KR20200010217A - Antibodies for the treatment of hepatitis B infections and related diseases - Google Patents

Abstract

Provided are antibodies against hepatitis B surface antigen, coding nucleic acids and preparations thereof, and pharmaceutical compositions comprising the antibodies. Also provided are the use of 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 the toxicity of HBV in the body of a subject (eg, humans), and HBV DNA And / or reduce serum levels of HBsAg.

Description

Antibodies for the treatment of hepatitis B infections and related diseases

The present invention relates to the field of molecular virology and immunology, in particular to the treatment of hepatitis B virus (HBV) infection. In particular, the present invention relates to antibodies against hepatitis B surface antigen (HBsAg), nucleic acid molecules encoding them, methods for their preparation, and pharmaceutical compositions containing the same. The pharmaceutical composition may be used for the prevention and / or treatment of HBV infection or a disease associated with HBV infection (e.g., hepatitis B), neutralization of the toxicity of HBV in a subject (e.g. human), or of 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 (particularly humanized antibodies thereof) and variants thereof in the manufacture of pharmaceutical compositions, wherein the pharmaceutical compositions are directed to diseases associated with HBV infection or HBV infection (eg hepatitis B). Prophylaxis and / or treatment, neutralizing the toxicity of HBV in a subject (eg, human), or reducing 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 the chronic hepatitis B virus, and these people eventually become infected with HBV infection-related liver disease. There is a risk of death from% to 25%, and more than 1 million people worldwide may die each year from the disease (Dienstag JL., Ibid; and Liaw YF et al., Ibid.).

Currently, therapeutic drugs for chronic HBV infection can be classified mainly as interferon (INF) and nucleoside or nucleotide (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 interferons (IFNs) and peg-interferons (also known as Peg-IFNs, also known as long-acting interferons), while achieving HBV inhibition and CHB therapeutic effects primarily through increased overall immunity in patients ; The latter are mainly lamivudine (LMV), adefovir difficile (ADV), entecavir (ETV), telbividin (LdT), tenofovir (tenofovir) which mainly inhibits HBV replication by directly inhibiting the polymerase activity of HBV. Etc. are mainly included. For HBV-infected patients (eg, CHB patients), treatment with the drug alone or in combination can effectively inhibit viral replication in vivo and significantly reduce HBV DNA levels; In particular, after 52 weeks of treatment or for an extended period of time, the response rate of virological responses in patients with HBV DNA levels below the lower limit of detection can reach 40-80% (Kwon H et al., Ibid.). However, therapies alone or in combination with the aforementioned drugs cannot completely eliminate HBV virus in infected patients, which usually results in less than 5% HBsAg negative conversion or HBsAg seroconversion (complete removal of HBV virus in infected patients). The response rate of Kwon H et al., Ibid. Therefore, there is an urgent need to develop innovative treatment methods and drugs for HBV-infected patients that can more effectively eliminate HBV virus, particularly HBsAg.

The development of new 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 performed by two methods: active immunotherapy (corresponding to drug forms such as vaccines) and passive immunotherapy (corresponding to drug forms such as antibodies and the like). 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 inhibiting or eliminating HBV, thereby causing a cellular immune response to HBV (CTL). Effects, etc.) and / or humoral immune responses (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 (such as with antibodies as an example) administers a therapeutic antibody to an HBV-infected individual, thus preventing naïve hepatocytes from HBV infection by antibody-mediated virus neutralization, or for antibody-mediated immunity clearance. By which viruses and infected hepatocytes are removed from the body, thereby achieving a therapeutic effect. Currently, anti-HBs polyclonal antibodies, ie high-titer hepatitis B immunoglobulin (HBIG), obtained by purification from serum / plasma of an immune responder to a prophylactic hepatitis B vaccine or from a survivor of HBV infection, from mother to infant It has been widely used to block HBV vertical infection, to prevent HBV reinfection after liver transplantation in chronic HBV infected patients, and to prevent infection in humans who accidentally exposed to HBV. However, the treatment of direct administration of HBIG to HBV-infected patients (eg CHB patients) is not very effective and has a number of limitations, such as a low source of high titer plasma, expensive, unstable properties, and potential safety issues.

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

In one aspect, the present invention

(a) the sequence by (i) SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VH CDR1 consisting of a sequence selected from a sequence different from;

(ii) a sequence that differs from the sequence by SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR2 consisting of a sequence selected from among; And

(iii) a sequence that differs from the sequence by SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or one or more amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR3 consisting of a sequence selected from among

One or more (eg 1, 2 or 3) heavy chain variable regions (VH) complementarity determining regions (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 the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR1 consisting of the selected sequence;

(v) a sequence selected from the sequence that differs from the sequence by SEQ ID NO: 10, SEQ ID NO: 16, or one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR2; And

(vi) SEQ ID NO: 11, SEQ ID NO: 17, or a sequence selected from the sequence that differs from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR3 consists of

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 that can specifically bind to HBsAg.

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

(a) a sequence selected from sequences different from SEQ ID NO: 7 by (i) SEQ ID NO: 7, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR2;

(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 or multiple amino acid substitutions, deletions or additions (e.g., one, two or three 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 a; And / or

(b) (iv) a sequence selected from SEQ ID NO: 9 or a sequence different from SEQ ID NO: 9 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 11 or a sequence different from SEQ ID NO: 11 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

Light chain variable region (VL) comprising a

It includes.

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

In some preferred embodiments, the VH CDR1 is selected from (a) SEQ ID NO: 6, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences of VH CDR1 contained in an amino acid sequence encoded by a 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 an antibody or antigen-binding fragment thereof of the invention is a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR3 as shown in SEQ ID NO: 8, and SEQ ID NO: 6, SEQ ID NO: 137 or sequence VH CDR1 having the amino acid sequence of number 138.

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

In some preferred embodiments, the VL CDR2 is selected from (a) SEQ ID NO: 10, or by one or multiple amino acid substitutions, deletions, or additions (eg, one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences 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 invention is a VL CDR1 as shown in SEQ ID NO: 9, a VL CDR3 as shown in SEQ ID NO: 11, and SEQ ID NO: 10, SEQ ID NO: 139 or sequence VL CDR2 having the amino acid sequence of SEQ ID NO: 140.

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

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

(a) (i) SEQ ID NO: 12 or a sequence selected from a sequence different from SEQ ID NO: 12 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;

(ii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 14 or a sequence different from SEQ ID NO: 14 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

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

(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

Light chain variable region (VL) comprising a

It includes.

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

In some preferred embodiments, the VH CDR2 is selected from (a) SEQ ID NO: 13, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences of VH CDR2 contained in an amino acid sequence encoded by a 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) VH CDR1 as shown in SEQ ID NO: 12, VH CDR3 as shown in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: VH CDR2 having an amino acid sequence selected from the group consisting of 141 and SEQ ID NO: 142.

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

In some preferred embodiments, the VL CDR2 is selected from (a) SEQ ID NO: 16, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences 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 VL of an antibody or antigen-binding fragment thereof of the invention comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and SEQ ID NO: 16, SEQ ID NO: 143 and a sequence. VL CDR2 having an amino acid sequence selected from the group consisting of number 144.

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

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

(a) (i) SEQ ID NO: 18 or a sequence selected from a sequence different from SEQ ID NO: 18 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;

(ii) a VH CDR2 consisting of a sequence selected from SEQ ID NO: 19 or a sequence different from SEQ ID NO: 19 by one or a plurality of amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

(iii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 20 or a sequence different from SEQ ID NO: 20 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions)

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

(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR2 consisting of a sequence selected from SEQ ID NO: 16 or a sequence different from SEQ ID NO: 16 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) , And

(vi) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions)

Light chain variable region (VL) comprising a

It includes.

In some preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as shown in SEQ ID NO: 18, a VH CDR2 as shown in SEQ ID NO: 19, and a VH CDR3 as shown in SEQ ID NO: 20. And the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR2 as shown in SEQ ID NO: 16, and a VL CDR3 as shown 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, an 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 97%, or at least 98% humanization. In some preferred embodiments, the non-CDR region of an antibody of the invention or antigen-binding fragment thereof 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 Amino acid residues of up to 6, up to 5, up to 4, or up to 3 non-human sources (eg, cynomolgus monkey sources).

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

(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., one, two or three amino acid substitutions, VH FR1 consisting of a sequence selected from sequences different from said sequence by deletion or addition);

(ii) by 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, one, two or three 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 (eg, one, two or three amino acid substitutions, deletions or additions) VH FR3 consisting of a sequence selected from a sequence different from said sequence; 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 (e.g., one, two or three amino acid substitutions, deletions or additions) VH FR4 consisting of a sequence selected from a sequence different from the sequence

One or more (eg, one, two, three or four) heavy chain variable region (VH) framework regions (FR) selected from among them; 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, one, two or three amino acid substitutions, deletions or additions) VL FR1 consisting of a sequence selected from a sequence different from said sequence;

(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 (e.g., one, two or three 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 (e.g., one, two or three 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 sequences different from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL FR4 made up

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

It includes.

In some preferred embodiments, an 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, an 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, an 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

(a) at least 80%, at least 85% relative to the heavy chain framework region contained in the heavy chain variable region selected from among the 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% Heavy chain framework regions; And / or

(b) at least 80%, at least 85%, at least compared to the light chain framework region contained in the 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 Light chain frame with sequence homology of 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% Work area

It includes.

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

(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 the light chain variable regions as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166

It includes.

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

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

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

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

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

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

And / or the VL of the antibody or antigen-binding fragment thereof

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

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

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

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

It 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 an amino acid sequence encoded by a human germline antibody gene), and the framework region Optionally comprises one or more reverse mutations from human residues to cynomolgus monkey residues.

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

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

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

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

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

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

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

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

It includes.

In some preferred embodiments, the amino acid sequence of 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 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 the 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 an antibody or antigen-binding fragment thereof of the 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% relative to the amino acid sequence of the light chain variable region selected from the group consisting of regions Have sequence homology of at least 97%, at least 98%, at least 99%, or 100%. 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, an 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 an antibody of the invention or antigen-binding fragment thereof 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 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

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

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

(3) a VH as shown in SEQ ID NO: 5 and a VL as shown in SEQ ID NO: 4;

(4) a VH as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 58;

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

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

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

(8) VH as shown in SEQ ID NO: 57 and VL as shown 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 shown in SEQ ID NO: 163 and VL as shown 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 shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 166

It 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 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 antibodies of the invention or antigen-binding fragments thereof are of the IgG class (eg IgG1, IgG2, IgG3 or IgG4 class).

In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention can specifically bind HBsAg in the subject, neutralize the toxicity of HBV, and / or serum levels of HBV DNA and / or HBsAg Can be reduced.

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, such as a radioisotope, fluorescent material, luminescent material, chromogenic material or enzyme (eg horseradish peroxidase).

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody according to the invention or an antigen binding fragment thereof, 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 present 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 that allow 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. Provided are methods for producing an antibody or antigen-binding fragment thereof according to the invention.

In another aspect, the invention provides a kit comprising an antibody according to the invention or an antigen binding fragment thereof. 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 or antigen binding fragment thereof according to the invention. 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 materials, luminescent materials, chromogenic materials 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 according to the invention or an antigen binding fragment thereof. 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 for diagnostic or non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).

In another aspect, the present invention provides a method of diagnosing whether a subject has HBV infection, comprising detecting the presence of HBsAg protein in a sample from a subject using an antibody according to the invention or an antigen-binding fragment thereof. . 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.

Antibodies or antigen-binding fragments thereof according to the invention can be used to prevent or treat HBV infection or a disease associated with HBV infection (eg hepatitis B) in a subject (e.g. human), in vitro or in a subject (e.g. Humans) to neutralize HBV toxicity and to reduce serum levels of HBV DNA and / or HBsAg in subjects (eg, humans).

Thus, in another aspect, the present invention provides a pharmaceutical composition comprising the 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 invention may further comprise an additional pharmaceutically active agent. In a preferred embodiment, the additional pharmaceutically active agent is an agent for the prevention or treatment of an HBV infection or a disease associated with HBV infection (eg hepatitis B), for example an interferon-type agent such as interferon or Pegylated interferon.

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

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, human), and neutralizes HBV toxicity in a subject (eg, a human). And / or to reduce serum levels of HBV DNA and / or HBsAg in a subject (eg, a human), the method comprising providing an effective amount of an antibody or antigen-binding fragment thereof according to the invention or Administering a pharmaceutical composition according to the invention.

The pharmaceutical and pharmaceutical compositions provided herein may be used alone or in combination, or additional pharmaceutically active agents (eg other antiviral agents, eg interferon-type agents such as 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 those skilled in the art will be given the following drawings and examples only to illustrate the present invention and not to limit the scope of the present invention. Will know. Various objects and advantageous aspects of the invention will be apparent to those skilled in the art from the following preferred embodiments and the detailed description of the accompanying drawings.

1 shows the results of an ELISA assay to determine the binding activity of three cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 and M3-13 to antigen HBsAg in Example 2, wherein The abscissa represents the antibody concentration (Log ₁₀ ng / ml) and the ordinate represents the OD value. The results showed that all three cynomolgus monkey-human chimeric monoclonal antibodies had good antigen-binding activity.
FIG. 2 shows competitive ELISA assay for cross-blocking binding of three cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 and M3-13 to antigen HBsAg by 6D11 mouse antibody in Example 2. Shows the result. 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 results of evaluation of the in vivo therapeutic effect of cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 in HBV transgenic mice in Example 2. FIG. FIG. 3A shows changes in HBsAg levels in serum of mice, where abscissa indicates days after injection of chimeric antibody, and ordinate indicates relative decrease level of HBsAg in serum of mice (Log ₁₀ IU / ml). Represent; FIG. 3B shows the change in HBV DNA levels in serum of mice, where abscissa represents days after injection of chimeric antibody, and ordinate represents relative decrease level of HBV DNA in serum of mouse (Log ₁₀ IU / ml ). The results showed that M1-23 and M3-23 can obviously remove HBsAg and HBV DNA in animals.
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 shows the results of a competitive ELISA assay for cross-blocking the binding of humanized antibodies M1D, M3D to antigen HBsAg by 6D11 mouse antibody in Example 4. FIG. The results showed that binding of humanized antibodies M1D, M3D to antigen HBsAg can be significantly inhibited by 6D11.
6A and 6B show measurement results of neutralizing activity of humanized antibody M1D against HBV virus in Example 4, where FIG. 6A shows a standard curve obtained using HBIG as a standard, and FIG. 6B is The results of the measurement of neutralizing activity in the sample are shown, where the ordinate indicates the number of standard units (mIU) of HBIG having neutralizing activity equivalent to neutralizing activity per mg of antibody. The results showed that the neutralizing activity of M1D against HBV is comparable for the reference antibody 162.
7 shows the results of evaluation of the in vivo therapeutic effect of humanized antibodies M1D and M3D and chimeric antibody M3-13 in HBV transgenic mice in Example 5. FIG. FIG. 7A shows changes in HBsAg levels in serum of mice, where abscissa indicates days after injection of humanized antibody, and ordinate indicates relative decrease level of HBsAg in serum of mouse (Log ₁₀ IU / ml). Represent; FIG. 7B shows the change in HBV DNA levels in serum of mice, where abscissa represents days after injection of chimeric antibody, and ordinate represents relative decrease level of HBV DNA in serum of mouse (Log ₁₀ IU / ml ). The results showed that all three antibodies can clearly remove HBsAg and HBV DNA in animals and their effects are better than that of reference antibody 162.
8A-8E show the results of ELISA analysis of the binding activity of an antibody containing a substitution of a CDR region to antigen HBsAg in Example 6 as compared to humanized antibody M1D, wherein the abscissa represents the antibody concentration ( Log ₁₀ ng / ml) and the ordinate represents the OD value. The results showed that substitution of HCDR1 and LCDR2 of antibody M1D did not affect binding activity to antigen HBsAg, indicating that the HCDR1 and LCDR2 regions of antibody M1D are not critical CDR regions for binding to antigen HBsAg.
9A-9E show the results of ELISA analysis of the binding activity of antibodies containing substitution of CDR regions to antigen HBsAg in Example 6 as compared to humanized antibody M3D, wherein the abscissa represents the antibody concentration ( Log ₁₀ ng / ml) and the ordinate represents the OD value. The results showed that substitution of heavy chain CDR2 (HCDR2) and light chain CDR2 (LCDR2) did not affect the binding activity to antigen HBsAg, which means that the HCDR2 and LCDR2 regions of antibody M3D are not critical CDR regions for binding to antigen HBsAg. Suggests.
10 shows the results of Western blot analysis for binding of antibodies M1D, M3D, 162 to various single-site mutated polypeptide fragments of HBsAg aa 113-135 in Example 8. FIG. The results indicate that antibody 162 loses binding activity to HBsAg when the sites in aa 120, aa 122 and aa 123 are mutated to alanine and its core epitope is CKTC (aa 121-124); Binding activity to HBsAg when the site in aa 118-124 is mutated to alanine and its core epitope is longer TGPCKTCT (aa 118-124) than in reference antibody 162 and in the forward position as compared to reference antibody 162 Loses; M3D lost binding activity to HBsAg when the site in aa 121-125 was mutated to alanine and its core epitope was CKTCT (aa 121-125) in the reverse position compared to 162.
11A and 11B show the results of ELISA analysis of the binding activity of humanized antibodies M1D, M3D against different subtypes of HBsAg-aa 113-135 in Example 9, where the abscissa represents the antibody concentration (Log ₁₀ ng / ml) and the ordinate represents the OD value. The results showed that M1D and M3D can 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 of humanized antibody M1D, M3D in Example 11 A curve graph for concentration is shown, where abscissa represents time (h) and ordinate represents blood concentration (ng / ml).
13A and 13B show the Tm values of humanized antibodies M1D, M3D in three different buffers in Example 12. The abscissa represents temperature (° C.) and the ordinate represents CP (cal / ° C.). The results showed that the Tm onset of the humanized antibody M1D was above 61.7 ° C. and that the Tm onset value of M3D was both above 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 QVQLQESGPGLVKPSEILSVTCSVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLRSRVTLSVDTSKNQLSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGLRVTVSS 2 EIVMTQSPSSLSASVGDRVTITCRATQDISSSLNWYQQKPGKAPQLLIYYANRLESGVPSRFRGSGSGTEFTLTISSLQPEDFTTYYCQQYHSLPLTFGGGTKVEIK 3 QLQLQESGPGLVKPSETLSLTCAVSGVSISSPYDWTWIRQPPGKGLEWIGRIHGNGGSTSYNPSLKNRVTISKDTSKNQFSLILSSVTAADTAVYYCVRDETWGQGVLVTVSS 4 DIQMSQSPDSLAVSLGERVTINCKSSQSLLYSSNNKNYLAWYQQKPGQAPKLLFYWASTRESGVPNRFSGSGSGTDFTLTIRGLQAEDVAVYYCQQHYTTPLTFGQGTKVEIK 5 QVQLQESGPRLVKSSETLPLTCAVSGASNSSNYWSWIRQPPGKGLEWIGRIYGNSGSTDYNPSLKSRVSISTDTSKNQFSLKLSSVTAADTAVYFCARGSVYTYSWGQGVLVTVSS 6 GGSITSNF 7 ISGSGTYT 8 ARSHDYGSNDYAFDF 9 QDISSS 10 YAN 11 QQYHSLPLT 12 GVSISSPYDW 13 IHGNGGST 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 DIQMTQSPSSLSASVGDRVTITCRAT 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'- GATGGGCCCTTGGTCGACGCtgaagagacggtgacGGTGGTcccttggccccagaaatc 70 5'- AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTCTCCATCC 71 5'- CAGCAAAAACCGGGGAAAGCCCCTAAGCTCCTGATCTATTATGC 72 5'- GAACCTTGATGGGACCCCACTTTGCAAACGATTTGCATAATAGATCAGGAGC 73 5'- CTGTCCCAGATCCACTGCCACTGAACCTTGATGGGACCCC 74 5'- GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAGG 75 5'- GCAGCCTGCAACCTGAGGATTTTGCAACTTATTACTGTCAAC 76 5'- GACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCAT 77 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC 78 5'- ATTCCCGGGTagatgcccaGTAAAAGAGCAGCTTAGGAGGCTGTCCTGGTTTCTGCTG 79 5'- ggtgtctccatcagcagtccttatgacTGGACCTGGGTCCGCCAGCCCCCAGGGAAG 80 5'- AACTGGTTCTTGGACTTGTCTACTGAAATGGTGACTCGACTCTTGAGGGAGGGGTTG 81 5'- AGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGC 82 5'- gaaaccTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC 83 5'- AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTCCAGACTCC 84 5'- CCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGAGAGAGGGCCACCATCAACTGCAAGTCCAG 85 5'- CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCTTTTACtgggcatctACCCGGGAAT 86 5'- GTGAAATCTGTCCCAGATCCACTGCCACTGAATCGGTCAGGGACCCCGGATTCCCGGGTagatgcc 87 5'- GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATG 88 5'- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC 89 5'-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGAGTCAGGC 90 5'- GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGAC 91 5'- AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCAGTC 92 5'- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCCC 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'- GATGGGCCCTTGGTCGACGCTGAGGAGACGGTGACCAG 115 5'- AGTAGCAACTGCAACCGGTGTACATTCTGACATCGTGATGACCCAGTCTC 116 5'- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTC 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 CTTGCATGGTGCCGTGCTGGTAGTTGATGTTCCTGG 189 CAGCACGGGAGCATGCAAGACCTGCACGATTCCTGC 190 GTCTTGCATGCTCCCGTGCTGGTAGTTGATGTTCCT 191 ACGGGACCATCCAAGACCTGCACGATTCCTGCTCAA 192 GCAGGTCTTGGATGGTCCCGTGCTGGTAGTTGATGT 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 that are commonly understood by one of ordinary skill in the art unless otherwise stated. Moreover, execution steps of cell culture, biochemistry, nucleic acid chemistry, immunology experiments, etc., as used herein, are conventional steps that are widely used in the corresponding art. In addition, related definitions and descriptions are provided below for a better understanding of the present invention.

As used herein, the term "antibody" typically refers to an immunoglobulin molecule consisting 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 the antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V _H ) and a heavy chain constant region (C _H ). The heavy chain constant region consists of three domains (C _H 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 (C _L ). The light chain constant region consists of one domain C _L. The constant region of the antibody may mediate the 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 highly variable regions (called complementarity determining regions (CDRs)) spaced apart from relatively conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino terminus to carboxy terminus. 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 various regions or domains can be found in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to an amino acid residue in the variable region of an antibody that is responsible for antigen binding, and 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 eg 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 of generating an antibody. 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 antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds and / or specifically binds to the antigen. 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”. In general, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), the entire contents of which are incorporated herein by reference for all purposes. By recombinant DNA techniques or by enzymatic or chemical cleavage of the complete antibody In some cases, antigen-binding fragments are Fab, Fab ', F (ab') ₂ , Fd, Fv, dAb and complementarity. A polypeptide comprising a fragment of a determining region (CDR), a single chain antibody (eg scFv), a chimeric antibody, a diabody, and at least a portion of an antibody sufficient to confer specific antigen binding capacity 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 disulfide bridges in the hinge region.

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

In some cases, the antigen-binding fragment of an antibody is a single chain antibody (eg scFv), where the V _L and V _H domains are paired, monovalent through a linker that allows them to produce a single polypeptide chain. To form a molecule (see, eg, Bird et al., Science 242: 423-426 (1988); Houston 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. Suitable linkers in the prior art may consist of a repeating amino acid sequence of GGGGS or a variant thereof. For example, linkers having an 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 the following literature: 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 the first heavy chain of the antibody to the second and third constant regions of the 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, the antigen-binding fragment of an antibody is a diabody, ie the V _L and V _H domains are expressed on a single polypeptide chain, but the linker used is too short to pair between two domains of the same chain, one domain Is a bivalent antibody that is forcibly paired with the complementary domain of another chain to create two antigen-binding sites (see, eg, 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 the antibodies (e.g., upper body fragments described above) may be obtained by using a conventional technique known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) Monoclonal antibodies provided herein) and can be screened for specificity in the same manner as for the complete antibody.

Herein, unless explicitly stated otherwise, when referring to the term "antibody", an antibody includes not only complete antibodies but also antigen-binding fragments of the antibody.

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 completely identical antibody molecules except for spontaneous mutations that may occur spontaneously. Refers to the fragment. Monoclonal antibodies have high specificity for a single epitope of 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 can also be obtained by recombinant DNA techniques (e.g., US patents). 4,816,567).

For example, monoclonal antibodies can be prepared as follows. First, non-human primates (eg cynomolgus monkeys) or other suitable host animals are immunized by injection of an immunogen (add an adjuvant, if necessary). Means for injection of the immunogen or adjuvant are generally subcutaneous multi-point injections or intraperitoneal injections. Pre-conjugation of an immunogen to some known protein (eg, serum albumin or soybean trypsin inhibitor) may promote 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. 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 propagated in the medium, and the culture medium preferably comprises one or more substances capable of inhibiting the proliferation of parental myeloma cells, which are not fused. For example, in the case of parental myeloma cells lacking hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), substances such as hypoxanthine, aminopterin and thymidine (HAT culture medium) may be added to the culture medium to Inhibits proliferation of HGPRT-deficient cells. Preferred myeloma cells should have high fusion rate, stable antibody secretion capacity, be sensitive to HAT culture medium, and the like. Culture medium for propagation of hybridoma cells is used to detect the production of monoclonal antibodies directed against specific antigens. The following specific 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 be. See, eg, Munson et al., Anal. Biochem. 107: 220 (1980)] can be used to determine the affinity of monoclonal antibodies. After measuring the specificity, affinity and reactivity of the antibodies produced by the hybridomas, the cell line of interest was described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996] can be subcloned by the standard restriction dilution method described. Suitable culture media may be DMEM or RPMI-1640 and the like. In addition, hybridoma cells can be propagated in the form of ascites tumors in an animal body. Cell cultures of monoclonal antibodies secreted by subclonal cells by using traditional methods for 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 recombination techniques. Nucleic acid primers that specifically bind to the genes of the MAb heavy and light chains are PCR amplified to obtain DNA molecules encoding the MAb heavy and light chains from hybridoma cells. The obtained DNA molecule is inserted into an expression vector, the host cell (e.g., E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) is transfected with the vector and by recombinant expression Incubate under conditions suitable to obtain the antibody of interest.

As used herein, the terms “antigenic epitope” and “epitope” refer to the portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitopes" are also known as "antigen determinants". Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and generally have specific three dimensional structures and specific charge characteristics. For example, epitopes are generally at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or in a unique conformational form (which may be "linear" or "stereomorphic"). 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 the interaction sites between the protein and the interacting molecule (eg an antibody) are linear 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 competitive advantage in binding to the same epitope by conventional techniques known by those skilled in the art. For example, studies on competition or cross-competition can be performed to obtain antibodies that compete or cross-competite with each other for binding to the antigen. Mass processing methods for obtaining antibody binding to the same epitope, based on cross-competition, are described in international patent application WO 03/48731.

As used herein, the term "germline antibody gene" or "germline antibody gene segment" does not undergo a maturation process that can induce gene rearrangements and mutations for the expression of specific immunoglobulins. Refers to a sequence present in the genome of a gene encoding an immunoglobulin. In the present invention, the expression "heavy chain germline gene" is a germline antibody gene encoding an immunoglobulin heavy chain comprising a V gene (variable), D gene (diversity), J gene (binding) and C gene (constant) or Refers to gene fragments; Similarly, the expression “light chain germline gene” refers to a germline antibody gene or gene fragment that encodes an immunoglobulin light chain, including the V gene (variable), J gene (binding) and C gene (constant). In the present invention, an amino acid sequence encoded by a germline antibody gene or a germline antibody gene fragment is also referred to as a "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 professional databases (eg IMGT, unswag, NCBI or VBASE2).

As used herein, the terms “specifically bind” and “specifically bind” refer to the binding of two molecules in a non-random manner, eg, the reaction between an antibody and an antigen thereon. do. In some embodiments, the antibody that specifically binds to the antigen (or antibody specific for the antigen) is less than about 10 ⁻⁵ M, for example about 10 ⁻⁶ M, 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻ An antibody that binds to an antigen with an affinity (K _D ) of less than or equal to 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 the antibody for antigen. The smaller the dissociation constant, the tighter the binding to the antibody and the higher the affinity between the antibody and antigen. In general, antibodies (eg antibodies according to the invention) are less than about 10 ⁻⁵ M, for example about 10 ⁻⁶ M, 10, as measured by surface plasmon resonance (SPR), for example in a BIACORE device. ^Binds to an antigen (eg HBsAg) with a K _D of less than or equal to ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, or 10 ⁻¹⁰ M.

As used herein, the term "immunogenic" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. This refers not only to the properties of the antigen that stimulates the activation, proliferation and differentiation of specific immune cells in order to finally produce immunological effector substances such as antibodies and sensitized lymphocytes, but also the antibody or sensitized T after stimulation of the antigen. It refers to a specific immune response in which lymphocytes can form in the immune system of an organism. When heterologous antibodies are applied to a subject, the immunogenicity of the heterologous antibodies in the subject is unnecessary. Such immunogenicity may lead to rejection of the heterologous antibody by the subject's immune system / immune cells, thereby producing reduced efficacy of the heterologous antibody in the subject or creating unwanted side effects in the subject. Thus, prior to administration to human subjects, heterologous antibodies (eg cynomolgus monkey-derived antibodies) generally need to be engineered to reduce their immunogenicity as much as possible.

As used herein, the term “chimeric antibody” means that as long as the antibody still retains the activity of binding to the antigen of interest, some of its light and / or heavy chains may be antibodies (origin from a particular species or specific antibody type). Or a different portion of the light and / or heavy chains thereof, which is derived from another antibody (which may originate from the same or different species or belong to the same or different antibody type or subtype). Antibodies such as (US 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 are comprised of primary antibodies (eg While the cynomolgus monkey-derived antibody) is derived, 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 whose amino acid sequence has been modified to increase its homology to the sequence of a human antibody. In general, some or all of the CDR regions of a humanized antibody are derived from non-human antibodies (donor antibodies), and some or all of the non-CDR regions (eg variable region FR and / or constant regions) are human immune. Derived from globulin (receptor antibody). Humanized antibodies typically retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to neutralize and / or eliminate viruses, and the like. Donor antibodies are mice, rats, rabbits or non-human primates (eg cynomolgus monkeys) having the expected properties (eg antigen specificity, affinity, reactivity, virus neutralizing ability, and / or virus eliminating ability). It may be an antibody of.

Humanized antibodies can retain the expected properties of non-human donor antibodies (eg cynomolgus monkey-derived antibodies) and are non-human donor antibodies (eg cynomolgus monkey-derived antibodies in human subjects). It is particularly advantageous because it is effective for reducing immunogenicity. However, due to the problem of congruence 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, virus neutralizing ability, and / or virus eliminating ability) are non- It is generally lower than for human donor antibodies (eg cynomolgus monkey-derived antibodies).

Thus, researchers in the field have made extensive studies of humanization of antibodies and made some advances (see, 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). Detailed guidance is provided in the prior art regarding how to humanize some donor antibodies enough to allow the resulting humanized antibodies to have as high humanization as possible while maintaining the expected properties of the donor antibody as much as possible. It is not. Those skilled in the art should investigate, explore and manipulate specific donor antibodies, and have a high degree of humanization (eg at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least Many creative studies have been conducted to obtain humanized antibodies that have a degree of humanization of 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) as well as retain the expected properties of the donor antibody. It must be done.

In the present invention, to prepare a humanized antibody which retains as much of the properties of the donor antibody as possible (including specificity, affinity, reactivity, virus neutralizing ability and / or virus eliminating ability), the humanized antibody of the present invention The framework region (FR) of may comprise both amino acid residues of a receptor 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 to reduce the immunogenicity caused by non-human amino acid residues as much as possible, some of the amino acid residues derived from the CDR regions of the donor antibodies in the humanized antibodies of the invention may be exemplified. For example, a corresponding amino acid residue or other amino acid residue in the CDR region of a human immunoglobulin.

In addition, in order to further improve or optimize the performance of humanized antibodies, some of the amino acid residues in the heavy and light chain variable regions of the humanized antibodies of the invention are not derived from, for example, recipient antibodies or from donor antibodies. And may be further substituted with amino acid residues.

Specifically, in the present application, the inventors first identified three cynomolgus monkey-human chimeric monoclonal antibodies (the heavy and light chain variable regions thereof) of excellent properties, named M1-23, M3-23 and M3-13, respectively. Are shown in SEQ ID NOs: 1-5, respectively). Three antibodies can specifically recognize / bind HBsAg and neutralize HBV toxicity, as well as serum levels of HBV DNA and / or HBsAg in subjects. Can be reduced and effectively remove HBV and cells infected by HBV from the body. Thus, M1-23, M3-23 and M3-13 antibodies have the 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 made extensive creative efforts to intensively study and modify two chimeric antibodies M1-23 and M3-23, thus developing 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 the expected properties that are substantially the same (or even superior) to the parent antibody (including but not limited to HBsAg binding activity, HBV neutralizing activity, biomarkers). Removal activity of HBV DNA or HBsAg in vivo, or removal activity of cells infected with HBV and HBV in vivo.

Thus, the antibodies (particularly humanized antibodies) of the present invention are very advantageous because they can retain the function and properties of the parental 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% humanization), and thus can be safely administered to human subjects without elevating the immunogenic response. Antibodies (particularly humanized antibodies) according to the invention may have significant clinical value.

In the present application, the expected properties of the antibodies of the present invention include the activity of specifically binding to HBsAg, HBV neutralizing activity, removal activity of HBV DNA or HBsAg in vivo, and / or removal activity of cells infected with HBV and HBV in vivo. It includes. The humanized antibody according to the present invention retains one or more of the above expected properties of its parental antibody (cynomolgus monkey-human chimeric antibody), preferably its parental antibody (cynomolgus monkey-human chimeric) All of the above expected properties of the antibody).

In the present application, the humanized antibody of the present invention is obtained by modifying the parent antibody (cynomolgus monkey-human chimeric antibody) of the present invention. For example, some amino acid residues in the FR of the antibody are substituted (eg conservative substitutions). Such substitutions may, for example, reduce (1) the sensitivity of the antibody to proteolysis; (2) reduce the sensitivity of the antibody to oxidation; (3) change (eg, enhance) the antigen binding affinity of the antibody; (4) change (eg, enhance) the HBV-neutralizing activity of the antibody; (5) change (eg, enhance) the HBV-removing activity of the antibody; (6) further increase 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 present in 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 the humanized antibody can be calculated, for example, by the following formula: degree of humanization = (number of amino acids in FR minus 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 can significantly reduce or completely inhibit the toxicity (eg, the ability to infect a cell) of a target virus. . In general, neutralizing antibodies can recognize and bind to the target virus and prevent the target virus from entering and / or infecting the cell in the subject. The antibody according to the invention is a neutralizing antibody.

However, of course, in this application, the virus-neutralizing ability of the antibody is not directly equivalent to the virus-eliminating ability of the antibody. As used herein, "virus neutralization" neutralizes the target virus's toxicity by inhibiting the target virus from entering and / or infecting the cells of the subject (ie, the toxicity of the target virus is significantly reduced or completely). Suppressed). As used herein, "virus removal" means removing the target virus (whether or not infected with cells) from the organism, thus returning the organism to a state prior to infection by the virus (e.g., For example, the serological test results of the virus are negative). Thus, in general, neutralizing antibodies do not necessarily have the ability to remove viruses. However, in the present application, the inventors surprisingly find that the antibodies according to the invention not only neutralize HBV, but can also remove viruses (ie, remove HBV DNA and / or HBsAg in vivo, and in vivo). HBV and HBV-infected cells can be removed), and thus have significant clinical value.

As used herein, the term “isolated” refers to a state obtained from natural state by artificial means. If several "isolated" substances or components are present in nature, this is possible because of their natural environment changing or because the substance is isolated from the 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 in high purity from such natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic materials, nor other impure materials that do not affect the activity of the isolated material.

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 called an expression vector. The vector may have a transported genetic material expressed in the 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 chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); Phages such as lambda phage or M13 phage 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), chickenpox virus, baculovirus, papillomavirus, papova Viruses (eg SV40). Vectors can include a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may comprise an origin of replication.

As used herein, the term "host cell" refers to a cell into which a vector may be introduced, such as but not limited to prokaryotic cells such as E. coli or Bacillus subtilis, and fungal cells, eg 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 Cell or human cell.

As used herein, the term “homology” refers to the degree of agreement between two polypeptides or between two nucleic acids. Two sequences for comparison have the same monomeric subunit of base or amino acid at a site (e.g., two separate DNA molecules have adenine at a certain site, or two separate polypeptides have a With lysine), the two molecules are identical at this site. The percent homology between two sequences is a function of the number x 100 of the same site shared by the two sequences over the total number of sites for comparison. For example, if six of the ten sites of the two sequences match, these two sequences have 60% homology. For example, the DNA sequences: CTGACT and CAGGTT share 50% homology (3 of 6 sites match up). In general, the comparison of the two sequences is performed in a manner that produces maximum homology. Such alignment is based on a computer program such as the Align program (DNAstar, Inc.) (based on the method of Needleman, et al., J. Mol. Biol. 48: 443-453, 1970). Can be used. The percent homology between the two amino acid sequences was also integrated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, 12 break length penalties, and 4 break penalties, using 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 the Blossum 62 matrix or the PAM250 matrix and the truncated weights 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) integrated into the GAP program of the GCG software package (available at http://www.gcg.com): 444-453 (1970).

As used herein, the terms "conservative substitutions" and "conservative amino acid substitutions" refer to amino acid substitutions that do not adversely affect or change the expected properties of the protein / polypeptide comprising the 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 forms another amino acid residue having a similar side chain to the corresponding amino acid residue, for example physically or functionally similar (eg, to form a similar size, shape, charge, covalent bond or hydrogen bond). Substitutions with residues having chemical properties, including the ability to do so. Families of amino acid residues with 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 Razine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains For example threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, corresponding amino acid residues are preferably substituted with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions are well known in the art (see, eg, Brumell 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 related to this specification are shown according to conventional methods. See, eg, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). In the present invention, "polypeptides" The terms "and" protein "have the same meaning and can be used interchangeably. Furthermore, in the present invention, amino acids are generally represented as 1-letter codes and 3-letter codes. For example, alanine is represented by A or In addition, as used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and may 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 carriers and / or excipients" refers to carriers and / or excipients which are pharmacologically and / or physiologically compatible with the subject and active agent, (See, eg, Remington's Pharmaceutical Sciences.Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), but are not limited to pH regulators, surfactants, adjuvant, ionic strength enhancers, Diluents, osmotic-modulators, absorption retardants, 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, for example parabens, chlorobutanol, phenol and sorbic acid. Osmotic regulators 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 antibody that can increase the immune response to an antigen or change the type of immune response in the organism when delivered to or in advance with the antigen. It refers to non-specific immunopotentiators. Various adjuvant such as but not limited to aluminum adjuvant (eg aluminum hydroxide), Freund adjuvant (eg Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parbium, lipo Polysaccharides, cytokines and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is 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, disorder or condition (eg, a disease associated with HBV infection or HBV infection) in a subject. do. As used herein, the term “treating / treating” refers to the method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes may be, but are not limited to, alleviating symptoms, reducing disease range, stabilizing (ie, not exacerbating) disease states, regardless of whether they can be detected or not. Delay or slowing disease progression, and alleviation of symptoms (partial or complete). "Treatment" also refers to prolonged survival compared to the expected survival (if not receiving treatment). In the present application, the antibodies according to the present invention have the ability to neutralize HBV and thus can be used to prevent / protect uninfected subjects or their cells from infection by HBV. In addition, the antibodies according to the invention have the ability to eliminate HBV (ie can remove HBV DNA and / or HBsAg in vivo, and can remove cells infected by HBV and HBV in vivo). It can be used to treat HBV infection or a disease associated with HBV infection in an infected subject.

As used herein, the term "subject" refers to a mammal, eg, a primate mammal, such as 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 the prevention of a disease (eg, a disease associated with HBV infection or a HBV infection) may be an amount effective for preventing, suppressing, or delaying the onset of the disease (eg, a disease associated with HBV infection or an HBV infection). Refers to. An amount effective for the treatment of a disease refers to an amount effective for the healing or at least partial blockade of the disease and its complications in the diseased patient. Measurement of such effective amounts is within the ability of those skilled in the art. For example, the amount effective for therapeutic use depends on the severity of the disease to be treated, the general condition of the immune system in the patient, the general condition of the patient, such as age, weight and sex, the means of administering the drug, and additional therapies used simultaneously. Change.

Antibodies of the Invention

In the present application, the inventors first identified three cynomolgus monkey-human chimeric monoclonal antibodies, each named M1-23, M3-23 and M3-13, each of which has a heavy chain and a light chain variable region in sequence 3 antibodies can specifically recognize / bind HBsAg and neutralize the toxicity of HBV, as well as reduce serum levels of HBV DNA and / or HBsAg in subjects. It is possible to effectively remove HBV and cells infected by HBV from the body. Thus, M1-23, M3-23 and M3-13 antibodies have the 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. Not only has a very high degree of humanization (the degree of humanization can be as high as 98%), but the expected properties that are substantially the same (or even superior) to the parent antibody (including but not limited to HBsAg binding activity, HBV neutralizing activity, in vivo HBV DNA or Removal activity of HBsAg, or removal activity of cells infected with HBV and HBV in vivo.

Thus, the antibodies (particularly humanized antibodies) of the present invention can retain the function and properties of the parental 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 immunogen response. Antibodies (particularly humanized antibodies) of the invention have significant clinical value.

Thus, in one aspect, the invention provides an antibody or antigen-binding fragment thereof that can specifically bind HBsAg, wherein the antibody or antigen-binding fragment thereof

(a) the sequence by (i) SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VH CDR1 consisting of a sequence selected from a sequence different from;

(ii) a sequence that differs from the sequence by SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR2 consisting of a sequence selected from among; And

(iii) a sequence that differs from the sequence by SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or one or more amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR3 consisting of a sequence selected from among

One or more (eg 1, 2 or 3) heavy chain variable regions (VH) complementarity determining regions (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 the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR1 consisting of the selected sequence;

(v) a sequence selected from the sequence that differs from the sequence by SEQ ID NO: 10, SEQ ID NO: 16, or one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR2; And

(vi) SEQ ID NO: 11, SEQ ID NO: 17, or a sequence selected from the sequence that differs from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR3 consists of

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

It includes.

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

(a) a sequence selected from sequences different from SEQ ID NO: 7 by (i) SEQ ID NO: 7, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR2;

(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 or multiple amino acid substitutions, deletions or additions (e.g., one, two or three 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 a; And / or

(b) (iv) a sequence selected from SEQ ID NO: 9 or a sequence different from SEQ ID NO: 9 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 11 or a sequence different from SEQ ID NO: 11 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

Light chain variable region (VL) comprising a

It includes.

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

In some preferred embodiments, the VH CDR1 is selected from (a) SEQ ID NO: 6, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences of VH CDR1 contained in an amino acid sequence encoded by a 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 an antibody or antigen-binding fragment thereof of the invention is a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR3 as shown in SEQ ID NO: 8, and SEQ ID NO: 6, SEQ ID NO: 137 or sequence VH CDR1 having the amino acid sequence of number 138.

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

In some preferred embodiments, the VL CDR2 is selected from (a) SEQ ID NO: 10, or by one or multiple amino acid substitutions, deletions, or additions (eg, one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences 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 invention is a VL CDR1 as shown in SEQ ID NO: 9, a VL CDR3 as shown in SEQ ID NO: 11, and SEQ ID NO: 10, SEQ ID NO: 139 or sequence VL CDR2 having the amino acid sequence of SEQ ID NO: 140.

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

VL of an antibody or antigen-binding fragment thereof includes VL CDR1 as shown in SEQ ID NO: 9, VL CDR3 as shown in SEQ ID NO: 11, and VL CDR2 having the amino acid sequence of SEQ ID NO: 10.

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

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

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

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

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

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

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

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

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

(a) (i) SEQ ID NO: 12 or a sequence selected from a sequence different from SEQ ID NO: 12 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;

(ii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 14 or a sequence different from SEQ ID NO: 14 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

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

(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

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

Light chain variable region (VL) comprising a

It includes.

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

In some preferred embodiments, the VH CDR2 is selected from (a) SEQ ID NO: 13, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences of VH CDR2 contained in an amino acid sequence encoded by a 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) VH CDR1 as shown in SEQ ID NO: 12, VH CDR3 as shown in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: VH CDR2 having an amino acid sequence selected from the group consisting of 141 and SEQ ID NO: 142.

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

In some preferred embodiments, the VL CDR2 is selected from (a) SEQ ID NO: 16, or by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Different sequences; Or (b) a sequence selected from the sequences 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 VL of an antibody or antigen-binding fragment thereof of the invention comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and SEQ ID NO: 16, SEQ ID NO: 143 and a sequence. VL CDR2 having an amino acid sequence selected from the group consisting of number 144.

In some preferred embodiments, the VH of an antibody of the invention or antigen-binding fragment thereof is included in the heavy chain variable region of VH CDR1 as shown in SEQ ID NO: 12, VH CDR3 as shown in SEQ ID NO: 14, and any immunoglobulin. A VH CDR2 consisting of a sequence selected from the sequence of 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 VL CDR1 as shown in SEQ ID NO: 15, VL CDR3 as shown in SEQ ID NO: 17, and VL CDR2 as shown in SEQ ID NO: 16.

In some preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a 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 shown in SEQ ID NO: 15, VL CDR3 as shown in SEQ ID NO: 17, and the light chain variable region (eg, kappa light chain variable region) of any immunoglobulin. A VL CDR2 consisting of a sequence selected from a sequence of VL CDR2 (eg, SEQ ID NO: 16, SEQ ID NO: 143, or SEQ ID NO: 144).

In some preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention consists of a VH CDR1 as shown in SEQ ID NO: 12, a VH CDR3 as shown in SEQ ID NO: 14, and a group consisting of SEQ ID NO: 13, 141 or 142 VH CDR2 having a sequence selected from among; The VL of an antibody or antigen-binding fragment thereof comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown 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 an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1 as shown in SEQ ID NO: 12, a VH CDR2 as shown in SEQ ID NO: 13, and a VH CDR3 as shown in SEQ ID NO: 14. Including; The VL of an antibody or antigen-binding fragment thereof includes VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and VL CDR3 as shown in SEQ ID NO: 17.

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

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

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

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

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

(a) (i) SEQ ID NO: 18 or a sequence selected from a sequence different from SEQ ID NO: 18 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;

(ii) a VH CDR2 consisting of a sequence selected from SEQ ID NO: 19 or a sequence different from SEQ ID NO: 19 by one or a plurality of amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And

(iii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 20 or a sequence different from SEQ ID NO: 20 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions)

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

(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;

(v) a VL CDR2 consisting of a sequence selected from SEQ ID NO: 16 or a sequence different from SEQ ID NO: 16 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) , And

(vi) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions)

Light chain variable region (VL) comprising a

It includes.

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

(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., one, two or three amino acid substitutions, VH FR1 consisting of a sequence selected from sequences different from said sequence by deletion or addition);

(ii) by 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, one, two or three 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 (eg, one, two or three amino acid substitutions, deletions or additions) VH FR3 consisting of a sequence selected from a sequence different from said sequence;

(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 (e.g., one, two or three amino acid substitutions, deletions or additions) VH FR4 consisting of a sequence selected from a sequence different from the sequence

One or more (eg, one, two, three or four) heavy chain variable region (VH) framework regions (FR) selected from among them; 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, one, two or three amino acid substitutions, deletions or additions) VL FR1 consisting of a sequence selected from a sequence different from said sequence;

(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 (e.g., one, two or three 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 (e.g., one, two or three 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 sequences different from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL FR4 made up

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

It includes.

In some preferred embodiments, an 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, an 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, an 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 shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH FR4 as shown in SEQ ID NO: 24;

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

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

It includes.

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

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

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

It includes.

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

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

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

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

It includes.

In some preferred embodiments, an 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 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, Have 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 regions of the antibodies of the invention or antigen-binding fragments thereof are no more than 19, no more than 15, no more than 14, no more than 13 of non-human (eg cynomolgus monkey) sources. 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 Or the non-CDR region does not comprise amino acid residues of a non-human (eg cynomolgus monkey) source. In some preferred embodiments, the FR regions of an antibody or antigen-binding fragment thereof of the invention comprise no more than 19, no more than 15, no more than 14, no more than 13, 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 Either a residue or the FR region does not comprise an amino acid residue of non-human (eg cynomolgus monkey) origin.

In some preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a framework region of human immunoglobulin, for example a framework region contained in an amino acid sequence encoded by a human germline antibody gene. In some preferred embodiments, the antibody or antigen-binding fragment thereof is contained in a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or 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 an antibody of the invention or antigen-binding fragment thereof 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 comprise conservative substitutions of one or more amino acid residues or corresponding residues that are reverse mutated to the corresponding residues of cynomolgus monkeys (mutations such as Is called reverse mutation).

In some preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain framework region comprised in an amino acid sequence encoded by the human heavy chain germline gene IGHV 4-4 ^* 08 and the heavy chain framework region is human Optionally one or more reverse mutations from the derived residues to cynomolgus monkey-derived residues. 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 in H17;

(02) L at H20; And

(03) H23 to T

VH FR1 different from SEQ ID NO: 21 by one or a plurality of amino acid substitutions (eg, one, two or three 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 in H64;

(02) I in H69; And

(03) H78 to F

VH FR3 different from SEQ ID NO: 23 by one or a plurality of amino acid substitutions (eg, one, two or three amino acid substitutions) selected from the group consisting of;

(iv) SEQ ID NO: 24, or

(01) T in H107; And

(02) H108 to T

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

Wherein all of the 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 shown in SEQ ID NO: 44, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 45, and SEQ ID NO: VH FR4 as shown in 46.

In some preferred embodiments, the VH of an antibody or antigen-binding fragment thereof comprises a heavy chain framework region comprised in an amino acid sequence encoded by the human heavy chain germline gene IGHV 4-4 ^* 02 and the heavy chain framework region is human Optionally one or more reverse mutations from the derived residues to cynomolgus monkey-derived residues. In some preferred embodiments, the amino acid sequence encoded by IGHV 4-4 ^* 02 is shown 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) H to V; And

(02) G from H16

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

(ii) SEQ ID NO: 30 or the following amino acid substitutions: VH FR2 different from SEQ ID NO: 30 by V in H37;

(iii) SEQ ID NO: 31 or

(01) S at H65;

(02) V in H71;

(03) K at H73; And

(04) H81 to K

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

(iv) a VH FR4 that is SEQ ID NO: 32 or the following amino acid substitution: differs from SEQ ID NO: 32 by T in H107

Wherein all of the 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 shown in SEQ ID NO: 50, VH FR2 as shown in SEQ ID NO: 51, VH FR3 as shown in SEQ ID NO: 52, and SEQ ID NO: VH FR4 as shown in 53.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof comprises a light chain framework region contained in an amino acid sequence encoded by human light chain germline gene IGKV1-39 ^* 01, wherein the light chain framework region is Optionally one or more reverse mutations from human-derived residues to cynomolgus monkey-derived residues. In some preferred embodiments, the amino acid sequence encoded by IGKV1-39 ^* 01 is shown 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) L to D; And

(02) Q in L3

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

(ii) SEQ ID NO: 26 or the following amino acid substitution: VL FR2 different from SEQ ID NO: 26 by K at L45;

(iii) SEQ ID NO: 27, or

(01) Q at L55;

(02) S at L63;

(03) D at L70; And

(04) A to 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 of the 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 shown in SEQ ID NO: 47, VL FR2 as shown in SEQ ID NO: 48, VL FR3 as shown in SEQ ID NO: 49, and SEQ ID NO: VL FR4 as shown in 28.

In some preferred embodiments, the VL of the antibody or antigen-binding fragment thereof comprises a light chain framework region contained in an amino acid sequence encoded by human light chain germline gene IGKV4-1 ^* 01, wherein the light chain framework region is Optionally one or more reverse mutations from human-derived residues to cynomolgus monkey-derived residues. In some preferred embodiments, the amino acid sequence encoded by IGKV4-1 ^* 01 is shown 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) V at L3;

(02) T at L5; And

(03) A to L19

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

(ii) SEQ ID NO: 34 or the amino acid substitution below: VL FR2 different from SEQ ID NO: 34 by V in L43;

(iii) SEQ ID NO: 35, or

(01) D at L60;

(02) S at L76; And

(03) S from L77

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

(iv) VL FR4 of SEQ ID NO: 36

Wherein the above-mentioned amino acid positions are numbered according to the Kabat numbering system.

In some exemplary embodiments, the VL of an antibody or antigen-binding fragment thereof is VL FR1 as shown in SEQ ID NO: 54, VL FR2 as shown in SEQ ID NO: 55, VL FR3 as shown 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

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

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

It includes.

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

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

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

It includes.

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

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

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

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

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

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

It includes.

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

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

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

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

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

It 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 of the invention or antigen-binding fragment thereof 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 ; Light chain variable regions include 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 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 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 to the amino acid sequence of the heavy chain variable region selected from the group consisting of: Have a sequence homology of%, at least 98%, at least 99%, or 100%.

In some preferred embodiments, the heavy chain variable region of an antibody of the invention or antigen-binding fragment thereof 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 an antibody of the invention or antigen-binding fragment thereof consists of the 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% of the amino acid sequences of the light chain variable regions selected from the group, Have sequence homology of at least 98%, at least 99%, or 100%.

In some preferred embodiments, the light chain variable region of an antibody of the invention or antigen-binding fragment thereof 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, the antibodies of the invention comprise 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 an antibody of the invention or antigen-binding fragment thereof 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 an antibody of the invention or antigen-binding fragment thereof 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 antibodies of the invention are

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

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

(3) a VH as shown in SEQ ID NO: 5 and a VL as shown in SEQ ID NO: 4;

(4) a VH as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 58;

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

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

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

(8) VH as shown in SEQ ID NO: 57 and VL as shown 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 shown in SEQ ID NO: 163 and VL as shown 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 shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 166

It includes.

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

Antibodies of the invention have high specificity and high affinity for HBsAg protein. For example, an antibody of the invention can bind HBsAg with a KD of less than 1 × 10 ⁻⁵ M; Preferably the KD value is less than 1 × 10 ⁻⁶ M; More preferably the KD value is less than 1 × 10 ⁻⁷ M; Most preferably the KD value is less than 1 × 10 ⁻⁸ M.

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

Antigen-binding fragments of the invention can be obtained by hydrolysis of complete antibody molecules (Morimoto et al., J. biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al. , Science 229: 81 (1985). On the one hand, these antigen-binding fragments can be produced 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 bound to form F (ab') ₂ fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Moreover, Fv, Fab or F (ab ') ₂ fragments can also be isolated directly from the culture of recombinant host cells. Other techniques for making these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in some preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a scFv, Fab, Fab ', (Fab') ₂ , Fv fragment, diabody, bispecific antibody, multispecific antibody, chimeric antibody, Or a group consisting of humanized antibodies. Especially preferably, the antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or a humanized antibody.

In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention can specifically bind HBsAg, neutralize the toxicity of HBV, and / or serum levels of HBV DNA and / or HBsAg in a subject. Can be reduced.

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

Fusion antibody

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

Derivatized Antibodies

Antibodies or antigen-binding fragments thereof according to the invention can be derivatized, eg, linked to another molecule (eg another polypeptide or protein). In general, derivatization (eg labeling) of an antibody or antigen binding fragment thereof will not adversely affect its binding to HBsAg. Accordingly, the antibodies or antigen-binding fragments thereof according to the invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof according to the invention may be prepared by one or more other molecular groups, such as another antibody (eg forming a bispecific antibody), a detection agent, a drug, and / or an antibody or Functionally (by chemical coupling, gene fusion, non-covalent binding or other means) linkage to a protein or polypeptide that can mediate the association of an antigen binding fragment with another molecule (eg avidin or polyhistidine-tag) You can.

One type of derivatized antibody (eg bispecific antibody) is generated by crosslinking two or more antibodies (belonging to or not belonging to the same type). Suitable crosslinkers include, for example, heterobifunctional crosslinkers (eg m-maleimidobenzoyl-N-hydroxysuccinimide esters) comprising two different reactors separated by suitable spacers; And allofunctional crosslinkers (disuccinimidyl suverate). Such crosslinkers can 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, monosyl chloride, phycoerythrin, lanthanide phosphors, and the like. In addition, antibodies can also be labeled with enzymes such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When the antibody is labeled by an enzyme, a labeled antibody can be detected by addition of a reagent that can be used by the enzyme to produce an identifiable signal or reaction product. For example, when horseradish peroxidase is used to label antibodies, hydrogen peroxide and diaminobenzidine can be added to produce a detectable chromogenic reaction product to determine the presence or amount of labeled antibody. In addition, antibodies can 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. The antibody can also be labeled with a tag that can be recognized by a second reporter molecule (eg, leucine zipper pair sequence, metal-binding domain, 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 can also be derivatized with chemical groups such as 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 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 according to the invention or an antigen binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some preferred embodiments, the isolated nucleic acid molecules according to the invention encode the antibodies according to the invention or antigen binding fragments thereof, or heavy and variable chain regions.

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 vectors according to the invention are for example plasmids, cosmids, phages and the like. In some preferred embodiments, the vector can express the antibody or antigen-binding fragment thereof according to the invention in a subject (eg a mammal, eg a human).

In another aspect, the present 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 Mouse cells and human cells). The cells according to the invention can be cell lines, for example 293T cells.

In another aspect, the host cell according to the present invention is cultured under conditions that allow expression of the antibody or antigen-binding fragment thereof according to the present invention, and the antibody or antigen-binding fragment thereof is recovered 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.

Diagnostic Methods and Kits

The antibody or antigen-binding fragment thereof according to the present invention can specifically bind 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 by HBV. .

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

According to the above-described method, the antibody or antigen-binding fragment or secondary antibody thereof according to the present invention can be labeled. For example, the antibody or antigen-binding fragment thereof according to the invention can be labeled with a detectable label. Such detectable labels well known by those skilled in the art include, but are not limited to radioisotopes, fluorescent materials, luminescent materials, chromogenic materials and enzymes (eg horseradish peroxidase) and the like. In addition, such detectable labels include, for example, radioisotopes, such as ¹²⁵ I; Fluorescent materials such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors; Enzymes capable of producing identifiable signals or reaction products such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase; And 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, detection agents (eg tags) can be linked to antibodies through linkers of different lengths to reduce potential steric hindrance.

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 according to the invention or an antigen binding fragment thereof. 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 can be used for diagnostic or non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).

In another aspect, the present invention provides a method for diagnosing a subject's infection by 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 a 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 by HBV.

Therapeutic Methods and Pharmaceutical Compositions

Antibodies or antigen-binding fragments thereof according to the invention can be used to prevent or treat HBV infection or a disease associated with HBV infection (eg hepatitis B) in a subject (e.g. human), in vitro or in a subject (e.g. Humans) to neutralize HBV toxicity and to reduce serum levels of HBV DNA and / or HBsAg in subjects (eg, humans).

Thus, in another aspect, the present invention provides a pharmaceutical composition comprising the 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 invention may further comprise an additional pharmaceutically active agent. In a preferred embodiment, the additional pharmaceutically active agent is an agent for the prevention or treatment of HBV infection or a disease associated with HBV infection (eg hepatitis B), for example other antiviral agents, for example interferon-type Agents, for example interferon or pegylated interferon.

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

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

The antibody or antigen-binding fragment thereof according to the invention or the pharmaceutical composition according to the invention may be administered by conventional routes, including but not limited to oral, buccal, sublingual, intraocular, topical, parenteral, rectal, vaginal, intracranial, inguinal, Administration may be in the bladder, topically (eg, powders, ointments or drops), or by the nasal route. Antibodies or antigen-binding fragments thereof according to the invention can be administered by multiple routes known in the art. However, for many therapeutic uses, the preferred route of administration / administration is parenteral administration (eg intravenous injection, subcutaneous injection, intraperitoneal injection, and intramuscular injection). Those skilled in the art understand that the route / mode of administration can be varied according to the expected purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention is administered by intravenous infusion or injection.

The antibodies according to the invention or antigen-binding fragments thereof or the pharmaceutical compositions according to the invention may be administered in several dosage forms, for example liquid, semisolid, and solid forms, for example solutions (eg injections), dispersions or suspensions, tablets, It can be prepared as powders, granules, emulsions, pills, syrups, powders, liposomes, capsules and suppositories. Preferred dosage forms vary depending on the route of administration expected and the therapeutic use.

For example, one preferred dosage form is injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: The required dose of the antibody or antigen-binding fragment thereof according to the invention is incorporated into a suitable solvent and optionally other anticipated components (including but not limited to pH adjusting agents). , Surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents or any combination thereof) are simultaneously incorporated, followed by filtration sterilization. In addition, sterile injectable solutions can be prepared as sterile powders for ease of storage and use (eg by vacuum drying or lyophilization). Such sterile powders may be dispersed in a suitable vehicle, eg, sterile pyrogen-free water, before use.

Another preferred dosage form is a dispersion. Dispersions can be prepared by the following methods: Antibodies or antigen-binding fragments thereof according to the invention can be prepared using a basic dispersion medium and optionally other anticipated components (including but not limited to pH adjusters, surfactants, adjuvant, ionic strength enhancers, Isotonic agents, preservatives, diluents or any combination thereof). In addition, absorption retardants 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 comprise (a) inert drug excipients (or vehicles) such as sodium citrate and calcium phosphate; (b) fillers such as starch, lactose, sucrose, mannose and silicic acid; (c) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (d) humectants, such as glycerol; (e) disintegrants such as agar, calcium carbonate, potato or tapioca starch; (f) retardants such as olefins; (g) absorption enhancers such as quaternary ammonium compounds; (h) humectants, such as cetyl alcohol and glyceryl monostearate; (i) adsorbents such as kaolin and bentonite; (j) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, 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 modifiers (ie, agents that can change drug release rates) can also be added to oral solid dosage forms to obtain modified or pulsed release dosage forms. Such release rate modifiers include, but are not limited to, carboxypropyl methylcellulose, methylcellulose, carboxymethyl cellulose sodium, ethyl cellulose, cellulose acetate, polyethylene oxide, xanthan gum, isoacyl amino copolymers, hydrogenated flavor oils, carnauba wax, Paraffin, cellulose acetate phthalate, carboxypropyl methylcellulose phthalate, methacrylic acid copolymer, or any combination thereof. Modified release or pulsed release dosage forms can include one or a group of release rate modifiers.

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 are inert solvents customarily used in the art, for example water or other solvents such as ethyl alcohol, isopropanol, propylene glycol, 1,3-butylene glycol, oils (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, such oral liquid dosage forms may further comprise moisturizers, emulsifiers, suspending agents, sweetening agents, flavoring agents, fragrances and the like.

In addition, the antibodies or antigen-binding fragments thereof according to the invention may be present in unit dosage form in pharmaceutical compositions for ease of administration. The pharmaceutical composition according to the invention should be sterile and stable under the conditions of manufacture and storage.

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

The pharmaceutical composition according to the invention may comprise an "therapeutically effective amount" or "prophylactically effective amount" of an antibody according to the invention or an antigen binding fragment thereof. A “prophylactically effective amount” refers to an amount sufficient to prevent, inhibit or delay the development of a disease (eg, a disease associated with HBV infection or a disease associated with HBV infection). A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a diseased patient. The therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the 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 Sex, mode of administration of the drug, additional therapies used simultaneously.

Dosage regimens may be adjusted to provide the optimum desired effect (eg, a therapeutic or prophylactic effect). For example, a single dose may be administered, or several 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.

In the case of an antibody or antigen-binding fragment thereof according to the invention, an exemplary non-limiting range for a therapeutically or prophylactically effective amount is from 0.025 to 50 mg / kg, more preferably from 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, 0.1 to 10 mg / kg. It should be understood that the dose may vary depending on the type and severity of the disease to be treated. In addition, one skilled in the art should, for any particular patient, adjust the 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 purposes of illustration only, rather than to limit the use or scope of the pharmaceutical compositions according to the invention.

Beneficial Effects of the Invention

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

(1) The antibodies of the present invention not only can specifically recognize HBsAg and / or neutralize the toxicity of HBV, but can also reduce the serum levels of HBV DNA and / or HBsAg in subjects, and in vivo, HBV And cells infected by HBV can be effectively removed. Thus, the antibodies of the invention can be used for the prevention and treatment of HBV infections and diseases associated with HBV infections, such as hepatitis B.

(2) Antibodies (particularly humanized antibodies) of the present invention retain the function and properties of parental monkey antibodies, 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 (particularly humanized antibodies) of the present invention have significant clinical value.

Concrete model for carrying out the invention

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

Unless otherwise specified, molecular biology experimental methods and immunoassays used in the present invention are basically 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 recommendations. 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 HBsAg

1.1 Immunization of Cynomolgus Monkeys

Using a previously developed recombinant HBV vaccine (recombinant vaccine is described in detail in Chinese patent application 201710085194.3), cynomolgus monkeys weighing 4 ± 1 kg over 6 months were immunized by intramuscular injection. 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 found that at 13, 17, 25 and 29 weeks, respectively (ie 3 weeks after each immunization and multiple memories). Peripheral blood 10 ml / monkey / time of the cynomolgus monkey was sampled) at the time the B cells were generated.

1.2 Isolation of Cynomolgus Monkey Peripheral Blood Monocytes

Isolation of cynomolgus monkey peripheral blood mononuclear cells was performed with reference to the description of SepMate ^™ . Mononuclear cells (MNCs) were separated by density gradient centrifugation from human peripheral whole blood and umbilical cord blood samples using SepMate ^™ tubes. Samples, PBS buffer containing 2% fetal bovine serum (FBS) (PBS + 2% FBS), density gradient medium, and centrifuge were stored at room temperature (15 ° C.-25 ° C.) prior to separation. 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 produce small bubbles after pipette use, which will not affect performance.

2) Samples were diluted with equal volume of PBS + 2% FBS and slowly mixed thoroughly.

3) The SepMate ^™ tube was held vertical and the diluted sample was added down the side of the tube using a pipette. The sample will be mixed with the density gradient medium on the insert. Note: The sample could be poured directly down the side of the tube. Note: The diluted sample should not be poured directly through the center hole.

4) Centrifugation was performed at 1200 x g for 10 minutes at room temperature without the need to stop the centrifuge brake. Note: For samples stored over 24 hours, a 20 minute centrifuge time was recommended.

5) The top layer containing the concentrated MNC was poured into a new tube. The SepMate ^TM tube should 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 ^™ insert, but this did not affect performance.

6) MNC was washed with PBS + 2% FBS. Washes were repeated once. Note: The cells should be washed under centrifugation at 1200 × g for 8 minutes at room temperature without the need to stop the centrifuge brake.

1.3 Isolation of Memory B Cells

Since there are no specific reagents for sorting memory B cells of cynomolgus monkeys on the market, their isolation was performed using the EasySep ^™ Human Pan-B Cell Concentration 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) The wash was repeated once with 10 ml of filtered MACS buffer.

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

4) it was added to a 50 ㎕ / ㎖ the EasySep Human ^TM Pan -B cell enrichment cocktail to (EasySep Human ^TM Pan-B Cell Enrichment Cocktail) cells.

5) After incubation for 10 minutes at room temperature, it was added 75 ㎕ / ㎖ of EasySep ^TM D magnetic particles ^(TM EasySep Magnetic Particles D) in the cells.

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

7) The tube was placed on a magnetic separator for 5 minutes, then the desired positive cells were transferred to a new tube, during which unwanted negative cells remained in the original tube.

1.4 In Vitro Stimulation and Culture

1.4.1 HEK293-CD154 probe

The irradiation dose was 40 Gy and specific implementation steps were described in the instructions of the RS 2000 Biological X-Ray Irradiator.

1.4.2 Cell Smearing

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 and then cultured and plated at 250 μl / well in 96-well plates. Cells were incubated at 37 ° C. in a CO ₂ incubator and incubated for 14 days for stimulation.

1.4.3 Cell Culture Positive Well Assay

A commercial reaction plate (purchased from Beijing Wantai) for HBsAg 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 five times with PBST, and then incubated at 37 ° C. for 30 minutes by adding 100 μl of GAH-HRP diluted 1: 5000. The ELISA plate was then washed 5 times with PBST and substrate TMB solution was added. After 15 minutes of color development, the color development reaction was stopped with H ₂ SO ₄ and the readings were measured at OD450 / 620. RPMI 1640 culture medium was used as a negative control.

1.5 Acquisition of Antibody Variable Region Genes in 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 cell supernatants were aspirated. 150 μl of lysis buffer (purchased from Beijing GenMagBio) was added, pipetted up and down several times and transferred to a 1.5 ml Eppendorf tube without RNase. Samples can be stored for one year at -80 ° C in a refrigerator.

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 μl of protease K, 4 μl of acrylic carrier, 300 μl of lysis buffer and 20 μl of magnetic beads (mix the magnetic beads evenly by flipping up-down or pipetting up and down before use) Add to ml nuclease-free eppendorf tubes; An additional 200 μl sample was added and each centrifuge tube was shaken up and down for 10 minutes at room temperature to ensure uniform mixing and sufficient binding between magnetic beads and nucleic acid.

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

3) Magnetic beads were resuspended by addition of 500 μl 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 addition of 500 μl Wash Buffer II and adsorbed by magnetic shelf, the liquid in the tube was removed after 1 minute, the centrifuge tubes were simultaneously held on the magnetic shelf, and the magnetic beads were continuously Adsorbed.

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

6) 50 μl of elution buffer is added to resuspend magnetic beads; Five minutes were performed in a water bath at 55 ° C., and two light shakings were performed while completely eluting the nucleic acid in the water bath.

7) The centrifuge tube was placed on a magnetic shelf for 1 minute to allow magnetic beads to adsorb and the liquid was transferred to a new 1.5 ml nuclease free centrifuge tube.

1.5.3 RT-PCR for Antibody Variable Regions of 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) is shown in Table 2.

Reverse Transcription Primer 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 preparation method:

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

(3) It was quickly put in an ice bath and kept for 5 minutes.

(4) Solution II was then prepared according to the following recipe.

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

(6) The mixture was uniformly mixed and reverse transcription was performed for 40 minutes in a 42 캜 water bath.

(7) Packed and stored, stored at -20 ° C for short term use or -80 ° C 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 the literature (Meng W, Li L, Xiong W, et al. of monoclonal antibodies from single rhesus macaque Antibody secreting cells [C] // mAbs.Taylor & Francis, 2015, 7 (4): 707-718), wherein during the primers for the second round of nested PCR homologous to the vector The contained sequence was replaced with a fragment of the eukaryotic expression vector (see Example 1.5.5.1) of our laboratory as a homologous sequence. PCR reaction systems were prepared according to Table 3 and each system was 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 amplification cycle was 35; The first round of amplification product was used as template in the second round of the nested reaction and the reaction conditions were 95 ° C., 5 minutes; 95 ° C., 30 seconds; 58 ° C., 60 seconds; 72 ° C., 90 seconds; 72 ° C., 7 minutes; The amplification cycle was 35. The amplification product was then 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 of first round PCR (μl) Volume of second round PCR (μl) DEPC count 12.5 μl 15.5 μl 10ХPCR buffer 2.5 μl 2.5 μl 2.5 mmol / L dNTP 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 is necessary to construct a set of light and heavy chain vectors capable of efficiently recombining antibodies. In the present invention, the existing eukaryotic expression vector pTT5 in our laboratory was specifically modified to constitute a set of light and heavy chain recombinant vectors for co-transfection of double plasmids. Signal peptides for the light and heavy chains are MGWSCIILFLVATATGVHS. Downstream of the signal peptide was linked with sequences encoding light or heavy chain constant regions of human antibodies to construct pTT5-CH, pTT5-Cκ and pTT5-Cλ eukaryotic expression vector sets to facilitate antibody recombination. The constructed eukaryotic expression vector was subjected to a PCR product (primary having a sequence homologous to the vector) of the antibody variable region gene recovered in 1.5.4 by using a laboratory-made Gibson assembly solution. Ligation yielded the recombinant vectors VH + pTT5-CH, VH + pTT5-Cκ and VH + pTT5-Cλ. 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 taken from the plates and sequenced, and the sequencing results were sorted using Igblast to confirm the validity of the genes and the pseudogenes were excluded.

1.5.5.2 Small and Mass Expression of Antibody Genes

Constitutive recombinant vectors VH + pTT5-CH and VH + pTT5-Cκ and VH + pTT5-Cλ were co-transfected into HEK293-cells and co-transfected with small amounts of expression double plasmid at 500 μl per well in 24-well plates. To; If the small-expressing cell supernatant had antigenic activity, the transfection system was scaled up to 100 mL (depending on the amount of antibody) to CHO-S suspension cells (cell density: about 2 × 10 ⁶ cells / ml). Transfected cells were cultured in shake flasks at 32 ° C. in a 5% CO ₂ incubator and supernatants were 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. The specific steps thereof 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 0.22 μm. Filtration through a filter membrane with pore size. 10 ml of Sepharose 4B medium conjugated with Protein A was charged to the column 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 scan rate of pump A / B was adjusted. The column was first washed with 100% B (pH 2.3) to remove protein impurities and the pH was balanced to 10% B (pH 8.0), at the detection wavelength the signal returned to zero after stabilization and then the sample was loaded It was. After the pass peak passed, 10% B was used for balance until the signal at the detection wavelength was reduced to zero and stabilized, eluting was performed using 70% B (pH 4.0), and the eluting peaks were collected. Elution peak samples were dialyzed into PBS buffer and concentration analysis and SDS-PAGE analysis were performed to determine the purity of the IgG antibody.

Example 2: Characterization of cynomolgus monkey-human chimeric monoclonal antibodies that specifically bind HBsAg

Three cynomolgus monkey-human chimeric monoclonal antibodies (named M1-23, M3-23 and M3-13) that specifically bind to HBsAg 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: 1-5). 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 and light chain variable regions of these antibodies are shown in Table 5 (SEQ ID NOS: 6-20).

Amino acid sequence of the 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 QVQLQESGPGLVKPSEILSVTCSVSGGSITSNFWSWIRQPPGKGLEWIGYISGSGTYTDYNPSLRSRVTLSVDTSKNQLSLKLSSVTAADTAVYYCARSHDYGSNDYAFDFWGQGLRVTVSS 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

Sequences of the CDRs of the 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 IHGNGGST 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 capacities of M1-23, M3-23 and M3-13 to HBsAg were detected by ELISA. Twelve concentration gradients were obtained by performing 3-fold concentration gradient dilutions starting from 10 μg / ml. Diluted antibody was then added to a commercial reaction plate (purchased from Beijing Wantai) for HBsAg, incubated at 37 ° C. for 1 hour, then washed five times with PBST and dried. Thereafter, GAH-HRP enzyme-labeled secondary antibody was added, incubated for 30 minutes, washed five times with PBST and dried. Substrate TMB solution was added. After 15 minutes of color development, the color response was stopped with H ₂ SO ₄ and the readings were measured at OD450 / 630. The results are shown in FIG. 1, where M1-23, M3-23 and M3-13 all have good antigen-binding activity.

Competitive blocking of binding of M1-23, M3-23 and M3-13 to HBsAg by mouse monoclonal antibody 6D11 (described in Chinese patent application 201610879693.5) recognizing HBsAg SA type epitope (HBsAg aa113-135) The study was conducted by ELISA method. See Example 4.2 for the specific steps of the competitive ELISA method. As shown in FIG. 2, the results showed that mouse mAb 6D11 markedly blocked binding of M1-23, M3-23 and M3-13 to HBsAg, which represented M1-23, M3-23 and M3- 13 and mouse monoclonal antibody 6D11 recognize the same HBsAg SA linear epitope.

The virus clearance of cynomolgus-human chimeric antibodies in animals was evaluated in HBV transgenic mice. Cynomolgus monkey-human chimeric antibodies were administered to HBV transgenic mice (6 HBV transgenic mice per group) as a single dose at a dose of 10 mg / kg by tail vein injection. The blood of the mice was then collected from the posterior eye and venous gun and the changes in HBsAg and HBV DNA levels in the serum of the mice were measured. Methods for measuring HBsAg levels and HBV DNA levels in mouse serum can be found in Examples 5.1 and 5.2. The results are M1-23, M3-23 and M3-13 (the results of M1-23, M3-23 are shown in Figures 3A and 3B; removal results of M3-13 for HBsAg are shown in Figures 7A and 7B). It has been shown to have potential for 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 immunogenicity caused by heterologous antibodies administered to human subjects, we humanized two antibodies, M1-23 and M3-23. Due to the high homology of the amino acid sequences between cynomolgus monkeys and human immunoglobulins, for the two antibodies M1-23 and M3-23, only a few variable region FR amino acids are required to make the two antibodies suitable for human treatment. Is required to replace with the corresponding amino acid in the human variable region FR sequence. However, although the antibody recognizes the antigen primarily by contacting the antigen via the CDRs, some of the residues in the FR region of the antibody may also be involved in antigen-antibody interactions, thereby affecting the conformation of the CDRs. For example, after replacing the FR region of a mouse antibody with the FR region of a human antibody, the conformation of the CDRs of the mouse antibody can often change, which significantly reduces the affinity of the humanized antibody for recognizing / binding the antigen. And even 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 humanization process of M1-23 and M3-23, the location of the point mutations in the variable region FR sequences 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) with experimental experience, and based on a number of analyzes and experiments, the inventors surprisingly 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- It was found to be particularly advantageous as a template for the humanization of 23 and the relevant germline gene sequences are listed in Table 6 below.

Amino acid sequence of the human germline gene Reproductive system
gene Equivalent
The name of the 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

In view of the fact that changes in amino acids in the FR region close to the CDRs, corresponding to germline gene sequences, may affect the affinity of a humanized antibody for recognizing / binding antigens, the inventors have identified the FR regions of two antibodies. To be modified, the following single point mutations were designed while retaining amino acids near the CDR regions. Humanized M1-23 and M3-23 were named M1D and M3D, respectively.

Single point mutation of amino acid residues in the FR region of antibody M1-23 No. ^※ originally
amino acid After humanization
amino acid No. ^※ originally
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 amino acid residues in the FR region of antibody M3-23 No. ^※ originally
amino acid After humanization
amino acid No. ^※ originally
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 the 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'- GAACCTTGATGGGACCCCACTTTGCAAACGATTTGCATAATAGATCAGGAGC 1-23K2-R2 73 5'- CTGTCCCAGATCCACTGCCACTGAACCTTGATGGGACCCC 1-23K-F2 74 5'- GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAGG 1-23K-F3 75 5'- GCAGCCTGCAACCTGAGGATTTTGCAACTTATTACTGTCAAC PTT5-1-23K-R 76 5'- GACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCAT 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'-CCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGAGAGAGGGCCACCATCAACTGCAAGTCCAG 3-23KV2-R1 85 5'- CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCTTTTACtgggcatctACCCGGGAAT 3-23KV1-R2 86 5'-GTGAAATCTGTCCCAGATCCACTGCCACTGAATCGGTCAGGGACCCCGGATTCCCGGGTagatgcc 3-23KV1-F2 87 5'- GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATG PTT5-3-23KV1-R 88 5'- ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC

Following the single point mutations listed in Table 7 and Table 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. As an example, considering the humanization of the M1-23 heavy chain variable region (M1-23-H), oligonucleotide primers PTT5-1-23H-F, 1-23H-F1, 1-23H-F2, 1-23H-R1, 1-23H-F3 and PTT5-1-23H-R, whose sequences are shown in the table above, were designed. The specific protocol of PCR was as follows: A gene encoding M1-23-H was used as template, primer pairs 1-23H-F1 / 1-23H-R1 and 1-23H-F3 / PTT5-1-23H- R was used for amplification to yield 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; Fragments 4 and 2 were ligated together by redundant extension PCR and then PTT5-1-23H-F / PTT5-1-23H-R were 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. .

The humanized gene fragments M1D-H and M3D-H as obtained were ligated with the eukaryotic expression vector pTT5-CH using lab-produced Gibson assembly liquid, respectively, and M1D-L and M3D-L, respectively, The recombinant vector was obtained by ligation with the expression vector pTT5-Cκ. Recombinant vectors were transformed into Escherichia coli DH5α strains, plated on LB plates and incubated overnight at 37 ° C. incubator. Monoclonal colonies were taken from the plates and sequenced. Plasmids with the correct sequence were selected and the expression and purification of the antibodies were performed according to the method of Example 1.

The amino acid sequences of the 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 preparation method. As shown in Table 10 below, after the humanization of the two antibodies, the degree of humanization increased from about 90% to about 98%.

Sequence Analysis of Humanized Antibodies Before humanization After humanization Antibody Name Humanization degree Antibody Name Humanization degree M1-23 90% M1D 98.33% M3-23 91.67% M3D 97.78%

Degree of humanization = (number of amino acids in the FR region-number of monkey-derived amino acids maintained 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 Equivalent
The name of the 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 antigen HBsAg was examined by ELISA method (enzyme-linked immunosorbent assay). Twelve concentration gradients were obtained by performing 3-fold concentration gradient dilutions starting from 10 μg / ml. Humanized antibody 162 (described in detail in Chinese patent application CN201610879693.5) was used as reference antibody. Subsequently, the diluted antibody was added to a commercial reaction plate (purchased from Beijing Wantai) for HBsAg, incubated at 37 ° C. for 1 hour, then washed five times with PBST and dried. Then, GAH-HRP enzyme-labeled secondary antibody was added, incubated for 30 minutes, washed five times with PBST, and dried. Subsequent substrate TMB solution was added. After 15 minutes of color development, the color response was stopped with H ₂ SO ₄ and the readings were measured at OD450 / 630. The results are shown in FIG. 4, where both humanized antibodies M1D and M3D had excellent antigen-binding activity, and their affinity for antigen HBsAg was better than that of reference antibody 162. The results indicated that not only did two humanized antibodies have a very high degree of humanization (greater than 97%), but also substantially retained the antigen-binding activity of their parental antibodies and even showed better antigen binding activity than their parental antibodies. .

4.2 Preliminary Epitope Identification of Antibodies

We performed preliminary identification of epitopes for antibodies to determine whether selected human-monkey chimeric antibodies and humanized antibodies recognize SA epitopes on HBsAg. Using 6D11, which recognizes high concentrations of SA type epitopes, cross-blocked the binding of the antibody to test to HBsAg, where there was a significant blocking (competitive) effect, the antibody to be tested was subjected to the same epitope as 6D11. Recognized. The specific steps were as follows:

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

(2) 6D11-HRP diluted 1: 20000 with ED-11 was added to the 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 measured at OD450 / 630.

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

4.3 Measurement of Neutralizing Activity of Antibodies

We used a laboratory-produced cell model HepaG2-NTCP to assess the ability of antibodies to neutralize HBV infection (with HBV infection titers of 100 MOI). A certain amount of HBV and gradient-diluted antibodies were co-cultured in HepaG2 cells, and HBV could replicate in cells by incorporating differentiated HepaG2 cells. With the addition of different antibodies and the change in the amount of antibody added, the invasion and replication of HBV was weakened or inhibited to a different degree, so different levels of HBV antigen (HBeAg) would have been present in the cell supernatant. Thus, by measuring the level of HBV antigen (HBeAg), the ability of different humanized antibodies to neutralize HBV could be measured.

Humanized antibodies were diluted to 2-fold gradients and 162 antibodies were used as positive controls. From 10 μg / ml, a total of 18 dilution gradients were made. The viral infection system was mixed separately with the antibody and pre-incubated for 1 hour. This was then added to the cell plates pre-plated with HepaG2, incubated at 37 ° C. for 24 hours, and the medium was replaced with fresh every two days. Seven days after infection, cell culture supernatants were harvested to determine HBeAg levels. The method of measurement included the following steps:

(1) Preparation of reaction plates: 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 a 96-well chemiluminescent plate 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 the 96-well chemiluminescent plate at 200 μl / well and blocked at 37 ° C. for 2 hours. The blocking solution was then discarded, the plate was left in a dry room and dried 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: 96-well chemiluminescent plates were 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 material was purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.

(5) Washing: 96-well chemiluminescent plates were washed 5 times with PBST and dried.

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

(7) Read 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 reference antibody 162.

Example 5 Determination of Therapeutic Effects of Humanized Antibodies M1D, M3D

The five antibodies were separately administered to HBV transgenic mice at a single dose of 10 mg / kg, each group containing six HBV transgenic mice. Blood was then collected from the post-orbital venous total of mice and the change in HBsAg levels and HBV DNA levels in serum of mice was measured.

5.1 Quantitative Measurement of HBsAg

(1) Preparation of reaction plates: 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 It was added to the chemiluminescent plate. Plates were coated for 16-24 hours at 6 ° C. to 8 ° C. and then at 37 ° C. for 2 hours, then washed once with PBST washes 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, the plates were placed in a dry room and dried and stored at 2 ° C. to 8 ° C. until use.

(2) Sample Dilution: Collected mouse blood was diluted in two gradients, 1:30 and 1: 150, with a PBS solution containing 20% NBS (baby serum) for subsequent quantitative detection.

(3) Denaturation 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. 90 μl of neutralization buffer (4% CHAPS, dissolved in 20 mM PB7.4) was then added and mixed well.

(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 five times with PBST and dried.

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

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

(7) Calculation of HBsAg Concentration in Mouse Serum Samples: Parallel experiments were performed on standard samples, and standard curves were prepared based on the measurement results of standard samples. 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 Measurement of HBV DNA

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

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

The above results show that the humanized antibodies of the present invention have a very high degree of humanization (up to 98%), which not only reduces the possibility of immunological rejection, but are equivalent to that of the monkey-human chimeric antibody and more than that of the reference antibody 162. Demonstrated good virus removal capacity. Such technical effects were remarkable and unexpected.

Example 6: Determination of Insignificant CDR Regions of Humanized Antibodies M1D, M3D

In the antibody variable region domains, the site that directly binds to the antigen is primarily in the CDR region, but many studies have shown that not all six CDRs of an antibody are important regions for antigen-antibody binding, and that non-critical CDR regions are used for CDR replacement methods. It was shown that it can be measured by. In the present invention, replacement sequences for the original CDRs were selected according to the principle of optimal agreement. The VH and VL amino acid sequences of humanized antibodies M1D and M3D were entered into a search box of the IMGT information system, and the corresponding amino acid sequences of the antibody light and heavy chains in the germline gene were then searched by the system. In accordance with the principle that the FR regions had the best homology and the CDR region sequences had the greatest difference, two replacement sequences were selected for each CDR region in the humanized antibody, except for the heavy chain CDR3. 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 IHGNGGST 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 of the M1D and M3D antibodies were replaced by PCR methods. Primers as designed are shown in the table below. As an example, considering the first substitution of the M1D heavy chain variable region CDR1 (M1D-HCDR1) (M1D-H-CDR1-A), the oligonucleotide primers M1D-H-CDR1-A R and M1D-H-CDR1 F are designed and (The sequence is shown in the table below.) The antibody after the above replacement of the CDRs was named M1D HCDR1-1. Naming of other replacement antibodies could be performed in the same manner. The specific protocol of PCR was as follows: The coding gene of M1D-H was used as template, primer pair 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 gene fragments with replacement of HCDR1 of M1D.

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

According to the method, the gene fragment of the heavy chain having the CDR replacement obtained is 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. The antigenic activity of the cell supernatant in small amounts was measured. Similarly, the gene fragment of the light chain with CDR replacement obtained is ligated separately to the eukaryotic expression vector pTT5-Cκ, and then co-transfected with the expression vector of the corresponding heavy chain without CDR replacement, and the antigen- of the transfection supernatant. Binding activity was measured.

After replacing several CDR regions of a humanized antibody, if the corresponding cell transfection supernatants show no antigen-binding activity or a significant decrease in antigen-binding activity, the CDR regions replaced are important for antibody-antigen binding. Could conclude that it is a realm; If the corresponding cell transfection supernatants showed substantially unchanged antigen-binding activity after replacement, the replaced CDR regions were not important for antibody-antigen binding.

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

Replacement results for M3D are shown in FIGS. 9A-9E. After the 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, while the other replacement (M3D-LCDR1-2) showed substantially no antigen-binding activity; After replacement of heavy chain CDR1 (HCDR1) and light chain CDR3 (LCDR3), there was substantially no antigen-binding activity. The results indicated that HCDR2 and LCDR2 of antibody M3D are not critical CDR regions for antigen binding and their replacement does not affect the binding activity of the antibody to the antigen.

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

To determine amino acid sites important for antibody-antigen interactions, we performed alanine scanning on the four CDR regions HCDR2, HCDR3, LCDR1, LCDR3, and M3D four CDR regions HCDR1, HCDR3, LCDR1, LCDR3. It was. In general, important amino acids are more or less conserved, and considering amino acid residues that maintain the conformation of the antibody and, for example, 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 post-mutant antibody was calculated (WT / mutant, relative value of the original antibody EC50 was 1 and 0 indicates complete loss of binding activity. ), Including amino acid residues such as D, E, R, H, L, W and Y, with post-mutation rEC50 <0.2 (ie, activity reduced by 5 fold) is indicated in blue color. The results are shown in Table 14. Acidic amino acids D, E and basic amino acids R and H could form salt bridges when the antibody binds to the antigen, thereby playing an important role in the antibody-antigen binding reaction, thus mutating these amino acids to alanine Will disrupt the formation of ionic bonds and will significantly reduce antibody-binding activity. Aliphatic amino acids L were hydrophobic amino acids, W and Y were aromatic amino acids, which played an important role in maintaining the antibody conformation. Among them, mutations of M1D to H56Y, H97D, H100cD, H100dL, 94L, L96L to alanine resulted in a significant decrease in activity, thus they were sites that should not be changed; Mutations of M3D to H33Y, H34D, H35W, H94R, H95D, H101E, L27dY, L32Y, L91H to alanine resulted in a significant decrease in activity and thus 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 epitope 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. 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 antibodies to polypeptides with different single point mutants was measured by the Elisa method, and the binding curves were generated using PRISM software. If post-mutation binding activity was lost, the mutation site was an important amino acid for the SA linear epitope.

Point mutant peptide sequence based on SEQ13-B Name of the 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 polypeptides are summarized in Table 16 below (> 500 indicated 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 In position; The core epitope for M3D binding was CKTCT (aa121-125), which was in the reverse position compared to the 162 case.

EC50 values for binding of M1D, M3D to single point mutant polypeptides based on HBsAg (aa113-135) EC50 (ng / mL) Name of the 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

In order to further identify an important site of the antibody for binding to the antigen, we demonstrated this experiment in the HBsAg antigen protein. The relevant primers were designed (Table 17) and 14 HBsAg gene fragments with point mutations were obtained by amplification via PCR. Fragments obtained by amplification were separately ligated using Gibson to PTT5 empty vectors subjected to double enzyme cleavage by EcoRI and BamHI and completed vector construction when the sequencing results were correct. The constructed plasmid was used for small transfection of HEK293T cells, and after 2 days transfection, cells were collected and confirmed by Western blot. The specific steps were as follows:

(1) cells were collected and washed twice with 5 ml of PBS and centrifuged for 5 min at 1000 rpm 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 quantitated by the BCA method), and lysis was performed at 4 ° C. for 2 hours, during which time with a shaker. Shaking was performed several times;

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

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

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

(6) washing was performed with PBST three times, once every 5 minutes;

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

(8) washes were performed with PBST three times, once every 5 minutes;

(9) development and photography;

(10) β-actin was used as internal reference, in step (4) the region corresponding to β-actin on the PVDF membrane was cut and HRP-conjugated mouse anti-β-actin antibody (1: 5000) was obtained. Direct incubation at 30 ° C. was followed by other steps 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 are completely consistent with the case of the linear polypeptide.

Primer 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 CTTGCATGGTGCCGTGCTGGTAGTTGATGTTCCTGG C-P120A-F 189 CAGCACGGGAGCATGCAAGACCTGCACGATTCCTGC C-P120A-R 190 GTCTTGCATGCTCCCGTGCTGGTAGTTGATGTTCCT C-C121S-F 191 ACGGGACCATCCAAGACCTGCACGATTCCTGCTCAA C-C121S-R 192 GCAGGTCTTGGATGGTCCCGTGCTGGTAGTTGATGT 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 the widespread activity of M1D and M3D

In order to further confirm the broad spectrum activity of M1D and M3D, each HBsAg fragment (aa113-135) corresponding to different subtypes of A to G (where F type included F1 and F2) (in their sequence (See Table 18 for details), and the reaction plates were coated, 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, then 3-fold dilution to obtain 12 gradients. The binding activity of M1D, M3D to different subtypes of HBsAg (aa113-135) was measured 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 the polypeptide SEQ ID NO: Sequence of 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: Measurement of affinity of M1D and M3D

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

Affinity to the antigens of M1D, M3D Analytes 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 Initial Toxicity of Antibodies M1D and M3D in Cynomolgus Monkeys After Single Intravenous Injection

Six male cynomolgus monkeys that did not experience any experiment involving macromolecules (> 2000 Daltons) were divided into two groups of three animals / group. Among them, doses of 20 mg / kg of solutions of antibodies M1D and M3D were 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 initial toxicity of M1D and M3D molecules group Experiment The number of animals Route of administration Dosage Animal code One M1D 3 IV bolus injection 20 mg / kg C1001,
C1002,
C1003 2 M3D 3 IV bolus injection 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). Physical examination of the experimental animals was performed prior to the experiment to confirm the health of the animals. On the day of dosing, the condition of the test animal was observed before and after each blood collection, including the general condition, behavior, activity, excretion, respiration and other abnormal symptoms of the test animal. The results indicated 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 tracked after administration. The results are shown in Tables 21-24.

Body weight of experimental animals after M1D administration time 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 21st 4.15 3.81 3.72 28 days 4.01 3.99 3.87

Body temperature of experimental animals after M1D administration time Body temperature (℃) C1001 C1002 C1003 Before dosing 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 M3D administration time 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 21st 3.68 3.33 3.7 28 days 3.95 3.39 3.81

Body temperature of experimental animals after M3D administration time 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 antibodies M1D and M3D did not cause any side effects in experimental animals after administration, whereby good safety of the two antibodies was previously demonstrated.

In addition, about 1.0 ml of whole blood was administered before (0 hour), 0.25 h (15 minutes), 0.5 h (30 minutes), 1 h, 2 h, 4 h, 10 h, 24 h (1 day), 48 h (2 days) after administration , 72h (3 days), 96h (4 days), 144h (6 days), 192h (8 days), 240h (10 days), 336h (14 days), 408h (17 days), 504h (21 days) and 672h On day 28, they were collected from the lumbar cortical vein of the experimental animals. Collected serum and whole blood were subjected to the following analysis.

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

12A shows a curve graph of 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 of humanized antibody M1D.

12B shows a curve graph of 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 of humanized antibody M3D.

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

The following parameters were calculated using the algebraic linear trapezoidal method: initial serum drug concentration (C0), final detectable time (T _last ), elimination half-life (T _1/2 ), apparent distribution volume (V _dss ), overall Removal (CL), mean residence time from time _zero (MRT _0-last ) to the final point at which concentration can be detected, mean residence time from time zero to infinity (MRT _0-inf ), concentration detected from time zero Area-time curve below serum concentration (AUC _0-last ) to the _{last possible} time point, and area-time curve below serum concentration from time zero to infinity (AUC _0-inf ).

The results showed that the CL of male cynomolgus monkeys against humanized antibody M1D was 0.00962 ± 0.00335 ml / min / kg after a single intravenous injection of 20 mg / kg of humanized antibody M1D. The mean clearance half-life (t _1/2 ) of the humanized antibody M1D was 81.3 ± 78.7 hours. The Vdss and AUC _0-inf values of 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 to humanized antibody M3D was 0.0049 ± 0.0025 ml / min / kg. The mean clearance half-life (t _1/2 ) of humanized antibody M3D was 134 ± 94.6 hours. The Vdss and AUC _0-inf values of 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 Table 25 and Table 26.

Main Pharmacokinetic Parameters of Humanized Antibody M1D in Serum of Male Cynomolgus Monkeys of Group 1 PK parameter C1001 C1002 C1003 Average IV SD CV (%) Rsq_adj 0.800 0.830 0.581 - ± - - The number of _views used for 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 AUCExtra (%) 0.00695 0.00181 2.51 0.840 ± 1.45 172 AUMCExtra (%) 0.0369 0.0245 23.2 7.74 ± 13.3 173

Main Pharmacokinetic Parameters of Humanized Antibody M3D in Serum of Male Cynomolgus Monkeys of Group 2 PK parameter C2001 C2002 C2003 Average PO SD CV (%) Rsq_adj 0.949 0.889 0.881 - ± - - T _1/2 Views used for 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 AUCExtra (%) 1.17 0.0323 17.4 6.21 ± 9.74 157 AUMCExtra (%) 5.27 0.280 49.3 18.3 ± 27.0 148

Conventional serum chemistry assays were also performed on the sera 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 ions, phosphorus, total Cholesterol, triglycerides, sodium ions, potassium ions, chloride ions and globulin levels) showed no apparent abnormality. These test results showed that a single intravenous injection of humanized antibody M1D or M3D was safe under the specified dosage conditions. Thus, 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 Medicinal Properties of M1D and M3D

1. Measurement of isoelectric points of humanized antibodies M1D and M3D

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

Isoelectric point of M1D, M3D Isoelectric point (pI) Peak area Antibodies 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 the 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). Set initial scan temperature to 10 ° C, end point scan temperature to 110 ° C, scan rate to 200 ° C / hour, cooling rate to Exp, and final temperature to be maintained by the instrument to 25 ° C The data acquisition frequency was set to 10 seconds and the capillary temperature before injection 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 Tm onset of the humanized antibody M1D was above 61.7 ° C. and the Tm onset value of M3D both exceeded 61.6 ° C., indicating that M1D and M3D had good thermal stability.

Tm value of M1D Buffer Tm start (℃) Tm1 (℃) Tm2 (℃) Acetate pH5.0 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 start (℃) 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 Of

Humanized antibodies M1D and M3D were also dissolved in histidine buffer at pH 6.0 at a concentration of 60 mg / ml, further dissolved in pH 7.2 phosphate buffer at a concentration of 2 mg / ml, and then stored at 40 ° C., respectively. . Physical and chemical properties (including appearance, pH, protein concentration, etc.) of the samples were monitored at Week 0 (T0), Week 1 (T1W), and Week 2 (T2W). The results are shown in Tables 30 and 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 30-31 showed no significant changes in the samples of the two buffers and 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 humanized antibodies M1D and M3D had no significant changes in pH and protein concentration after two weeks of storage at 40 ° C. in different buffers.

SEC-HPLC Exam

Samples of humanized antibodies M1D and M3D with different storage conditions were subjected to Agilent 1260 Infiniti, 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) and analyzed by SEC-HPLC. The experimental results are shown in Tables 30 and 31 above. The results showed that the main peaks of the samples containing humanized antibodies M1D and M3D all exceed 95% under different storage conditions (different buffers, different storage times). This indicated that the humanized antibodies M1D and M3D are stable.

cIEF test

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

3. Solubility Analysis of Humanized Antibody M1D and M3D Variants

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

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

While specific embodiments of the 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 such changes are within the scope of the invention. The entirety of the present invention is provided by the appended claims and any equivalents thereof.

<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 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 variable 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 Gln 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   One <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> Artificial Sequence <220> <223> M3-23 / M3-13 LCDR2 <400> 16 Trp Ala Ser   One <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 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 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 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 Ile 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 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> 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 Phe 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 cagatttcaa 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 caggagtcag gcccaggact ggtgaagcct tcggagaccc 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 tgtagt 56 <210> 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 gcaaccggtg tacattctga catacagatg acgcagtctc catcc 55 <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 ggtgcagctg caggagtcgg gcccaggact 60 ggtgaagcc 69 <210> 78 <211> 58 <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 aactggttct tggacttgtc tactgaaatg gtgactcgac tcttgaggga ggggttg 57 <210> 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 gctgtgtctc tgggagagag ggccaccatc aactgcaagt 60 ccag 64 <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 caccgtacgt 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> 42 <212> 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> Artificial 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 agctagttgc 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 <212> 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 49 <210> 114 <211> 38 <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 38 <210> 117 <211> 54 <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 ccagttacta ctgctgatgg agccaccaga gacagcgcag gtgagggaca g 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 ccatcactat gcaggaggct ctggctggac ttgcagttga tggtggc 57 <210> 126 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 126 cagagcctcc tgcatagtga tggaaagacc tatttagcct ggtaccagca gaaac 55 <210> 127 <211> 45 <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 cggtgcatcc 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 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGHV4-4 * 08) for M1D HCDR-1 <400> 137 Gly Gly Ser Ile 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 <139> <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-A (IGKV1-39 * 01) for M1D LCDR-2 <400> 139 Ala Ala Ser   One <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   One <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   One <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   One <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 Ile 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 Val Thr Ala Ala Asp 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 Gln Gly Thr Thr Val Val 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 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> 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 Gln 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 Ile 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 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 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 Thr 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 <175> 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> Artificial 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 acgggaccat ccaagacctg cacgattcct gctcaa 36 <210> 192 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 192 gcaggtcttg gatggtcccg tgctggtagt tgatgt 36 <210> 193 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 193 gggaccatgc cggacctgca cgattcctgc tcaagg 36 <210> 194 <211> 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 <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 <206> 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> Artificial 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 ttccctcttg ttgctg 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> 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> 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> 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> 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> 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 Gln 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 15 Gln Gly Asn Ser Met Tyr Pro              20

Claims (26)

An antibody or antigen-binding fragment thereof that can specifically bind HBsAg,
(a) the sequence by (i) SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VH CDR1 consisting of a sequence selected from a sequence different from;
(ii) a sequence that differs from the sequence by SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR2 consisting of a sequence selected from among; And
(iii) a sequence that differs from the sequence by SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or one or more amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions) VH CDR3 consisting of a sequence selected from among
One or more (eg 1, 2 or 3) heavy chain variable regions (VH) complementarity determining regions (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 the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR1 consisting of the selected sequence;
(v) a sequence selected from the sequence that differs from the sequence by SEQ ID NO: 10, SEQ ID NO: 16, or one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR2; And
(vi) SEQ ID NO: 11, SEQ ID NO: 17, or a sequence selected from the sequence that differs from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL CDR3 consists of
One or more (eg 1, 2 or 3) light chain variable region (VL) CDRs selected from the group consisting of
An antibody or antigen-binding fragment thereof comprising a.
The method of claim 1,
(a) a sequence selected from sequences different from SEQ ID NO: 7 by (i) SEQ ID NO: 7, or one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR2;
(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 or multiple amino acid substitutions, deletions or additions (e.g., one, two or three 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 a; And / or
(b) (iv) a sequence selected from SEQ ID NO: 9 or a sequence different from SEQ ID NO: 9 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;
(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 11 or a sequence different from SEQ ID NO: 11 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And
(vi) a VL CDR2 consisting of a sequence selected from the sequences of VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin
Light chain variable region (VL) comprising a
It includes;
Preferably, VH CDR1 is the sequence of VH CDR1 comprised in the heavy chain variable region of human immunoglobulin;
Preferably, the VH CDR1 is (a) a sequence that differs from SEQ ID NO: 6 by SEQ ID NO: 6 or one or multiple amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions); Or (b) a sequence selected from the sequence of VH CDR1 contained in the amino acid sequence encoded by the human heavy chain germline gene;
Preferably, the human heavy chain germline gene is selected from the group consisting of IGHV4-4 ^* 08 and IGHV4-61 ^* 01;
Preferably, the VH comprises a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR3 as shown in SEQ ID NO: 8, and a VH CDR1 having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 137 or SEQ ID NO: 138;
Preferably, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of the human immunoglobulin;
Preferably, the VL CDR2 is (a) a sequence different from SEQ ID NO: 10 by SEQ ID NO: 10 or one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions); Or (b) a sequence selected from the sequences of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene;
Preferably, the human light chain germline gene is selected from the group consisting of IGKV1-39 ^* 01 and IGKV1-5 ^* 03;
Preferably, the VL comprises a VL CDR1 as shown in SEQ ID NO: 9, a VL CDR3 as shown in SEQ ID NO: 11, and a VL CDR2 having an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 139 or SEQ ID NO: 140;
Preferably, the VH comprises a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR3 as shown in SEQ ID NO: 8, and a VH CDR1 having an amino acid sequence of SEQ ID NO: 6, 137 or 138; The VL comprises a VL CDR1 as shown in SEQ ID NO: 9, a VL CDR3 as shown in SEQ ID NO: 11, and a VL CDR2 having an amino acid sequence of SEQ ID NO: 10, 139 or 140
Antibodies or antigen-binding fragments thereof.
The method of claim 2,
(i) 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 sequences of VH CDR1 (eg, SEQ ID NO: 6, 137 or 138) contained in the heavy chain variable region of any immunoglobulin; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR3 as shown in SEQ ID NO: 11, and VL CDR2 as shown in SEQ ID NO: 10; or
(ii) 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 sequence of VL CDR2 contained in a VL CDR1 as shown in SEQ ID NO: 9, a VL CDR3 as shown in SEQ ID NO: 11, and a light chain variable region (eg κ light chain variable region) of any immunoglobulin (eg VL CDR2 consisting of a sequence selected from SEQ ID NO: 10, 139 or 140)
It includes;
Preferably, VH CDR1 is the sequence of VH CDR1 contained in the heavy chain variable region of human immunoglobulin;
Preferably, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of the human immunoglobulin;
Preferably,
(1) VH CDR1 as shown in SEQ ID NO: 6, VH CDR2 as shown in SEQ ID NO: 7, and VH CDR3 as shown in SEQ ID NO: 8; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and VL CDR3 as shown in SEQ ID NO: 11;
(2) VH CDR1 as shown in SEQ ID NO: 137, VH CDR2 as shown in SEQ ID NO: 7, and VH CDR3 as shown in SEQ ID NO: 8; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and VL CDR3 as shown in SEQ ID NO: 11;
(3) VH CDR1 as shown in SEQ ID NO: 138, VH CDR2 as shown in SEQ ID NO: 7, and VH CDR3 as shown in SEQ ID NO: 8; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and VL CDR3 as shown in SEQ ID NO: 11;
(4) VH CDR1 as shown in SEQ ID NO: 6, VH CDR2 as shown in SEQ ID NO: 7, and VH CDR3 as shown in SEQ ID NO: 8; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 139, and VL CDR3 as shown in SEQ ID NO: 11; or
(5) VH CDR1 as shown in SEQ ID NO: 6, VH CDR2 as shown in SEQ ID NO: 7, and VH CDR3 as shown in SEQ ID NO: 8; And VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 140, and VL CDR3 as shown in SEQ ID NO: 11.
An antibody or antigen-binding fragment thereof comprising a.
The method of claim 1,
(a) (i) SEQ ID NO: 12 or a sequence selected from a sequence different from SEQ ID NO: 12 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;
(ii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 14 or a sequence different from SEQ ID NO: 14 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And
(iii) a VH CDR2 consisting of a sequence selected from the sequences of VH CDR2 contained in the heavy chain variable region of any immunoglobulin
A heavy chain variable region (VH) comprising a; And / or
(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;
(v) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And
(vi) a VL CDR2 consisting of a sequence selected from the sequences of VL CDR2 contained in the light chain variable region (eg, κ light chain variable region) of any immunoglobulin
Light chain variable region (VL) comprising a
It includes;
Preferably, VH CDR2 is the sequence of VH CDR2 contained in the heavy chain variable region of human immunoglobulin;
Preferably, the VH CDR2 is (a) a sequence that differs from SEQ ID NO: 13 by SEQ ID NO: 13 or one or multiple amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions); Or (b) a sequence selected from the sequence of VH CDR2 contained in the amino acid sequence encoded by the human heavy chain germline gene;
Preferably, the human heavy chain germline gene is selected from the group consisting of IGHV4-30-4 ^* 07 and IGHV4-4 ^* 01;
Preferably, the VH has a amino acid sequence selected from the group consisting of (a) VH CDR1 as shown in SEQ ID NO: 12, VH CDR3 as shown in SEQ ID NO: 14, and SEQ ID NO: 13, SEQ ID NO: 141 and SEQ ID NO: 142. Comprises CDR2;
Preferably, VL CDR2 is the sequence of VL CDR2 contained in the light chain variable region of human immunoglobulin;
Preferably, the VL CDR2 is (a) a sequence different from SEQ ID NO: 16 by SEQ ID NO: 16 or one or multiple amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions); Or (b) a sequence selected from the sequences of VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene;
Preferably, the human light chain germline gene is selected from the group consisting of IGKV2-28 ^* 01 and IGKV3-15 ^* 01;
Preferably, the VL comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 143, and SEQ ID NO: 144 and;
Preferably, the VH comprises a VH CDR1 as shown in SEQ ID NO: 12, a VH CDR3 as shown in SEQ ID NO: 14, and a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 141 and SEQ ID NO: 142. And VL comprises a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 143, and SEQ ID NO: 144
Antibodies or antigen-binding fragments thereof.
The method of claim 4, wherein
(i) a sequence of VH CDR1 as shown in SEQ ID NO: 12, VH CDR3 as shown in SEQ ID NO: 14, and a sequence of VH CDR2 contained in the heavy chain variable region of any immunoglobulin (eg SEQ ID NO: 13, SEQ ID NO: 141) Or VH CDR2 consisting of the sequence selected from SEQ ID NO: 142); And VL CDR1 as shown in SEQ ID NO: 15, VL CDR3 as shown in SEQ ID NO: 17, and VL CDR2 as shown in SEQ ID NO: 16; or
(ii) VH CDR1 as set forth in SEQ ID NO: 12; VH CDR3 as set forth in SEQ ID NO: 14; And VH CDR2 as set forth in SEQ ID NO: 13; And a sequence of a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and a VL CDR2 contained in the light chain variable region of any immunoglobulin (eg, SEQ ID NO: 16, SEQ ID NO: 143 or sequence). VL CDR2 consisting of a sequence selected from No. 144)
It includes;
Preferably, VH CDR2 is the sequence of VH CDR2 contained in the heavy chain variable region of human immunoglobulin;
Preferably, the VL CDR2 is the sequence of the VL CDR2 contained in the light chain variable region (eg κ light chain variable region) of the human immunoglobulin;
Preferably,
(1) VH CDR1 as shown in SEQ ID NO: 12, VH CDR2 as shown in SEQ ID NO: 13, and VH CDR3 as shown in SEQ ID NO: 14; And VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and VL CDR3 as shown in SEQ ID NO: 17;
(2) VH CDR1 as shown in SEQ ID NO: 12, VH CDR2 as shown in SEQ ID NO: 141, and VH CDR3 as shown in SEQ ID NO: 14; And VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and VL CDR3 as shown in SEQ ID NO: 17;
(3) VH CDR1 as shown in SEQ ID NO: 12, VH CDR2 as shown in SEQ ID NO: 142, and VH CDR3 as shown in SEQ ID NO: 14; And VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and VL CDR3 as shown in SEQ ID NO: 17;
(4) VH CDR1 as shown in SEQ ID NO: 12, VH CDR2 as shown in SEQ ID NO: 13, and VH CDR3 as shown in SEQ ID NO: 14; And VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 143, and VL CDR3 as shown in SEQ ID NO: 17; or
(5) VH CDR1 as shown in SEQ ID NO: 12, VH CDR2 as shown in SEQ ID NO: 13, and VH CDR3 as shown in SEQ ID NO: 14; And VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 144, and VL CDR3 as shown in SEQ ID NO: 17.
An antibody or antigen-binding fragment thereof comprising a.
The method of claim 1,
(a) (i) SEQ ID NO: 18 or a sequence selected from a sequence different from SEQ ID NO: 18 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) Consisting of VH CDR1;
(ii) a VH CDR2 consisting of a sequence selected from SEQ ID NO: 19 or a sequence different from SEQ ID NO: 19 by one or a plurality of amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) ; And
(iii) a VH CDR3 consisting of a sequence selected from SEQ ID NO: 20 or a sequence different from SEQ ID NO: 20 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions)
A heavy chain variable region (VH) comprising a; And / or
(b) (iv) a sequence selected from SEQ ID NO: 15 or a sequence different from SEQ ID NO: 15 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions). Consisting of VL CDR1;
(v) a VL CDR2 consisting of a sequence selected from SEQ ID NO: 16 or a sequence different from SEQ ID NO: 16 by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) , And
(vi) a VL CDR3 consisting of a sequence selected from SEQ ID NO: 17 or a sequence different from SEQ ID NO: 17 by one or multiple amino acid substitutions, deletions or additions (eg, one, two or three amino acid substitutions, deletions or additions)
Light chain variable region (VL) comprising a
It includes;
Preferably, the VH comprises VH CDR1 as shown in SEQ ID NO: 18, VH CDR2 as shown in SEQ ID NO: 19, and VH CDR3 as shown in SEQ ID NO: 20, and VL CDR1 as shown in SEQ ID NO: 15 , VL CDR2 as set forth in SEQ ID NO: 16, and VL CDR3 as set forth in SEQ ID NO: 17
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 6,
Humanized;
Preferably have a degree of humanization of 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%, or at least 98%;
Preferably, the non-CDR region 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, 6 or less, 5 or less, 4 or less, or 3 or less. Comprising amino acid residues of a non-human source of (e.g., cynomolgus monkey source)
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 7,
Further comprises a framework region FR;
Preferably, (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 VH FR1 consisting of a sequence selected from sequences differing from said sequence by amino acid substitution, deletion or addition);
(ii) by 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, one, two or three 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 (eg, one, two or three amino acid substitutions, deletions or additions) VH FR3 consisting of a sequence selected from a sequence different from said sequence; 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 (e.g., one, two or three amino acid substitutions, deletions or additions) VH FR4 consisting of a sequence selected from a sequence different from the sequence
One or more (eg 1, 2, 3 or 4) heavy chain variable region (VH) framework regions (FR) selected from among those; 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, one, two or three amino acid substitutions, deletions or additions) VL FR1 consisting of a sequence selected from a sequence different from said sequence;
(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 (e.g., one, two or three 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 (e.g., one, two or three 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 sequences different from the sequence by one or multiple amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) VL FR4 made up
One or more (eg 1, 2, 3 or 4) light chain variable region (VL) framework regions (FR) selected from
It includes;
Preferably, (a) at least 80% of the heavy chain framework region contained in the heavy chain variable region selected from among the heavy chain variable regions as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164, 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% Heavy chain framework regions having homology; And / or
(b) at least 80%, at least 85%, at least compared to the light chain framework region contained in the 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 Light chain frame with sequence homology of 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% Work area
It includes;
Preferably, (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 the light chain variable regions as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166
Containing
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 8,
Further comprising a framework region of a human immunoglobulin (eg, a framework region contained in an amino acid sequence encoded by a human germline antibody gene), wherein the framework region is one or more from human residues to cynomolgus monkey residues Optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 reverse mutations;
Preferably, a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene;
Preferably 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, the heavy chain framework region and And / or the light chain framework region optionally comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reverse mutations from human residues to cynomolgus monkey residues. and;
Preferably 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, wherein the heavy chain framework region and And / or the light chain framework region optionally comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reverse mutations from human residues to cynomolgus monkey residues. doing
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 9,
At least 80% of the amino acid sequence of the heavy chain variable region as compared to the amino acid sequence of the heavy chain variable region selected from the group consisting of heavy chain variable regions as set forth in SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164, 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% Have the same sex;
Preferably, the heavy chain variable region 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
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 10,
At least 80% of the amino acid sequence of the light chain variable region compared to the amino acid sequence of the light chain variable region selected from the group consisting of the light chain variable regions as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 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% of the sequence Homology,
Preferably, the light chain variable region 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.
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 11,
An antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region according to claim 11 and a light chain variable region according to claim 12.
The method according to any one of claims 1 to 12,
(a) the heavy chain variable region 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 is a light chain variable region as set forth in any one of SEQ ID NOs: 2, 58, 159 and 160; or
(b) the heavy chain variable region 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 is a light chain variable region as set forth in any one of SEQ ID NOs: 4, 60, 165 and 166
Antibodies or antigen-binding fragments thereof.
The method according to any one of claims 1 to 13,
Antibodies
(1) VH as shown in SEQ ID NO: 1 and VL as shown in SEQ ID NO: 2;
(2) VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO: 4;
(3) a VH as shown in SEQ ID NO: 5 and a VL as shown in SEQ ID NO: 4;
(4) a VH as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 58;
(5) VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 60;
(6) VH as shown in SEQ ID NO: 157 and VL as shown in SEQ ID NO: 58;
(7) VH as shown in SEQ ID NO: 158 and VL as shown in SEQ ID NO: 58;
(8) VH as shown in SEQ ID NO: 57 and VL as shown 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 shown in SEQ ID NO: 163 and VL as shown 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 shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 166
An antibody or antigen-binding fragment thereof comprising a.
The method according to any one of claims 1 to 14,
scFv, Fab, Fab ', (Fab') ₂ , Fv fragments, diabodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, and humanized antibodies; Preferably, the antibody or antigen-binding fragment thereof, wherein the antibody is a chimeric antibody or a humanized antibody.
The method according to any one of claims 1 to 15,
An antibody or antigen-binding fragment thereof, wherein the antibody is of the IgG class (eg, IgG1, IgG2, IgG3 or IgG4 class).
The method according to any one of claims 1 to 16,
An antibody or antigen-binding fragment thereof that can specifically bind HBsAg, neutralize the toxicity of HBV, and / or reduce serum levels of HBV DNA and / or HBsAg in a subject.
The method according to any one of claims 1 to 17,
Labeled; Preferably, the antibody or antigen-binding fragment thereof labeled with a detectable label, such as a radioisotope, fluorescent material, luminescent material, chromogenic material or enzyme (eg horseradish peroxidase).
An isolated nucleic acid molecule encoding the antibody according to any one of claims 1 to 18 or an antigen binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.
20. An isolated nucleic acid molecule according to claim 19; Preferably, the vector is a cloning vector or expression vector.
A host cell comprising the isolated nucleic acid molecule of claim 19 or the vector of claim 20.
The host cell of claim 1, comprising culturing the host cell according to claim 21 under conditions permitting the 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 18.
A pharmaceutical composition comprising the antibody according to any one of claims 1 to 18 or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient.
Use of an antibody according to any one of claims 1 to 18 or an antigen-binding fragment thereof or a pharmaceutical composition according to claim 23 in the manufacture of a medicament, wherein the medicament is used for HBV infection or HBV in a subject (eg human). Prevent or treat diseases associated with infection (eg, hepatitis B), neutralize the toxicity of HBV in vitro or in subjects (eg humans), and HBV DNA and / or in subjects (eg humans) Use to reduce serum levels of HBsAg.
Used to prevent or treat HBV infection or a disease associated with HBV infection (e.g. hepatitis B) in a subject (e.g. human), and to neutralize the toxicity of HBV in vitro or in the subject (e.g. human) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, which is used for and / or is used to reduce serum levels of HBV DNA and / or HBsAg in a subject (eg, a human). Or a pharmaceutical composition according to claim 23.
Prevent or treat HBV infection or a disease associated with HBV infection (e.g. hepatitis B) in a subject (e.g. human), neutralize the toxicity of HBV in a subject (e.g. human), and / or A method for reducing serum levels of HBV DNA and / or HBsAg, for example in a human), wherein the subject has an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or 23 A method comprising administering a pharmaceutical composition according to claim.

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