffer (15% SDS, dissolved in 20 mM PB7.4), and reacted at 37° C. for 1 hour. Then, 90 μl of neutralization buffer (dissolved in 4% CHAPS, 20 mM PB7.4) was added and thoroughly mixed.

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

(5) Enzyme labeling reaction: HBs-A6A7-HRP reaction solution (100 μl/well) was added to a chemiluminescent plate, and reacted at 37° C. for 1 hour. The plate was then washed 5 times with PBST and dried.

(6) Luminescence reaction and measurement: A luminescent liquid (100 μl/well) was added to a chemiluminescence plate, and light intensity measurement was performed.

(7) Calculation of HBsAg concentration in mouse serum sample: A parallel experiment was performed on a standard sample, and a standard curve was prepared based on the measurement results of the standard sample. The light intensity values of the mouse serum samples were then inserted into the standard curve to calculate the HBsAg concentration in the serum samples to be tested.

5.2 Quantitative determination of HBV DNA

Quantitative measurement of HBV DNA was performed according to the instructions of the HBV DNA Real-Time Fluorescence Quantitation Kit (the kit was purchased from Shanghai Kehua Bioengineering Co., Ltd.).

In this example, the virus clearance ability of humanized antibodies M1D, M3D, cynomolgus-monkey chimeric antibody M3-13, and reference antibody 162 in animals was measured. The results are shown in Figures 7a and 7b. The results show that the humanized antibodies M1D, M3D and the cynomolgus monkey-human chimeric antibody M3-13 have good virus clearance, and their ability to clear HBsAg and HBV DNA in animals is better than that of the reference antibody 162. seemed

The above results show that the humanized antibody of the present invention has a very high degree of humanization (up to 98%), which not only reduces the likelihood of immunological rejection, but is also equivalent to that of the monkey-human chimeric antibody and higher than that of the reference antibody 162. It was demonstrated that it exhibits excellent virus removal ability. Such technical effects were remarkable and unexpected.

Example 6: Determination of non-critical CDR regions of humanized antibodies M1D, M3D

In the antibody variable region domain, the site for direct antigen binding is mainly in the CDR region, but a number of studies have shown that not all six CDRs of an antibody are important regions for antigen-antibody binding, and non-critical CDR regions are used in the CDR replacement method. showed that it can be measured by In the present invention, replacement sequences for the original CDRs were selected according to the principle of best match. The VH and VL amino acid sequences of the humanized antibodies M1D and M3D were entered into the search box of the IMGT information system, and then the corresponding amino acid sequences of the antibody light and heavy chains in the germline genes were searched by the system. Following the principle that the FR regions had the greatest homology and the CDR region sequences had the greatest differences, two replacement sequences were selected for each CDR region in the humanized antibody, with the exception of the heavy chain CDR3. The replacement sequences are shown in the table below:

CDR replacement sequences for humanized antibody M1D, M3D number original CDR Replacement CDR-A Replacement CDR-A germline gene Replacement CDR-B Replacement CDR-B germline gene M1D-H-CDR1 GGSITSNF GGSISSYY IGHV4-4*08 GGSVSSGSYY IGHV4-61*01 M1D-H-CDR2 ISGSGTYT IYTSGST IGHV4-4*08 ISSSSSYT IGHV3-11*03 M1D-L-CDR1 QDISSS QSVSSSY IGKV3D-7*01 SSNIGSNT IGLV1-44*01 M1D-L-CDR2 YAN AAS IGKV1-39*01 KAS IGKV1-5*03 M1D-L-CDR3 QQYHSLPLT LQDYNYPLT IGKV1-6*01 HQSSSLPLT IGKV6-21*02 M3D-H-CDR1 GVSISSPYDW GGSISSGGYS IGHV4-30-4*07 GGSISSSNW IGHV4-4*01 M3D-H-CDR2 IHGNGST IYHSGST IGHV4-4*02 ISGSGGST IGHV3-23*02 M3D-L-CDR1 QSLLYSSNNKNY QSLLHSDGKTY IGKV2-29*02 QSVSSSY IGKV3D-7*01 M3D-L-CDR2 WAS LGS IGKV2-28*01 GAS IGKV3-15*01 M3D-L-CDR3 QQHYTTPLT QQYYSTPWT IGKV4-1*01 MQALQTPWT IGKV2-28*01

Relevant primers were designed according to the CDR replacement sequences described in the table above, and the corresponding CDR regions in M1D and M3D antibodies were replaced by PCR method. Primers as designed are shown in the table below. Considering the first substitution (M1D-H-CDR1-A) of the M1D heavy chain variable region CDR1 (M1D-HCDR1) as an example, the oligonucleotide primers M1D-H-CDR1-A R and M1D-H-CDR1 F were designed and (The sequence is shown in the table below), and the antibody after the replacement of the CDRs was named M1D HCDR1-1. The naming of other replacement antibodies could be done in the same way. The specific protocol of PCR was as follows: the coding gene of M1D-H was used as a template, and the primer pairs pTT5-M1D-HF/M1D-H-CDR1-A R and M1D-H-CDR1-A F/pTT5-M1D -HR was used for amplification to obtain upstream and downstream fragments, respectively; The fragments were ligated together by overlap extension PCR and then amplified with pTT5-M1D-HF/pTT5-M1D-HR as upstream and downstream primers to obtain a gene fragment in which HCDR1 of M1D was replaced.

Primer sequence for CDR replacement Primer name SEQ ID NO: primer sequence pTT5-M1D-H-F 89 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCAGGC pTT5-M1D-H-R 90 5'-GATGGCCCTTGGTCGACGCTGAAGAGACGGTGAC pTT5-M1D-L-F 91 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTC pTT5-M1D-L-R 92 5'-ATGGTGCAGCCACCGTACTTTGATTTCCACCTTGGTCCC M1D-H-CDR1-1 R 93 5'-GTAGTAACTACTGATGGAGCCACCAGAGACAGTGCAGGTGAG M1D-H-CDR1-1 F 94 5'-GgtggctccatcagtagttactacTGGAGCTGGATCCGCCAG M1D-H-CDR1-2R 95 5'-ATAGTAACTACCACTGCTGACGGAGCCACCAGAGACAGTGCAGGTGAG M1D-H-CDR1-2F 96 5'-GgtggctccgtcagcagtggtagttactATTGGAGCTGGATCCGCCAG M1D-H-CDR2-1 R 97 5'-GGTGCTCCCACTGGTATAGATATACCCAATCCACTCCAG M1D-H-CDR2-1 F 98 5'-AtctataccagtgggagcaccGACTACAACCCCTCCCTC M1D-H-CDR2-2R 99 5'-TGTGTAACTACTACTACTACTAATATACCCAATCCACTCCAG M1D-L-CDR2-2F 100 5'-AttagtagtagtagtagttacacaGACTACAACCCCTCCCTC M1D-L-CDR1-1 R 101 5'-GTAGCTGCTGCTAACACTCTGAGTTGCCCGGCAAGTGATGG M1D-L-CDR1-1 F 102 5'-CagagtgttagcagcagctacTTAAATTGGTATCAGC M1D-L-CDR1-2R 103 5'-AGTATTACTTCCGATGTTGGAGCTAGTTGCCCGGCAAGTGATGG M1D-L-CDR1-2F 104 5'- AgctccaacatcggaagtaatactTTAAATTGGTATCAGC M1D-L-CDR2-1 R 105 5'-GACCCCACTTTGCAAACGGGATGCAGCATAGATCAGGAGCTTAGG M1D-L-CDR2-1 F 106 5'-CCTAAGCTCCTGATCTATGctgcatccCGTTTGCAAAGTGGGGTC M1D-L-CDR2-2R 107 5'-GACCCCACTTTGCAAACGAGACGCCTTATAGATCAGGAGCTTAGG M1D-L-CDR2-2F 108 5'-CCTAAGCTCCTGATCTATAaggcgtctCGTTTGCAAAGTGGGGTC M1D-L-CDR3-1R 109 5'-AGTGAGAGGGTAATTGTAATCTTGTAGACAGTAATAAGTTGCAAAATC M1D-L-CDR3-1F 110 5'-CtacaagattacaattaccctCTCACTTTCGGCGGAGGGACCAAG M1D-L-CDR3-2R 111 5'-AGTGAGAGGTAAACTACTACTCTGATGACAGTAATAAGTTGCAAAATC M1D-L-CDR3-2F 112 5'-catcagagtagtagtttacctCTCACTTTCGGCGGAGGGACCAAG pTT5-M3D-H-F 113 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCG pTT5-M3D-H-R 114 5'-GATGGCCCTTGGTCGACGCTGAGGAGACGGTGACCAG pTT5-M3D-L-F 115 5'-AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTC pTT5-M3D-L-R 116 5'-ATGGTGCAGCCACCGTACTTTGATTTCCACCTTGGTC M3D-H-CDR1-1 R 117 5'-GGAGTAACCACCACTGCTGATGGAGCCACCAGAGACAGCGCAGGTGAGGGACAG M3D-H-CDR1-1 F 118 5'-ggtggctccatcagcagtggtggttactccACCTGGGTCCGCCAGCCC M3D-H-CDR1-2R 119 5'-CCAGTTACTACTGCTGATGGAGCCACCAGAGACAGCGCAGGTGAGGGACAG M3D-H-CDR1-2F 120 5'-GgtggctccatcagcagtagtaactggACCTGGGTCCGCCAGCCC M3D-H-CDR2-1 R 121 5'-GGTGCTCCCACTATGATAGATTCGTCCAATCCACTCCAG M3D-H-CDR2-1 F 122 5'- AtctatcatagtgggagcaccAGCTACAACCCCTCCCTCAAG M3D-H-CDR2-2R 123 5'-TGTGCTACCACCACTACCACTAATTCGTCCAATCCACTCCAG M3D-L-CDR2-2F 124 5'- attagtggtagtggtggtagcacaAGCTACAACCCCTCCCTCAAG M3D-L-CDR1-1 R 125 5'-ATAGGTCTTTCCATCACTATGCAGGAGGCTCTGGCTGGACTTGCAGTTGATGGTGGC M3D-L-CDR1-1 F 126 5'-cagagcctcctgcatagtgatggaaagacctatTTAGCCTGGTACCAGCAGAAAC M3D-L-CDR1-2R 127 5'-GTAGCTGCTGCTAACACTCTGGCTGGACTTGCAGTTGATGGTGGC M3D-L-CDR1-2F 128 5'-CagagtgttagcagcagctacTTAGCCTGGTACCAGCAGAAAC M3D-L-CDR2-1 R 129 5'- GGGACCCCGGATTCCCGGGTAGAACCCAAGTAAAAGAGCAGCTTAGGAGG M3D-L-CDR2-1 F 130 5'-CCTCCTAAGCTGCTCTTTTACTtgggttctACCCGGGAATCCGGGGTCCC M3D-L-CDR2-2R 131 5'- GGGACCCCGGATTCCCGGGTGGATGCACCGTAAAAGAGCAGCTTAGGAGG M3D-L-CDR2-2F 132 5'-CCTCCTAAGCTGCTCTTTTACGgtgcatccACCCGGGAATCCGGGGTCCC M3D-L-CDR3-1R 133 5'-CGTCCAAGGAGTACTATAATATTGCTGACAGTAATACACTGCCACATC M3D-L-CDR3-1F 134 5'-CagcaatattatagtactccttggacgTTCGGCCAAGGGACCAAG M3D-L-CDR3-2R 135 5'-CGTCCAAGGAGTTTGTAGAGCTTGCATACAGTAATACACTGCCACATC M3D-L-CDR3-2F 136 5'-AtgcaagctctacaaactccttggacgTTCGGCCAAGGGACCAAG

According to the above method, the obtained gene fragment of the heavy chain with CDR replacement was separately ligated into the eukaryotic expression vector pTT5-CH, and then co-transfected in small amounts into HEK293-cells with the expression vector of the corresponding light chain without CDR replacement. and the antigen activity of the cell supernatant with small expression was measured. Similarly, the obtained gene fragment of the light chain with CDR replacement was separately ligated into the eukaryotic expression vector pTT5-Cκ, then co-transfected with the expression vector of the corresponding heavy chain without CDR replacement, and antigen- Binding activity was measured. After replacing several CDR regions of a humanized antibody, if the corresponding cell transfection supernatant showed no antigen-binding activity or a significant decrease in antigen-binding activity, the replaced CDR regions were replaced with the antibody. - it was possible to conclude that it is a CDR region that is important for antigen binding; If the corresponding cell transfection supernatants showed substantially unchanged antigen-binding activity after replacement, then the replaced CDR regions were not critical for antibody-antigen binding.

The replacement results for M1D are shown in FIGS. 8A to 8E . After replacement of the heavy chain CDR1 (M1D-H-CDR1) and light chain CDR2 (M1D-L-CDR2) of M1D, the antigen-binding activity for HBsAg was substantially unchanged; After replacement of heavy chain CDR2 and light chain CDR1, antigen-binding activity was significantly reduced; After replacement of light chain CDR3, antigen-binding activity was completely lost. The above results indicated that HCDR1 and LCDR2 of the antibody M1D are not important CDR regions for binding to the antigen HBsAg, and their replacement does not affect the antigen-binding activity of the antibody.

The replacement results for M3D are shown in FIGS. 9A to 9E . After replacement of the heavy chain CDR2 (HCDR2) and light chain CDR2 (LCDR2) of M3D, the antigen-binding activity was substantially unchanged; After replacement of the light chain CDR1, one replacement (M3D-LCDR1-1) showed a significant decrease in antigen-binding activity, whereas the other replacement (M3D-LCDR1-2) showed substantially no antigen-binding activity; After replacement of heavy chain CDR1 (HCDR1) and light chain CDR3 (LCDR3), antigen-binding activity was substantially absent. The above results indicated that HCDR2 and LCDR2 of antibody M3D are not important CDR regions for antigen binding, and that their replacement does not affect the antigen-binding activity of the antibody.

Example 7: Determination of important amino acids in humanized antibodies M1D, M3D for binding to antigen

To determine the amino acid regions critical for antibody-antigen interaction, we performed alanine scanning on the four CDR regions HCDR2, HCDR3, LCDR1, LCDR3 of M1D, and the four CDR regions HCDR1, HCDR3, LCDR1, LCDR3 of M3D. did. In general, important amino acids are somewhat conserved, and given the amino acid residues that maintain the conformation of the antibody and eg buried amino acid residues, mutation of these amino acids to alanine will render the antibody inactive. The mutated antibody was tested for binding-activity to HBsAg by the Elisa method and the rEC50 of the antibody after mutation was calculated (WT/mutant, the relative value of the original antibody EC50 was 1, 0 indicates complete loss of binding activity) ), where amino acid residues such as D, E, R, H, L, W and Y, with rEC50<0.2 (ie, the activity was reduced by 5-fold) after mutation, are indicated in blue color. The results are shown in Table 14. The acidic amino acids D, E and the basic amino acids R and H can form salt bridges when the antibody binds to the antigen, thereby playing an important role in the antibody-antigen binding reaction, and thus the mutation of these amino acids to alanine will disrupt the formation of ionic bonds and significantly reduce the antibody-binding activity. The aliphatic amino acids L were hydrophobic amino acids and W and Y were aromatic amino acids, which played an important role in the maintenance of the antibody conformation. Among these, mutations of H56Y, H97D, H100cD, H100dL, 94L, L96L of M1D to alanine resulted in a significant decrease in activity, and thus these were sites that should not be changed; Mutations of H33Y, H34D, H35W, H94R, H95D, H101E, L27dY, L32Y, L91H of M3D to alanine resulted in a significant decrease in activity, and thus these were sites that should not be changed.

Example 8: Identification of core epitopes recognized by M1D and M3D

M1D and M3D recognized SA linear epitopes. To further measure the core epitopes recognized by the two antibodies, we performed a single point mutation in aa113-135 (SEQ ID NO: 168) of HBsAg and synthesized 14 polypeptides as shown in the table below. Polypeptide was diluted to CB, 100 ng/ml for plate coating, antibody was diluted to 1000 ng/ml with 20% NBS solution, followed by 3-fold dilution to obtain 12 gradients. The binding activity of antibodies to polypeptides with different single point mutants was measured by the Elisa method, and binding curves were generated using PRISM software. In the case of loss of binding activity after mutation, the site of mutation was an important amino acid for the SA linear epitope.

Point mutant peptide sequence based on SEQ13-B Name of Polypeptide SEQ ID NO: order SEQ13-B (aa113-135) 168 SSTTSTGPCKTCTTPAQGTSMFP S13-S117A 169 SSTTATGPCKTCTTPAQGTSMFP S13-T118A 170 SSTTSAGPCKTCTTPAQGTSMFP S13-G119A 171 SSTTSTAPCKTCTTPAQGTSMFP S13-P120A 172 SSTTSTGACKTCTTPAQGTSMFP S13-C121S 173 SSTTSTGPSKTCTTPAQGTSMFP S13-K122A 174 SSTTSTGPCATCTTPAQGTSMFP S13-T123A 175 SSTTSTGPCKACTTPAQGTSMFP S13-C124S 176 SSTTSTGPCKTSTTPAQGTSMFP S13-T125A 177 SSTTSTGPCKTCATPAQGTSMFP S13-T126A 178 SSTTSTGPCKTCTAPAQGTSMFP S13-P127A 179 SSTTSTGPCKTCTTAAQGTSMFP S13-Q129A 180 SSTTSTGPCKTCTTPAAGTSMFP S13-T131A 181 SSTTSTGPCKTCTTPAQGASMFP S13-S132A 182 SSTTSTGPCKTCTTPAQGTAMFP

The EC50 values of the binding of M1D, M3D and reference antibody 162 to the single point mutant polypeptide are summarized in Table 16 below (>500 indicates complete loss of binding activity). From the results, the core epitope for 162 binding was CKTC (aa121-124), whereas the core epitope for M1D binding was TGPCKTCT (aa118-124), which was longer than for reference antibody 162 and forward compared to aa113-135. was in position; The core epitope for M3D binding was CKTCT (aa121-125), which was in a reverse position relative to that of 162.

EC50 values for binding of M1D, M3D to single point mutant polypeptides based on HBsAg (aa113-135) EC50 (ng/mL) Name of Polypeptide 162 M1D M3D SEQ13-B 39.8 58.1 33.34 S13-S117A 31.1 32.5 33.93 S13-T118A 31.4 >500 37.42 S13-G119A 38.5 >500 41.7 S13-P120A 53.6 >500 67.88 S13-C121S >500 >500 >500 S13-K122A 111 >500 >500 S13-T123A >500 >500 >500 S13-C124S >500 >500 >500 S13-T125A 48.8 353 >500 S13-T126A 41.9 40.8 33.89 S13-P127A 40.4 33.2 69.39 S13-Q129A 39.8 58.1 33.34 S13-T131A 60.1 51.7 62.08 S13-S132A 39.8 58.1 33.34

To further identify the critical sites of the antibody for binding to antigen, we demonstrated this experiment on the HBsAg antigen protein. Relevant primers were designed (Table 17), and 14 HBsAg gene fragments with point mutations were obtained by amplification through PCR. The fragment obtained by amplification was separately ligated using Gibson to the PTT5 empty vector subjected to double enzymatic digestion with EcoRI and BamHI, and the vector construction was completed when the sequencing result was correct. The constructed plasmid was used for small-scale transfection of HEK293T cells, and after 2 days of transfection, cells were collected and confirmed by Western blot. The specific steps were as follows: (1) cells were collected, washed twice with 5 ml of PBS, and centrifuged at 1000 rpm for 5 minutes after cell counting;

(2) 30 μl of DDM/1×10 ⁶ cells were added to lyse the cells (the amount of cellular protein could be quantified by the BCA method), and the lysis was performed at 4° C. for 2 hours with a shaker during this time. Shaking was performed several times;

(3) centrifugation was performed at 13300 rpm for 10 minutes, the supernatant was aspirated as a sample, and reduced SDS-PAGE gel electrophoresis was performed on 30 μl thereof;

(4) electrophoretic transfer by means of a mobile device under a constant current of 300 mA for 90 minutes, and blocking using a commercial blocking solution at 37° C. for 1 hour;

(5) 1 mg/ml of antibodies M1D, M3D and 162 were separately diluted 1000-fold with ED-11 and incubated at 37° C. for 1 hour;

(6) washing with PBST was performed once every 5 minutes for a total of 3 times;

(7) enzyme-labeled secondary antibody GAH-HRP (1:5000) was added and incubated at 37° C. for 30 minutes;

(8) washing with PBST was performed once every 5 minutes for a total of 3 times;

(9) development and photography;

(10) β-actin was used as an internal reference, and in step (4) the region corresponding to β-actin on the PVDF membrane was cleaved, and HRP-conjugated mouse anti-β-actin antibody (1:5000) was 37 Direct incubation at ℃ for 30 minutes, other steps were the same as above. From the results shown in FIG. 10 , it was found that the core epitopes of the three antibodies identified by the HBsAg antigen protein were completely consistent with those of the linear polypeptide.

Primers for C-HBsAg single point mutation Primer name SEQ ID NO: primer sequence C-S117A-F 183 ATCAACTACCGCCACGGGACCATGCAAGACCTGCAC C-S117A-R 184 GGTCCCGTGGCGGTAGTTGATGTTCCTGGAAGTAGA C-T118A-F 185 AACTACCAGCGCGGGACCATGCAAGACCTGCACGAT C-T118A-R 186 CATGGTCCCGCGCTGGTAGTTGATGTTCCTGGAAGT C-G119A-F 187 ACCAGCACGGCACCATGCAAGACCTGCACGATTCCT C-G119A-R 188 CTTGCATGGTGCCGTGCTGGTAGTTGATGTTTCCTGG C-P120A-F 189 CAGCACGGGAGCATGCAAGACCTGCACGATTCCTGC C-P120A-R 190 GTCTTGCATGCTCCCGTGCTGGTAGTTGATGTTTCCT C-C121S-F 191 ACGGGACCATCCAAGACCTGCACGATTCCTGCTCAA C-C121S-R 192 GCAGGTCTTGGATGGTCCCGTGCTGGTAGTTGATGTGT C-K122R-F 193 GGGACCATGCCGGACCTGCACGATTCCTGCTCAAGG C-K122S-R 194 GTGCAGGTCCGGCATGGTCCCGTGCTGGTAGTTGAT C-K122A-F 195 GGGACCATGCGCGACCTGCACGATTCCTGCTCAAGG C-K122A-R 196 GTGCAGGTCGCGCATGGTCCCGTGCTGGTAGTTGAT C-T123A-F 197 ACCATGCAAGGCCTGCACGATTCCTGCTCAAGGAAC C-T123A-R 198 ATCGTGCAGGCCTTGCATGGTCCCGTGCTGGTAGTT C-C124S-F 199 TGCAAGACCTCCACGATTCCTGCTCAAGGAACCTCT C-C124S-R 200 AGGAATCGTGGAGGTCTTGCATGGTCCCGTGCTGGT C-T125A-F 201 CAAGACCTGCGCGATTCCTGCTCAAGGAACCTCTAT C-T125A-R 202 GCAGGAATCGCGCAGGTCTTGCATGGTCCCGTGCTG C-I126A-F 203 GACCTGCACGGCTCCTGCTCAAGGAACCTCTATGTT C-I126A-R 204 TGAGCAGGAGCCGTGCAGGTCTTGCATGGTCCCGTG C-P127A-F 205 CTGCACGATTGCTGCTCAAGGAACCTCTATGTTTCC C-P127A-R 206 CCTTGAGCAGCAATCGTGCAGGTCTTGCATGGTCCC C-Q129A-F 207 GATTCCTGCTGCAGGAACCTCTATGTTTCCCTCTTG C-Q129A-R 208 GAGGTTCCTGCAGCAGGAATCGTGCAGGTCTTGCAT C-T131A-F 209 TGCTCAAGGAGCCTCTATGTTTCCCTCTTGTTGCTG C-T131A-R 210 AACATAGAGGCTCCTTGAGCAGGAATCGTGCAGGTC C-S132A-F 211 TCAAGGAACCGCTATGTTTCCCTCTTGTTGCTGTAC C-S132A-R 212 GGAAACATAGCGGTTCCTTGAGCAGGAATCGTGCAG

Example 9: Identification of broad spectrum activity of M1D and M3D

To further confirm the broad spectrum activity of M1D and M3D, each of the HBsAg fragments (aa113-135) corresponding to different subtypes of A to G (where F type included F1 and F2) (in their sequence For coating reaction plates, see Table 18), where the polypeptide was diluted to 100 ng/ml CB for plate coating. The antibody was diluted to 1000 ng/ml with 20% NBS solution, followed by 3-fold dilution to obtain 12 gradients. The binding activity of M1D, M3D to different subtypes of HBsAg (aa113-135) was determined by the Elisa method. Binding curves were created using PRISM software. As a result as shown in FIGS. 11A and 11B , both M1D and M3D were able to bind most of the HBV subtypes.

Amino acid sequences of different subtypes of HBsAg-aa113-135 Name of Polypeptide SEQ ID NO: the sequence of the polypeptide SEQ13-A 213 STTTSTGPCKTCTTPAQGNSMFP SEQ13-B 168 SSTTSTGPCKTCTTPAQGTSMFP SEQ13-C 214 TSTTSTGPCKTCTIPAQGTSMFP SEQ13-D 215 SSTTSTGPCRTCTTPAQGTSMYP SEQ13-E 216 SSTTSTGPCRTCTTLAQGTSMFP SEQ13-F/H 217 STTTSTGPCKTCTTLAQGTSMFP SEQ13F1 218 STTTSTGPCKTCTALAQGTSMFP SEQ13F2 219 STTTSTGPCRTCTTLAQGTSMLP SEQ13-G 220 SSTTSTGPCKTCTTPAQGNSMYP

Example 10: Affinity Measurement of M1D and M3D

The coating process was carried out on a Biacore 3000 device with 5 μg/ml of HBsAg dissolved in sodium acetate (pH 4.5), and HBsAg was coated on CM5 chips with a coating amount of 2400 RU. The analytes were diluted in a 2-fold gradient from 100 nM to obtain 7 sample concentrations. The affinity measurement procedure was run on a Biacore 3000 instrument, where the flow rate was 50 μl/min, the association time was 90 seconds, the dissociation time was 600 seconds, the sample chamber temperature was 10 °C, and the regeneration solution was 50 mM NaOH , the regeneration flow rate was 50 μl/min, and the regeneration time was 60 seconds. Table 19 shows the affinity of the M1D molecule and the M3D molecule for the antigen HBsAg, 1.06 nM and 1.12 nM, respectively.

Affinity for antigens of M1D and M3D analyte Ka (/Ms) Kd(/s) KD(M) M1D 1.02E06 1.08E03 1.06E-09 M3D 5.27E05 5.89E04 1.12E-09

Example 11: Evaluation of Pharmacokinetics and Early Toxicity of Antibodies M1D and M3D in Cynomolgus Monkeys after Single Intravenous Injection

Six male cynomolgus monkeys that had not undergone any experiments involving macromolecules (>2000 daltons) were divided into 2 groups of 3 animals/group. Among them, a dose of 20 mg/kg of a solution of antibodies M1D and M3D was administered to cynomolgus monkeys of groups 1 and 2, respectively, by intravenous bolus injection. Pharmacokinetic properties and initial toxicity were evaluated in cynomolgus monkeys after a single intravenous injection of antibodies M1D and M3D. The specific experimental design is shown in Table 20.

Experimental design for evaluation of pharmacokinetic properties and early toxicity of M1D and M3D molecules group experimental agent number of animals route of administration Dosage animal code One M1D 3 IV bolus 20 mg/kg C1001,
C1002,
C1003 2 M3D 3 IV bolus 20 mg/kg C2001,
C2002,
C2003

Prior to dosing (0 hours), the health and appearance of the experimental animals were observed twice a day (9:30 am and 4:00 pm). A physical examination of the experimental animals was performed prior to the experiment to confirm the health status of the animals. On the day of administration, the condition of the experimental animal, including general condition, behavior, activity, excretion, respiration, and other abnormal symptoms, was observed before and after each blood sampling time point. The results showed that all animals did not show any abnormal response during and after dosing. In addition, the body weight and body temperature of the experimental animals were also followed after administration. The results are shown in Tables 21 to 24.

Body weight of experimental animals after administration of M1D hour Weight (kg) C1001 C1002 C1003 0 days 4.12 3.92 3.66 7 days 4.02 3.88 3.70 14 days 4.10 3.75 3.80 21 days 4.15 3.81 3.72 28 days 4.01 3.99 3.87

Body temperature of experimental animals after M1D administration hour Body temperature (℃) C1001 C1002 C1003 before administration 37.8 38.0 37.9 1 hours 38.4 37.9 38.4 6 hours 38.6 38.4 38.2 10 hours 38.1 37.8 37.6 24 hours 38.1 38.2 38.4

Body weight of experimental animals after administration of M3D hour Weight (kg) C2001 C2002 C2003 0 days 3.6 3.27 3.6 7 days 3.58 3.19 3.58 14 days 3.73 3.33 3.7 21 days 3.68 3.33 3.7 28 days 3.95 3.39 3.81

Body temperature of experimental animals after administration of M3D hour Body temperature (℃) C2001 C2002 C2003 before administration 38.6 38.7 38.2 1 hours 38.5 38.4 37.7 6 hours 39 39 38.2 10 hours 36.3 36.5 36.7 24 hours 38.2 38.1 37.4

The experimental results showed that the antibodies M1D and M3D did not cause any side effects in the experimental animals after administration, thereby proving good safety of the two antibodies in advance. In addition, about 1.0 ml of whole blood was administered before administration (0). time), 0.25h (15min), 0.5h (30min), 1h, 2h, 4h, 10h, 24h (1 day), 48h (2 days), 72h (3 days), 96h (4 days) after administration , at 144h (6 days), 192h (8 days), 240h (10 days), 336h (14 days), 408h (17 days), 504h (21 days), and 672h (28 days) of experimental animals. was collected from The following analyzes were performed on the collected serum and whole blood.

The concentrations of antibodies M1D and M3D in cynomolgus monkey serum were determined by chemiluminescence immunoassay (CLIA). The lower limit of quantitation (LLOQ) for humanized antibodies M1D and M3D in serum was 0.063 ng/ml and the upper limit of quantitation (ULOQ) was 4.0 ng/ml. The test results are shown in FIGS. 12A and 12B.

FIG. 12A depicts a curve graph for the blood concentration of humanized antibody M1D in the serum of each cynomolgus monkey (group 1) as a function of time after a single intravenous injection of 20 mg/kg humanized antibody M1D.

FIG. 12B depicts a curve graph for the blood concentration of humanized antibody M3D in the serum of each cynomolgus monkey (group 2) as a function of time after a single intravenous injection of 20 mg/kg humanized antibody M3D.

Experimental data of humanized antibodies M1D, M3D were processed using the pharmacokinetic software WinNonlinTM Version 6.2.1 (Pharsight, Mountain View, CA) with an IV bolus infusion model.

The logarithmic linear trapezoidal method was used to calculate the following parameters: initial serum drug concentration (C0), final detectable time (T _last ), elimination half-life (T _1/2 ), apparent distribution volume (V _dss ), total Clearance (CL), mean residence time from time zero to the last time point at which concentrations can be detected (MRT _0-last ), mean residence time from time zero to infinity (MRT _0-inf ), and concentration from time zero detectable area-time curve under serum concentration from time point zero to infinity (AUC ₀ -inf ), and area-time curve under serum concentration from time point zero to infinity (AUC _0-inf ).

The results showed that the CL of male cynomolgus monkeys for humanized antibody M1D was 0.00962±0.00335 ml/min/kg after a single intravenous injection of 20 mg/kg humanized antibody M1D. The mean elimination half-life (t _1/2 ) of the humanized antibody M1D was 81.3±78.7 hours. The Vdss and AUC _0-inf values of the humanized antibody M1D in cynomolgus monkey serum were 0.05±0.0209 L/kg and 38053±14915 μg·h/ml, respectively.

After a single intravenous injection of 20 mg/kg of humanized antibody M3D, the CL of male cynomolgus monkeys for humanized antibody M3D was 0.0049±0.0025 ml/min/kg. The mean elimination half-life (t _1/2 ) of the humanized antibody M3D was 134±94.6 hours. The Vdss and AUC _0-inf values of the humanized antibody M3D in cynomolgus monkey serum were 0.0497±0.0143 L/kg and 79419±33500 μg·h/ml, respectively.

The experimental results are also summarized in Tables 25 and 26.

Key Pharmacokinetic Parameters of Humanized Antibody M1D in Serum of Group 1 Male Cynomolgus Monkeys PK parameters C1001 C1002 C1003 Average IV SD CV (%) Rsq_adj 0.800 0.830 0.581 -- ± -- -- Number of time points used in T _1/2
(No. points) 17.0 17.0 14.0 ND ± -- -- C0 (ng/ml) 593886 520570 359594 491350 ± 119848 24.4 T _1/2 (h) 172 32.8 39.0 81.3 ± 78.7 96.8 Vdss(L/kg) 0.0583 0.0406 0.0472 0.0487 ± 0.00894 18.3 Cl (ml/min/kg) 0.00670 0.0128 0.00969 0.00971 ± 0.00303 31.2 T _last (h) 672 672 672 672 ± -- -- AUC _0-last (ng.h/ml) 49777730 26142490 33521840 36480687 ± 12092238 33.1 AUC _0-inf (ng.h/ml) 49781190 26142960 34385180 36769777 ± 11998175 32.6 MRT _0-last (h) 145 53.1 64.0 87.4 ± 50.3 57.5 MRT _0-inf (h) 145 53.1 81.2 93.2 ± 47.2 50.6 AUC Extra (%) 0.00695 0.00181 2.51 0.840 ± 1.45 172 AUMCExtra(%) 0.0369 0.0245 23.2 7.74 ± 13.3 173

Key Pharmacokinetic Parameters of Humanized Antibody M3D in Serum of Group 2 Male Cynomolgus Monkeys PK parameters C2001 C2002 C2003 average PO SD CV (%) Rsq_adj 0.949 0.889 0.881 -- ± -- -- T _1/2 number of points used in 16.0 16.0 11.0 ND ± -- -- C0 (ng/ml) 740468 562963 674855 659429 ± 89752 13.6 T _1/2 (h) 119 47.2 235 134 ± 94.6 70.7 Vdss(L/kg) 0.0428 0.0401 0.0660 0.0497 ± 0.0143 28.7 Cl (ml/min/kg) 0.00381 0.00782 0.00308 0.00490 ± 0.00255 52.0 T _last (h) 672 672 672 672 ± -- -- AUC _0-last (ng.h/ml) 86427070 42620190 89311120 72786127 ± 26164236 35.9 AUC _0-inf (ng.h/ml) 87449170 42633960 108174500 79419210 ± 33500012 42.2 MRT _0-last (h) 179 85.3 219 161 ± 68.8 42.7 MRT _0-inf (h) 187 85.5 357 210 ± 137 65.4 AUC Extra (%) 1.17 0.0323 17.4 6.21 ± 9.74 157 AUMCExtra(%) 5.27 0.280 49.3 18.3 ± 27.0 148

In addition, routine serum chemistry analysis was performed on the serum of cynomolgus monkeys. Numerous indicators of cynomolgus monkey serum (bilirubin, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, alkaline phosphatase, γ-glutamyltransferase, glucose, urea, creatinine, calcium ion, phosphorus, total cholesterol, triglycerides, sodium ions, potassium ions, chloride ions and globulin levels) showed no apparent abnormalities. The results of these tests indicated that a single intravenous injection of the humanized antibody M1D or M3D was safe under the specified dosage conditions. Accordingly, the humanized antibody M1D or M3D of the present invention can be administered to a subject (eg, a human) to prevent and/or treat HBV infection or a disease associated with HBV infection (eg, hepatitis B).

Example 12: Evaluation of the medicinal properties of M1D and M3D

1. Determination of Isoelectric Points of Humanized Antibodies M1D and M3D

The isoelectric point of the humanized antibodies M1D and M3D was determined by capillary isoelectric point electrophoresis (cIEF). The experimental results are shown in Table 27. The results showed that the humanized antibody M1D had a pI value of 8.8 and M3D had a pI of 9.0.

M1D, M3D isoelectric point isoelectric point (pI) peak area antibody main peak Basic peak (%) Main peak (%) Acid peak (%) M1D 8.8 3.8 64.7 31.5 M3D 9.0 12.1 58.7 29.2

2. Determination of Stability of Humanized Antibodies M1D and M3D Tm values are typically used to describe the stability of antibody molecules. The higher the Tm value, the better the thermal stability of the antibody molecule. Humanized antibodies M1D and M3D were separately dissolved in three buffers, diluted to 1 mg/ml and tested by differential scanning calorimetry (DSC). The initial scan temperature is set to 10℃, the end-point scan temperature is set to 110℃, the scan rate is 200℃/hour, the cooling rate is set to Exp, and the final temperature to be maintained by the equipment is set to 25℃ and the data acquisition frequency was set to 10 seconds, and the pre-injection capillary temperature was set to 30°C. The experimental results are shown in FIGS. 13A and 13B and Tables 28 and 29. The results showed that the humanized antibody M1D had a Tm onset greater than 61.7°C, and that of M3D both exceeded 61.6°C, indicating that M1D and M3D had good thermal stability.

Tm value of M1D buffer Tm onset (°C) Tm1(℃) Tm2(℃) Acetate pH 5.0 61.7 68.9 95.6 Histidine pH6.0 63.8 71.0 96.1 PBS 7.2 65.6 72.9 92.4

Tm value of M3D buffer Tm onset (°C) Tm1(℃) Tm2(℃) Acetate 5.0 61.6 68.8 82.2 Histidine 6.0 62.8 71.1 82.3 PBS7.2 65.7 78.5 /

In addition, the humanized antibodies M1D and M3D were dissolved in histidine buffer at pH 6.0 at a concentration of 60 mg/ml, further dissolved in a phosphate buffer at pH 7.2 at a concentration of 2 mg/ml, and then stored at 40°C, respectively. . Physical and chemical properties of the samples (including appearance, pH, protein concentration, etc.) were monitored at week 0 (T0), week 1 (T1W) and week 2 (T2W). The results are shown in Table 30 and Table 31.

monitoring of appearance

The sample vial was washed and the color and transparency of the sample was observed using a transparency detector (black background and white background). The results in Tables 30 to 31 did not show significant changes in the samples of the two buffers and the humanized antibodies M1D and M3D at 40°C storage conditions.

Monitoring of pH and protein concentration

The absorbance of the sample was measured at A280 and the protein concentration in the sample was calculated using the Beer-Lambert method. In addition, the pH of the sample was measured using a pH meter, the measurement was repeated twice, and the average value was considered as the final result. The experimental results are shown in Tables 30 and 31 above. The results showed that the humanized antibodies M1D and M3D had no significant changes in pH and protein concentration after storage for 2 weeks at 40°C in different buffers.

SEC-HPLC test

Samples of humanized antibodies M1D and M3D subjected to different storage conditions were run on an Agilent 1260 Infinity, TSK G3000SWXL gel column (5 μm, 7.8 mm×300 mm), where the mobile phase was 50 mM PB+300 mM NaCl, pH 7.0±0.2 ; flow rate was 1.0 ml/min; detection wavelength was 280 nm; sample concentration was 10 mg/ml; injection volume was 10 μl). The experimental results are shown in Tables 30 and 31 above. The results showed that under different storage conditions (different buffers, different storage times) the main peaks of samples containing humanized antibodies M1D and M3D both exceeded 95%. This indicated that the humanized antibodies M1D and M3D were stable.

cIEF exam

Samples were analyzed by capillary electrophoresis. The results showed that the main peak of cIEF decreased by 6.0% after storage of the M1D molecule in histidine buffer at 40° C. for 2 weeks, whereas the main peak decreased by 24.9% after storage for 2 weeks in PBS buffer. For M3D, the cIEF main peak decreased by 13.7% after storage in histidine buffer for 2 weeks at 40°C, whereas the main peak in PBS buffer decreased by 30.6% after 2 weeks storage at 40°C.

3. Solubility Analysis of Humanized Antibody M1D and M3D Variants

Humanized antibodies M1D and M3D were separately dissolved in 25 mM histidine solution (pH 6.0) containing 5% sucrose and 0.02% PS80 at a concentration of 60 mg/ml, and the liquid level of the sample solution was increased with increasing centrifugation time. Concentrate by centrifugal ultrafiltration (centrifugation temperature was 5° C., speed was 4850 ppm) until they no longer fell significantly together. Samples were carefully collected with a pipette, at which point the properties of the samples were observed. The results showed that the solution sample was yellow.

1000 μl of the solution sample was then pipetted, transferred to a 1.5 ml EP tube, and centrifuged at 10,000 rpm for 20 minutes. The centrifugation result showed that there was no stratification and sedimentation in the sample and the liquid was clear. After centrifugation, the upper and lower layers of the sample were carefully pipetted with a pipette, and the protein concentration was measured by UV280. The result is that the solubility of M1D is 171 mg/ml; The solubility of M3D was shown to be 170 mg/ml. Additionally, the viscosities of humanized antibody M1D and M3D variants separately dissolved in 25 mM histidine solution (pH 6.0) containing 5% sucrose and 0.02% PS80 were measured. In these buffer systems, the viscosity of the humanized antibody M1D at a concentration of 171 mg/ml is 13.2 cp (1 cP = 1 mPa.s), whereas M3D has a viscosity of 19.8 cp at a concentration of 170 mg/ml and 100 mg/ml. It had a viscosity of 11.5 cp in ml.

While specific embodiments of the present invention have been described in detail, those skilled in the art will recognize that many modifications and variations in detail are possible in light of the overall teachings of the disclosure, and that such variations are within the scope of the present invention. The entire invention is provided by the appended claims and any equivalents thereof.

SEQUENCE LISTING <110> XIAMEN UNIVERSITY YANG SHENG TANG COMPANY, LTD. <120> Antibodies for treatment of hepatitis B infection and related diseases <130> IEC170022PKR <150> CN 201710223992.8 <151> 2017-04-07 <160> 220 <170> PatentIn version 3.5 <210> 1 <211> 122 <212 > PRT <213> Artificial Sequence <220> <223> M1-23 heavy chain variable region <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Ile Leu Ser Val Thr Cys Ser Val Ser Gly Gly Ser Ile Thr Ser Asn 20 25 30 Phe Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Gly Ser Gly Thr Tyr Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Arg Ser Arg Val Thr Leu Ser Val Asp Thr Ser Lys Asn Gln Leu Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Leu Arg Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> M1-23 light chain variab le region <400> 2 Glu Ile Val Met 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 Thr Gln Asp Ile Ser Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45 Tyr Tyr Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Thr Thr Tyr Tyr Cys Gln Gln Tyr His Ser Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3-23 heavy chain variable region <400> 3 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Val Ser Ile Ser Ser Pro 20 25 30 Tyr Asp Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Arg Ile His Gly Asn Gly Gly Ser Thr Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Asn Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Ile Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Asp Glu Thr Trp Gly Gln Gly Val Leu Val Thr Val Ser 100 105 110 Ser <210 > 4 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3-23/M3-13 light chain variable region <400> 4 Asp Ile Gln Met Ser Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Arg Gly Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 5 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> M3-13 heavy chain variable region <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Arg Leu Val Lys Ser Ser Glu 1 5 10 15 Thr Leu Pro Leu Thr Cys Ala Val Ser Gly Ala Ser Asn Ser Ser Asn 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Gly Asn Ser Gly Ser Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Ser Ile Ser Thr Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ser Val Tyr Thr Tyr Ser Trp Gly Gly Gly Val Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 6 <211 > 8 <212> PRT <213> Artificial Sequence <220> <223> M1-23 HCDR1 <400> 6 Gly Gly Ser Ile Thr Ser Asn Phe 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> M1-23 HCDR2 <400> 7 Ile Ser Gly Ser Gly Thr Tyr Thr 1 5 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> M1 -23 HCDR3 <400> 8 Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp Phe 1 5 10 15 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> M1-23 LCDR1 <400> 9 Gln Asp Ile Ser Ser Ser 1 5 <210> 10 <211 > 3 <212> PRT <213> Artificial Sequence <220> <223> M1-23 LCDR2 <400> 10 Tyr Ala Asn 1 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> < 223> M1-23 LCDR3 <400> 11 Gln Gln Tyr His Ser Leu Pro Leu Thr 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> M3-23 HCDR1 <400 > 12 Gly Val Ser Ile Ser Ser Pro Tyr Asp Trp 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> M3-23 HCDR2 <400> 13 Ile His Gly Asn Gly Gly Ser Thr 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> M3-23 HCDR3 <400> 14 Val Arg Asp Glu Thr 1 5 <210> 15 <211 > 12 <212> PRT <213> Artificial Sequence <220> <223> M3-23/M3-13 LCDR1 <400> 15 Gln Ser Leu Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 16 <211 > 3 <212> PRT <213> Ar tificial Sequence <220> <223> M3-23/M3-13 LCDR2 <400> 16 Trp Ala Ser 1 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> M3-23 /M3-13 LCDR3 <400> 17 Gln Gln His Tyr Thr Thr Pro Leu Thr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> M3-13 HCDR1 <400> 18 Gly Ala Ser Asn Ser Ser Asn Tyr 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> M3-13 HCDR2 <400> 19 Ile Tyr Gly Asn Ser Gly Ser Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> M3-13 HCDR3 <400> 20 Ala Arg Gly Ser Val Tyr Thr Tyr Ser 1 5 <210> 21 <211 > 25 <212> PRT <213> Artificial Sequence <220> <223> M1-23 HFR1 <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Ile Leu Ser Val Thr Cys Ser Val Ser 20 25 <210> 22 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> M1-23/M1D HFR2 <400> 22 Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 15 Tyr <210> 23 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> M1-23 HFR3 <400> 23 Asp Tyr Asn Pro Ser Leu Arg Ser Arg Val Thr Leu Ser Val Asp Thr 1 5 10 15 Ser Lys Asn Gln Leu Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220 > <223> M1-23 HFR4 <400> 24 Trp Gly Gln Gly Leu Arg Val Thr Val Ser 1 5 10 <210> 25 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> M1 -23 LFR1 <400> 25 Glu Ile Val Met 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 Thr 20 25 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> M1-23 LFR2 <400> 26 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile 1 5 10 15 Tyr <210> 27 <211> 36 < 212> PRT <213> Artificial Sequence <220> <223> M1-23 LFR3 <400> 27 Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Thr 20 25 30 Thr Tyr Tyr Cys 35 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223 > M1-23/M1D LFR4 <400> 28 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 29 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> M3-23 HFR1 <400> 29 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser 20 25 <210> 30 <211> 16 <212> PRT <213 > Artificial Sequence <220> <223> M3-23 HFR2 <400> 30 Thr Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile Gly Arg 1 5 10 15 <210> 31 <211> 38 <212> PRT < 213> Artificial Sequence <220> <223> M3-23 HFR3 <400> 31 Ser Tyr Asn Pro Ser Leu Lys Asn Arg Val Thr Ile Ser Lys Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Ile Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> M3- 23/M3-13 HFR4 <400> 32 Trp Gly Gln Gly Val Leu Val Thr Val Ser Ser 1 5 10 <210> 33 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> M3-23 LFR1 <400> 33 Asp Ile Gln Met Ser Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Asn Cys Lys Ser Ser 20 25 <210> 34 <211> 17 <212> PRT < 213> Artificial Sequence <220> <223> M3-23 LFR2 <400> 34 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Phe 1 5 10 15 Tyr <210> 35 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> M3-23 LFR3 <400> 35 Thr Arg Glu Ser Gly Val Pro Asn Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Arg Gly Leu Gln Ala Glu Asp Val Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> M3-23/M3D LFR4 <400> 36 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 37 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> M3-13 HFR1 <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Arg Leu Val Lys Ser Ser Glu 1 5 10 15 Thr Leu Pro Leu Thr Cys Ala Val Ser 20 25 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223 > M3-13 HFR2 <400> 38 Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 15 Arg <210> 39 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> M3-13 HFR3 <400> 39 Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Ser Ile Ser Thr Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Phe Cys 35 <210> 40 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> IGHV4-4*08 <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg <210> 41 <211> 95 <212> PRT <213> Artificial Sequence <220 > <223> IGKV1-39*01 <400> 41 Asp Ile Gln Met Thr Gln Ser Pro 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 85 90 95 <210> 42 <211> 98 <212> PRT <213> Artificial Sequence <220> <223 > IGHV4-4*02 <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 43 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> IGKV4-1*01 <400> 43 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro 100 <210> 44 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> M1D HFR1 <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 45 <211 > 38 <212> PRT <213> Artificial Sequence <220> <223> M1D HFR3 <400> 45 Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> M1D HFR4 <400> 46 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 47 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> M1D LFR1 <400> 47 Asp Ile Gln Met 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 Thr 20 25 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> M1D LFR2 <400 > 48 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 49 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> M1D LFR3 <400 > 49 Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Il e Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 50 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> M3D HFR1 <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser 20 25 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223 > M3D HFR2 <400> 51 Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Arg 1 5 10 15 <210> 52 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> M3D HFR3 <400> 52 Ser Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 53 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> M3D HFR4 <400> 53 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 54 <211 > 26 <212> PRT <213> Artificial Sequence <220> <223> M3D LFR1 <400> 54 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser 20 25 <210> 55 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> M3D LFR2 <400> 55 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Phe 1 5 10 15 Tyr <210> 56 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> M3D LFR3 <400> 56 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 57 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> M1D heavy chain variable region <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Asn 20 25 30 Phe Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Gly Ser Gly Thr Tyr Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Ph e Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 58 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> M1D light chain variable region <400> 58 Asp Ile Gln Met 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 Thr Gln Asp Ile Ser Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Asn Arg 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 Tyr His Ser Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 59 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3D heavy chain variable region <400> 59 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Val Ser Ile Ser Ser Pro 20 25 30 Tyr Asp Trp Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Arg Ile His Gly Asn Gly Gly Ser Thr Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Asp Glu Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 60 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3D light chain variable region <400> 60 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Thr Thr Pro Leu Thr P he Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 ggacagcckg gaaggtgtgc 20 <210> 62 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 gaggcagttc cagattcaa 20 <210> 63 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 63 ccgcgtactt gttgttgctc tgt 23 <210> 64 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 agtagcaact gcaaccggtg tacattctca ggtgcagctg caggagtcag gcccag 56 <210> 65 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 cagagtcag gcccaggact ggtgaagcct tcggagccc tgtccctcac ctgcac 56 <210> 66 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 aagccttcgg agaccctgtc cctcacctgc actgtctctg gtggctccat c 51 <210> 67 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 gttcttggac gtgtctacgg atatggtgac tcgactcttg agggaggggt tg 68 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 tccgtagaca cgtccaagaa ccagttctcc ctgaaactga gctc 44 <210> 69 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 gatgggccct tggtcgacgc tgaagagacg gtgacggtgg tcccttggcc ccagaaatc 59 <210> 70 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 agtagcaact gcaaccctg cat cctactt ga <210> 71 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 cagcaaaaac cggggaaagc ccctaagctc ctgatctatt atgc 44 <210> 72 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 gaaccttgat gggaccccac tttgcaaacg atttgcataa tagatcagga gc 52 <210> 73 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 ctgtcccaga tccactgcca ctgaaccttg atgggacccc 40 <210> 74 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcaacct gagg 54 <210> 75 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 gcagcctgca acctgaggat tttgcaactt attactgtca ac 42 <210> 76 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 gaccaaggtg gaaatcaaac gtacggtggc tgcaccat 38 <210> 77 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 agtagcaact gcaaccggtg tacattctca ggtgtgcagctg caggagtcgg <211> <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 attcccgggt agatgcccag taaaagagca gcttaggagg ctgtcctggt ttctgctg 58 <210> 79 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 ggtgtctcca tcagcagtcc ttatgactgg acctgggtcc gccagccccc agggaag 57 <210> 80 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 aactggttgact tggacttgtc tactgaatt tgacttgagg ga ggacttgtc tactgaatt gtgactc> 81 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 agacaagtcc aagaaccagt tctccctgaa gctgagctct gtgaccgccg c 51 <210> 82 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 gaaacctggg gccagggaac cctggtcacc gtctcctcag cgtcgaccaa gggcccatc 59 <210> 83 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 agtagcaact gcaaccggtg tacattctga catcgtgatg acccagtctc cagactcc 58 <210> 84 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 cccagtctcc agactccctg gctgtgtctct 60 tgggagagag < 210> 85 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 cagcagaaac caggacagcc tcctaagctg ctcttttact gggcatctac ccgggaat 58 <210> 86 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 gtgaaatctg tcccagatcc actgccactg aatcggtcag ggaccccgga ttcccgggta 60 gatgcc 66 <210> 87 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 ggcagtggat ctgggacaga tttcactctc accatcagca gcctgcaggc tgaagatg 58 <210> 88 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 atggtgcagc caccgtac gt ttgatttcca ccttggtcc 39 <210> 89 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 agtagcaact gcaaccggtg tacattctca ggtgcagctg caggagtcag gc 52 <210> 90 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 gatgggccct tggtcgacgc tgaagagacg gtgac 35 <210> 91 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 agtagcaact gcaaccggtg tacattctga catacagatg acgcagtc 48 <210> 92 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 atggtgcagc caccgtacgt ttgatttcca ccttggtccc 40 <210> 93 <211> DNA <213> Artificial Sequence <220> <223> primer <400> 93 gtagtaacta ctgatggagc caccagagac agtgcaggtg ag 42 <210> 94 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 ggtggctcca tcagtagtta ctactggagc tggatccgcc ag 42 <210> 95 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 atagtaacta ccactgctga cggagccacc agagacagtg caggtgag 48 <210> 96 <211> 48 212> DNA <213> Art ificial Sequence <220> <223> primer <400> 96 ggtggctccg tcagcagtgg tagttactat tggagctgga tccgccag 48 <210> 97 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 ggtgctccca ctggtataga tatacccaat ccactccag 39 <210> 98 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 atctatacca gtgggagcac cgactacaac ccctccctc 39 <210> 99 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 99 tgtgtaacta ctactactac taatataccc aatccactcc ag 42 <210> 100 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 attagtagta gtagtagtta cacagactac aacccctccc tc 42 <210> 101 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 gtagctgctg ctaacactct gagttgcccg gcaagtgatg g 41 <210> 102 <211> 37 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 102 cagagtgtta gcagcagcta cttaaattgg tatcagc 37 <210> 103 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 103 agtattactt ccgatgttgg agctatttgc ccggcaagtg atgg 44 <210> 104 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 104 agctccaaca tcggaagtaa tactttaaat tggtatcagc 40 <210> 105 <211> 45 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 105 gaccccactt tgcaaacggg atgcagcata gatcaggagc ttagg 45 <210> 106 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 106 cctaagctcc tgatctatgc tgcatcccgt ttgcaaagtg gggtc 45 <210> 107 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 107 gaccccactt tgcaaacgag acgccttata gatcaggagc ttagg 45 <210> 108 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 108 cctaagctcc tgatctataa ggcgtctcgt ttgcaaagtg gggtc 45 <210> 109 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 109 agtgagaggg taattgtaat cttgtagaca gtaataagtt gcaaaatc 48 <210> 110 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 110 ctacaagatt acaattaccc tctcactttc ggcggaggga ccaag 45 <210> < 111 <211> 48 > DNA <213> Artificial Sequence <220> <223> primer <400> 111 agtgagaggt aaactactac tctgatgaca gtaataagtt gcaaaatc 48 <210> 112 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 112 catcagagta gtagtttacc tctcactttc ggcggaggga ccaag 45 <210> 113 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 113 agtagcaact gcaaccggtg tacattctca ggtgcagctg caggagtcg 38 <210> 212> DNA <213> Artificial Sequence <220> <223> primer <400> 114 gatgggccct tggtcgacgc tgaggagacg gtgaccag 38 <210> 115 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400 > 115 agtagcaact gcaaccggtg tacattctga catcgtgatg acccagtctc 50 <210> 116 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 116 atggtgcagc caccgtacgt ttgatttcca ccttggtc <211> 54 < 117 212> DNA <213> Artificial Sequence <220> <223> primer <400> 117 ggagtaacca ccactgctga tggagccacc agagacagcg caggtgaggg acag 54 <210> 118 <211> 48 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 118 ggtggctcca tcagcagtgg tggttactcc acctgggtcc gccagccc 48 <210> 119 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 119 ccagggtacta ctgctgatgg agccaccaga gacagcgcag 51 <210> 120 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 120 ggtggctcca tcagcagtag taactggacc tgggtccgcc agccc 45 <210> 121 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 121 ggtgctccca ctatgataga ttcgtccaat ccactccag 39 <210> 122 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 122 atctatcata gtgggagcac cagctacaac ccctccctca ag 42 <210> 123 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 123 tgtgctacca ccactaccac taattcgtcc aatccactcc ag 42 <210> 124 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 124 attagtggta gtggtggtag cacaagctac aacccctccc tcaag 45 <210> 125 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 125 ataggtcttt ccatcact at gcaggaggct ctggctggac ttgcagttga tggtggc 57 <210> 126 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 126 cagagcctcc tgcatagtga tggaaagacc tatttagcct ggtaccagca 127 <211> 45 <210> 212> DNA <213> Artificial Sequence <220> <223> primer <400> 127 gtagctgctg ctaacactct ggctggactt gcagttgatg gtggc 45 <210> 128 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 128 cagagtgtta gcagcagcta cttagcctgg taccagcaga aac 43 <210> 129 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 129 gggaccccgg attcccgggt agaacccaag taaaagagca gcttaggagg 50 <210> 130 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 130 cctcctaagc tgctctttta cttgggttct acccgggaat ccggggtccc 50 <210> 131 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 131 gggaccccgg attcccgggt ggatgcaccg taaaagagca gcttaggagg 50 <210> 132 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 132 cctcctaagc tgctctttta c ggtgcatcc acccgggaat ccggggtccc 50 <210> 133 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 133 cgtccaagga gtactataat attgctgaca gtaatacact gccacatc 48 <210> 134 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 134 cagcaatatt atagtactcc ttggacgttc ggccaaggga ccaag 45 <210> 135 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 135 cgtccaagga gtttgtagag cttgcataca gtaatacact gccacatc 48 <210> 136 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 136 atgcaagctc tacaaactcc ttggacgttc ggccaaggga ccaag 45 <210> 137 > PRT <213> Artificial Sequence <220> <223> CDR-A (IGHV4-4*08) for M1D HCDR-1 <400> 137 Gly Gly Ser Ile Ser Ser Ser Tyr Tyr 1 5 <210> 138 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGHV4-61*01) for M1D HCDR-1 <400> 138 Gly Gly Ser Val Ser Ser Gly Ser Tyr Tyr 1 5 10 <210> 139 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV1 -39*01) for M1D LCDR-2 <400> 139 Ala Ala Ser 1 <210> 140 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGKV1-5*03 ) for M1D LCDR-2 <400> 140 Lys Ala Ser 1 <210> 141 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGHV4-4*02) for M3D HCDR -2 <400> 141 Ile Tyr His Ser Gly Ser Thr 1 5 <210> 142 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGHV3-23*02) for M3D HCDR-2 <400> 142 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 143 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV2-28*01) for M3D LCDR-2 <400> 143 Leu Gly Ser 1 <210> 144 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGKV3-15*01) for M3D LCDR- 2 <400> 144 Gly Ala Ser 1 <210> 145 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGHV4-4*08) for M1D HCDR-2 <400> 145 Ile Tyr Thr Ser Gly Ser Thr 1 5 <210> 146 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGHV3-11*03) for M1D HCDR-2 <400 > 146 Ile Ser Ser Ser Ser Ser Tyr Thr 1 5 <210> 147 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV3D-7*01) for M1D LCDR-1 < 400> 147 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 148 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGLV1-44*01) for M1D LCDR-1 <400> 148 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 149 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV1-6*01) for M1D LCDR -3 <400> 149 Leu Gln Asp Tyr Asn Tyr Pro Leu Thr 1 5 <210> 150 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV6-21*02) for M1D LCDR-3 <400> 150 His Gln Ser Ser Ser Leu Pro Leu Thr 1 5 <210> 151 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGHV4-30 -4*07) for M3D HCDR-1 <400> 151 Gly Gly Ser Ile Ser Ser Gly Gly Tyr Ser 1 5 10 <210> 152 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGHV4-4*01) for M3D HCDR-1 <400> 152 Gly Gly Ser I le Ser Ser Ser Asn Trp 1 5 <210> 153 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV2-29*02) for M3D LCDR-1 <400> 153 Gln Ser Leu Leu His Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 154 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGKV3D-7*01) for M3D LCDR -1 <400> 154 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 155 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV4-1*01) for M3D LCDR-3 <400> 155 Gln Gln Tyr Tyr Ser Thr Pro Trp Thr 1 5 <210> 156 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-B (IGKV2-28*01 ) for M3D LCDR-3 <400> 156 Met Gln Ala Leu Gln Thr Pro Trp Thr 1 5 <210> 157 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> M1D (HCDR1-A) heavy chain variable region <400> 157 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Gly Ser Gly Thr Tyr Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 158 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> M1D (HCDR1-B) heavy chain variable region <400> 158 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30 Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Ser Gly Ser Gly Thr Tyr Thr Asp Tyr Asn Pro 50 55 60 Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 65 70 75 80 Phe Ser Leu Lys Leu Ser Ser Ser Val Thr Ala Ala A sp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp 100 105 110 Phe Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 159 <211> 107 < 212> PRT <213> Artificial Sequence <220> <223> M1D (LCDR2-A) light chain variable region <400> 159 Asp Ile Gln Met 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 Thr Gln Asp Ile Ser Ser Ser 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 Arg 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 Tyr His Ser Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 < 210> 160 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> M1D (LCDR2-B) light chain variable region <400> 160 Asp Ile Gln Met 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 Thr Gln Asp Ile Ser Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Se r 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 Tyr His Ser Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 161 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> M1D (HCDR2-B) heavy chain variable region <400> 161 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Asn 20 25 30 Phe Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Asp Tyr Gly Ser Asn Asp Tyr Ala Phe Asp Phe Trp 100 105 110 Gly Gly Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 162 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> M1D (LCDR1-B) light chain variable region <400> 162 Asp Ile Gln Met 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 Thr Ser Ser Asn Ile Gly Ser 20 25 30 Asn Thr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Tyr Ala Asn Arg Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Leu 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 163 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> M3D (HCDR2-A) heavy chain variable region <400> 163 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Val Ser Ile Ser Ser Pro 20 25 30 Tyr Asp Trp Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Arg Il e Tyr Thr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Glu Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 164 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3D (HCDR2 -B) heavy chain variable region <400> 164 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Val Ser Ile Ser Ser Pro 20 25 30 Tyr Asp Trp Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Arg Ile Ser Gly Ser Gly Gly Ser Thr Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Asp Glu Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 165 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3D (LCDR2-A) light chain variable region <400> 165 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Phe Tyr Leu Gly Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 166 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> M3D (LCDR2 -B) light chain variable region <400> 166 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Lys Leu Leu Phe Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 167 <211> 112 <212 > PRT <213> Artificial Sequence <220> <223> M3D (LCDR1-A) light chain variable region <400> 167 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His 85 90 95 Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 168 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HBsAg-aa113-135 (SEQ13-B) <400> 168 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 169 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-S117A < 400> 169 Ser Ser Thr Thr Ala Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 170 <211> 23 <212> PRT <213> Artificial Sequence <220 > <223> S13-T118A <400> 170 Ser Ser Thr Thr Ser Ala Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 171 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-G119A <400> 171 Ser Ser Thr Thr Ser Thr Ala Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 172 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-P120A <400> 172 Ser Ser T hr Thr Ser Thr Gly Ala Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 173 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13 -C121S <400> 173 Ser Ser Thr Thr Ser Thr Gly Pro Ser Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 174 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-K122A <400> 174 Ser Ser Thr Thr Ser Thr Gly Pro Cys Ala Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 175 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-T123A <400> 175 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Ala Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 176 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-C124S <400> 176 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Ser Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 177 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-T125A <400> 177 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Ala Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 178 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-T126A <400> 178 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ala Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 179 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-P127A <400> 179 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Ala Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 180 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-Q129A <400> 180 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Ala Gly Thr Ser Met Phe Pro 20 <210> 181 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> S13-T131A <400> 181 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Ala Ser Met Phe Pro 20 <210> 182 <211> 23 <212> PRT <213> A rtificial Sequence <220> <223> S13-S132A <400> 182 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ala Met Phe Pro 20 <210> 183 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 183 atcaactacc gccacgggac catgcaagac ctgcac 36 <210> 184 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 184 ggtcccgtgg cggtagttga tgttcctgga agtaga 36 <210> 185 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 185 aactaccagc gcgggaccat gcaagacctg cacgat 36 <210> 186 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 186 catggtcccg cgctggtagt tgatgttcct ggaagt 36 <210> 187 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 187 accagcacgg caccatgcaa gacctgcacg attcct 36 <210> 188 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 188 cttgcatggt gccgtgctgg tagttgatgt tcctgg 36 <210> 189 <211> 36 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 189 cagcacggga gcatgcaaga cctgcacgat tcctgc 36 <210> 190 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 190 gtcttgcatg ctcccgtgct ggtagttgat gttcct 36 <210> 191 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 191 aggggaccat ccaagacctg cacgattcct gctcaa 36 <210> 192 <211> 36 <212> DNA <213 > Artificial Sequence <220> <223> p rimer <400> 192 gcaggtcttg gatggtcccg tgctggtagt tgatgt 36 <210> 193 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 193 gggaccatgc cggacctgca cgattcctgc tcaag < 36 <210> 194 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 194 gtgcaggtcc ggcatggtcc cgtgctggta gttgat 36 <210> 195 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 195 gggaccatgc gcgacctgca cgattcctgc tcaagg 36 <210> 196 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 196 gtgcaggtcg cgcatggtcc cgtgctggta gttgat 36 <210> 197 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 197 accatgcaag gcctgcacga ttcctgctca aggaac 36 <210> 198 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 198 atcgtgcagg ccttgcatgg tcccgtgctg gtagtt 36 <210> 199 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 199 tgcaagacct ccacgattcc tgctcaagga acctct 36 <210> 200 <211> 36 < 212> DNA <213> Artificial Sequence ce <220> <223> primer <400> 200 aggaatcgtg gaggtcttgc atggtcccgt gctggt 36 <210> 201 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 201 caagacctgc gcgattcctg ctcaaggaac ctctat 36 <210> 202 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 202 gcaggaatcg cgcaggtctt gcatggtccc gtgctg 36 <210> 203 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 203 gacctgcacg gctcctgctc aaggaacctc tatgtt 36 <210> 204 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 204 tgagcaggag ccgtgcaggt cttgcatggt cccgtg 36 < 210> 205 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 205 ctgcacgatt gctgctcaag gaacctctat gtttcc 36 <210> 206 <211> 36 <212> DNA <213> Artificial Sequence < 220> <223> primer <400> 206 ccttgagcag caatcgtgca ggtcttgcat ggtccc 36 <210> 207 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 207 gattcctgct gcaggaacct ctatgtttcc ctcttg 36 <210 > 208 <211> 36 <212> DNA <213> A rtificial Sequence <220> <223> primer <400> 208 gaggttcctg cagcaggaat cgtgcaggtc ttgcat 36 <210> 209 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 209 tgctcaagga gcctctatgt ttccctctg t 36 <210> 210 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 210 aacatagagg ctccttgagc aggaatcgtg caggtc 36 <210> 211 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 211 tcaaggaacc gctatgtttc cctcttgttg ctgtac 36 <210> 212 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 212 ggaaacatag cggttccttg agcaggaatc gtgcag 36 <210> 213 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-A <400> 213 Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Asn Ser Met Phe Pro 20 <210> 214 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-C <400> 214 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 215 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-D <400> 215 Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala 1 5 10 15 Gln Gly Thr Ser Met Tyr Pro 20 <210> 216 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-E <400> 216 Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Thr Thr Leu Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 217 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-F/H <400> 217 Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Leu Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 218 <211> 23 <212> PRT <213> Artificial Sequence <220> <223 > SEQ13F1 <400> 218 Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ala Leu Ala 1 5 10 15 Gln Gly Thr Ser Met Phe Pro 20 <210> 219 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13F2 <400> 219 Ser Thr Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Thr Thr Leu Ala 1 5 10 15 Gl n Gly Thr Ser Met Leu Pro 20 <210> 220 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SEQ13-G <400> 220 Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 1 5 10 15Gln Gly Asn Ser Met Tyr Pro 20

Claims (17)

An antibody or antigen-binding fragment thereof capable of specifically binding to HBsAg, comprising:
The antibody or antigen-binding fragment is
(a) (i) VH CDR2 consisting of a sequence selected from among SEQ ID NO: 7, or a sequence different from SEQ ID NO: 7 by one conservative amino acid substitution with alanine, said conservative amino acid substitution according to the Kabat numbering system for heavy chains which does not occur at position 56;
(ii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 8, or a sequence different from SEQ ID NO: 8 by one conservative amino acid substitution with alanine, said conservative amino acid substitution being 97, 100c according to the Kabat numbering system for heavy chains and not occurring at the 100d position; and
(iii) a VH CDR 1 consisting of a sequence selected from: a) SEQ ID NO: 6; or b) a sequence of VH CDR1 contained in the amino acid sequence encoded by a human heavy chain germline gene, wherein the human heavy chain germline gene is selected from the group consisting of IGHV4-4 ^* 08 and IGHV4-61 ^* 01. One;
a heavy chain variable region (VH) comprising; and
(b) (iv) a VL CDR1 consisting of a sequence selected from SEQ ID NO: 9, or a sequence different from SEQ ID NO: 9 by one conservative amino acid substitution with alanine;
(v) VL CDR3 consisting of SEQ ID NO: 11, or a sequence selected from a sequence different from SEQ ID NO: 11 by one conservative amino acid substitution with alanine, said conservative amino acid substitutions being 94 and 96 according to the Kabat numbering system for light chains which does not occur at the burn position; and
(vi) a VL CDR 2 consisting of a sequence selected from: a) SEQ ID NO: 10; or b) a sequence of VL CDR2 contained in the amino acid sequence encoded by a human light chain germline gene, wherein the human light chain germline gene is selected from the group consisting of IGKV1-39 ^* 01 and IGKV1-5 ^* 03. 2 ;
A light chain variable region (VL) comprising
includes,
wherein one conservative amino acid substitution with alanine is no more than one in the entire CDR. The method of claim 1,
An antibody or antigen thereof, wherein the VH comprises a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and a VH CDR1 having the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 137 or SEQ ID NO: 138- binding fragment. The method of claim 1,
vl
VL CDR1 as set forth in SEQ ID NO: 9,
VL CDR3 as set forth in SEQ ID NO: 11, and
VL CDR2 having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 139 or SEQ ID NO: 140
comprising, an antibody or antigen-binding fragment thereof. According to claim 1,
(i) a VH CDR2 as set forth in SEQ ID NO:7; VH CDR3 as set forth in SEQ ID NO:8; and VH CDR1 as defined in claim 1 ; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR3 as set forth in SEQ ID NO: 11, and VL CDR2 as set forth in SEQ ID NO: 10; or
(ii) a VH CDR2 as set forth in SEQ ID NO:7; VH CDR3 as set forth in SEQ ID NO:8; and VH CDR1 as set forth in SEQ ID NO:6; and a VL CDR1 as set forth in SEQ ID NO: 9, a VL CDR3 as set forth in SEQ ID NO: 11, and a VL CDR2 as defined in claim 1 .
comprising, an antibody or antigen-binding fragment thereof. The method of claim 1,
(1) VH CDR1 as set forth in SEQ ID NO: 6, VH CDR2 as set forth in SEQ ID NO: 7, and VH CDR3 as set forth in SEQ ID NO: 8; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR2 as set forth in SEQ ID NO: 10, and VL CDR3 as set forth in SEQ ID NO: 11;
(2) a VH CDR1 as set forth in SEQ ID NO: 137, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR2 as set forth in SEQ ID NO: 10, and VL CDR3 as set forth in SEQ ID NO: 11;
(3) a VH CDR1 as set forth in SEQ ID NO: 138, a VH CDR2 as set forth in SEQ ID NO: 7, and a VH CDR3 as set forth in SEQ ID NO: 8; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR2 as set forth in SEQ ID NO: 10, and VL CDR3 as set forth in SEQ ID NO: 11;
(4) VH CDR1 as set forth in SEQ ID NO: 6, VH CDR2 as set forth in SEQ ID NO: 7, and VH CDR3 as set forth in SEQ ID NO: 8; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR2 as set forth in SEQ ID NO: 139, and VL CDR3 as set forth in SEQ ID NO: 11; or
(5) VH CDR1 as set forth in SEQ ID NO: 6, VH CDR2 as set forth in SEQ ID NO: 7, and VH CDR3 as set forth in SEQ ID NO: 8; and VL CDR1 as set forth in SEQ ID NO: 9, VL CDR2 as set forth in SEQ ID NO: 140, and VL CDR3 as set forth in SEQ ID NO: 11
An antibody or antigen-binding fragment thereof comprising a. According to claim 1,
An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is humanized. According to claim 1,
Antibody
(1) VH as set forth in SEQ ID NO: 1 and VL as set forth in SEQ ID NO: 2;
(2) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 58;
(3) VH as set forth in SEQ ID NO: 157 and VL as set forth in SEQ ID NO: 58;
(4) VH as set forth in SEQ ID NO: 158 and VL as set forth in SEQ ID NO: 58;
(5) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 159; or
(6) VH as set forth in SEQ ID NO: 57 and VL as set forth in SEQ ID NO: 160
An antibody or antigen-binding fragment thereof comprising a. According to claim 1,
wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, Fab, Fab', (Fab') ₂ , Fv fragment, diabody, bispecific antibody, multispecific antibody, chimeric antibody, and humanized antibody Antibodies or antigen-binding fragments thereof. According to claim 1,
An antibody or antigen-binding fragment thereof, wherein the antibody is of the IgG class. According to claim 1,
An antibody or antigen-binding fragment thereof, wherein the antibody is of class IgG1, IgG2, IgG3 or IgG4. According to claim 1,
An antibody or antigen-binding fragment thereof capable of specifically binding to HBsAg, neutralizing toxicity of HBV, and/or reducing serum levels of HBV DNA and/or HBsAg in a subject. According to claim 1,
An antibody or antigen-binding fragment thereof labeled with a detectable label. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or a heavy chain variable region and/or a light chain variable region thereof. A vector comprising the isolated nucleic acid molecule according to claim 13 . A host cell comprising an isolated nucleic acid molecule according to claim 13 or a vector comprising said isolated nucleic acid molecule. 13. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or a heavy chain variable region and/or a light chain variable region thereof, or a vector comprising said isolated nucleic acid molecule culturing the host cell under conditions permissive for expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell;
A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. A pharmaceutical for preventing or treating HBV infection or a disease associated with HBV infection in a subject comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier and/or excipient composition.

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