U.S. patent application number 17/613392 was filed with the patent office on 2022-08-11 for anti-hepatitis b virus antibodies and use thereof.
This patent application is currently assigned to XIAMEN UNIVERSITY. The applicant listed for this patent is XIAMEN UNIVERSITY, YANG SHENG TANG COMPANY, LTD.. Invention is credited to Xiaoqing CHEN, Yichao JIANG, Wenxin LUO, Jixian TANG, Ningshao XIA, Chao YU, Quan YUAN, Tianying ZHANG.
Application Number | 20220251173 17/613392 |
Document ID | / |
Family ID | 1000006336371 |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220251173 |
Kind Code |
A1 |
LUO; Wenxin ; et
al. |
August 11, 2022 |
ANTI-HEPATITIS B VIRUS ANTIBODIES AND USE THEREOF
Abstract
Antibodies (especially humanized antibodies) against the
hepatitis B surface antigen (HBsAg), a nucleic acid molecule
encoding same, a method for preparing same, and a pharmaceutical
composition containing same. The anti-HBsAg antibodies have a
higher binding affinity to HBsAg at a neutral pH than at an acidic
pH, thereby significantly enhancing virus clearance efficiency and
prolonging virus inhibition time. The antibodies and pharmaceutical
composition may be used to prevent and/or treat HBV infections or
diseases related to HBV infection (such as hepatitis B) for use in
neutralizing the virulence of HBV in the body of a subject (such as
a human) to reduce a serum level of HBV DNA and/or HBsAg in the
body of the subject, or to activate a humoral immune response of a
subject (such as a person infected with chronic HBV or a patient
who has chronic hepatitis B) against HBV.
Inventors: |
LUO; Wenxin; (Xiamen,
CN) ; JIANG; Yichao; (Xiamen, CN) ; YU;
Chao; (Xiamen, CN) ; CHEN; Xiaoqing; (Xiamen,
CN) ; TANG; Jixian; (Xiamen, CN) ; YUAN;
Quan; (Xiamen, CN) ; ZHANG; Tianying; (Xiamen,
CN) ; XIA; Ningshao; (Xiamen, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
XIAMEN UNIVERSITY
YANG SHENG TANG COMPANY, LTD. |
Xiamen
Hangzhou |
|
CN
CN |
|
|
Assignee: |
XIAMEN UNIVERSITY
Xiamen
CN
YANG SHENG TANG COMPANY, LTD.
Hangzhou
CN
|
Family ID: |
1000006336371 |
Appl. No.: |
17/613392 |
Filed: |
May 22, 2020 |
PCT Filed: |
May 22, 2020 |
PCT NO: |
PCT/CN2020/091890 |
371 Date: |
November 22, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/52 20130101; C07K 2317/565 20130101; C07K 2317/24
20130101; A61P 31/20 20180101; C07K 2317/76 20130101; C07K 2317/622
20130101; C07K 2317/567 20130101; A61K 2039/505 20130101; A61K
2039/575 20130101; C07K 16/082 20130101 |
International
Class: |
C07K 16/08 20060101
C07K016/08; A61P 31/20 20060101 A61P031/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2019 |
CN |
201910432602.7 |
Claims
1. An antibody or antigen-binding fragment thereof capable of
specifically binding to HBsAg, wherein the antibody or
antigen-binding fragment thereof binds to HBsAg with higher
affinity at neutral pH than at acidic pH, and the antibody or
antigen-binding fragment thereof comprises: (a) a heavy chain
variable region (VH) comprising the following 3 CDRs: (i) HCDR1
with a sequence of X.sub.1X.sub.2YHX.sub.3N (SEQ ID NO: 26),
wherein X.sub.1 is selected from Y or H, X.sub.2 is selected from G
or R, X.sub.3 is selected from W or Y; (ii) HCDR2 with a sequence
of YIX.sub.4X.sub.5DGSVX.sub.6YNPSLEN (SEQ ID NO: 27), wherein
X.sub.4 is selected from S, N or H, X.sub.5 is selected from Y or
H, X.sub.6 is selected from L, H or Q; and (iii) HCDR3 with a
sequence of GFDH (SEQ ID NO: 13); and/or, (b) a light chain
variable region (VL) comprising the following 3 CDRs: (iv) LCDR1
with a sequence of RSSQSLVHSYGDX.sub.7YLH (SEQ ID NO: 28), wherein
X.sub.7 is selected from T or N; (v) LCDR2 with a sequence of
KVSNRFS (SEQ ID NO: 15); and (vi) LCDR3 with a sequence of
SQNTHX.sub.8PYT (SEQ ID NO: 29), wherein X.sub.8 is selected from
V, L or H.
2. The antibody or antigen-binding fragment thereof according to
claim 1, wherein the antibody or antigen-binding fragment thereof
comprises: (a) a heavy chain variable region (VH) comprising the
following 3 CDRs: (i) HCDR1, consisting of a sequence selected from
the following: SEQ ID NOs: 17, 21, 24; (ii) HCDR2, consisting of a
sequence selected from: SEQ ID NOs: 12, 18, 20, 22; and (iii)
HCDR3, consisting of a sequence shown in SEQ ID NO: 13; and/or, (b)
a light chain variable region (VL) comprising the following 3 CDRs:
(iv) LCDR1, consisting of a sequence selected from the following:
SEQ ID NOs: 14, 25; (v) LCDR2, consisting of a sequence shown in
SEQ ID NO: 15; and (vi) LCDR3, consisting of a sequence selected
from the following: SEQ ID NOs: 16, 19, 23.
3. The antibody or antigen-binding fragment thereof according to
claim 1 or 2, wherein the antibody or antigen-binding fragment
thereof comprises: (1) a VH comprising the following 3 CDRs: HCDR1
shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown
in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1
shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown
in SEQ ID NO: 23; (2) a VH comprising the following 3 CDRs: HCDR1
shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown
in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1
shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown
in SEQ ID NO: 19; (3) a VH comprising the following 3 CDRs: HCDR1
shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown
in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1
shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown
in SEQ ID NO: 16; (4) a VH comprising the following 3 CDRs: HCDR1
shown in SEQ ID NO: 24, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown
in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1
shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown
in SEQ ID NO: 16; (5) a VH comprising the following 3 CDRs: HCDR1
shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown
in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1
shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown
in SEQ ID NO: 23; or (6) a VH comprising the following 3 CDRs:
HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3
shown in SEQ ID NO: 13; and, a VL comprising the following 3 CDRs:
LCDR1 shown in SEQ ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3
shown in SEQ ID NO: 16.
4. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 3, wherein the antibody or antigen-binding
fragment thereof further comprises a framework region of a human
immunoglobulin (for example, 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 (for example,
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues
to murine residues; preferably, the antibody or antigen-binding
fragment thereof comprises: a heavy chain framework region
contained in an amino acid sequence encoded by a human heavy chain
germline gene, and/or a light chain framework region contained in
an amino acid sequence encoded by a human light chain germline
gene; preferably, the antibody or antigen-binding fragment thereof
comprises: a heavy chain framework region contained in an amino
acid sequence encoded by human heavy chain germline gene 4-28-02
(SEQ ID NO: 38), and a light chain framework region contained in an
amino acid sequence encoded by human light chain germline gene
2D-28-01 (SEQ ID NO: 39), and the heavy chain framework region
and/or the light chain framework region optionally comprises one or
more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations
from human residues to murine residues; preferably, the VH of the
antibody or antigen-binding fragment thereof comprises: VH FR1 as
shown in SEQ ID NO: 30, VH FR2 as shown in SEQ ID NO: 31, VH FR3 as
shown in SEQ ID NO: 32, and VH FR4 shown in SEQ ID NO: 33;
preferably, the VL of the antibody or antigen-binding fragment
thereof comprises: VL FR1 as shown in SEQ ID NO: 34, VL FR2 as
shown in SEQ ID NO: 35, VL FR3 as shown in SEQ ID NO: 36, and VL
FR4 shown in SEQ ID NO: 37.
5. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 4, wherein the antibody or antigen-binding
fragment thereof comprises: (a) a heavy chain variable region (VH),
which comprises an amino acid sequence selected from the following:
(i) a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; (ii) a
sequence with substitution, deletion or addition of one or several
amino acids (for example, substitution, deletion or addition of 1,
2, 3, 4 or 5 amino acids) as compared with a sequence shown in any
one of SEQ ID NOs: 3, 5, 6, 8; or (iii) a sequence with a sequence
identity of at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% as compared
with a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; and (b)
a light chain variable region (VL), which comprises an amino acid
sequence selected from the following: (iv) a sequence shown in any
one of SEQ ID NOs: 2, 4, 7, 9, 10; (v) a sequence with
substitution, deletion or addition of one or several amino acids
(for example, substitution, deletion or addition of 1, 2, 3, 4 or 5
amino acids) as compared with a sequence shown in any one of SEQ ID
NOs: 2, 4, 7, 9, 10; or (vi) a sequence with a sequence identity of
at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% as compared with a
sequence shown in any one of SEQ ID NOs: 2, 4, 7, 9, 10;
preferably, the substitution described in (ii) or (v) is a
conservative substitution.
6. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 5, wherein the antibody or antigen-binding
fragment thereof comprises: (1) a VH with a sequence shown in SEQ
ID NO: 3 and a VL with a sequence shown in SEQ ID NO: 4; (2) a VH
with a sequence shown in SEQ ID NO: 5 and a VL with a sequence
shown in SEQ ID NO: 2; (3) a VH with a sequence shown in SEQ ID NO:
6 and a VL with a sequence shown in SEQ ID NO: 7; (4) a VH with a
sequence shown in SEQ ID NO: 8 and a VL with a sequence shown in
SEQ ID NO: 9; (5) a VH with a sequence shown in SEQ ID NO: 3 and a
VL with a sequence shown in SEQ ID NO: 10; or (6) a VH with a
sequence shown in SEQ ID NO: 3 and a VL with a sequence shown in
SEQ ID NO: 9.
7. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 6, wherein the antibody or antigen-binding
fragment thereof further comprises a constant region derived from a
human immunoglobulin; preferably, the heavy chain of the antibody
or antigen-binding fragment thereof comprises a heavy chain
constant region derived from a human immunoglobulin (for example,
IgG1, IgG2, IgG3 or IgG4), and the light chain of the antibody or
antigen-binding fragment thereof comprises a light chain constant
region derived from a human immunoglobulin (for example, .kappa. or
.lamda.); preferably, the antibody or antigen-binding fragment
thereof comprises a light chain constant region (CL) as shown in
SEQ ID NO: 41.
8. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 7, wherein the antibody or antigen-binding
fragment thereof comprises a variant of a human IgG1 heavy chain
constant region, the variant has the following substitution as
compared to a wild-type sequence from which it is derived: (i)
M252Y, N286E, N434Y; or, (ii) K326D, L328Y; wherein the
above-mentioned amino acid positions are positions according to the
Kabat numbering system; preferably, the antibody or antigen-binding
fragment thereof comprises a heavy chain constant region (CH) as
shown in SEQ ID NO: 42 or 43.
9. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 7, wherein the antibody or antigen-binding
fragment thereof comprises a heavy chain constant region (CH) as
shown in SEQ ID NO: 40.
10. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 9, wherein the antibody or antigen-binding
fragment thereof comprises: (1) a heavy chain comprising a VH shown
in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain
comprising a VL shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO:
41; (2) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a
CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown
in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (3) a heavy chain
comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO:
48, and a light chain comprising a VL shown in SEQ ID NO: 4 and a
CL shown in SEQ ID NO: 41; (4) a heavy chain comprising a VH shown
in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 40, and a light chain
comprising a VL shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO:
41; (5) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a
CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown
in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41; (6) a heavy chain
comprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO:
48, and a light chain comprising a VL shown in SEQ ID NO: 2 and a
CL shown in SEQ ID NO: 41; (7) a heavy chain comprising a VH shown
in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 40, and a light chain
comprising a VL shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO:
41; (8) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a
CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown
in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (9) a heavy chain
comprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO:
48, and a light chain comprising a VL shown in SEQ ID NO: 7 and a
CL shown in SEQ ID NO: 41; (10) a heavy chain comprising a VH shown
in SEQ ID NO: 8 and a CH shown in SEQ ID NO: 40, and a light chain
comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO:
41; (11) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a
CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown
in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (12) a heavy chain
comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO:
48, and a light chain comprising a VL shown in SEQ ID NO: 9 and a
CL shown in SEQ ID NO: 41; (13) a heavy chain comprising a VH shown
in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain
comprising a VL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO:
41; (14) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a
CH shown in SEQ ID NO: 47, and a light chain comprising a VL shown
in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (15) a heavy
chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ
ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 10
and a CL shown in SEQ ID NO: 41; (16) a heavy chain comprising a VH
shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 40, and a light
chain comprising a VL shown in SEQ ID NO: 9 and a CL shown in SEQ
ID NO: 41; (17) a heavy chain comprising a VH shown in SEQ ID NO: 3
and a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; or (18) a
heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in
SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID
NO: 9 and a CL shown in SEQ ID NO: 41.
11. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 10, wherein the antibody or antigen-binding
fragment thereof is selected from the group consisting of scFv,
Fab, Fab', (Fab').sub.2, Fv fragment, diabody, bispecific antibody,
multispecific antibody, probody, chimeric antibody or humanized
antibody; preferably, the antibody is a chimeric antibody or a
humanized antibody.
12. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 10, wherein the antibody or antigen-binding
fragment thereof is able to specifically bind to HBsAg, neutralize
a virulence of HBV, and/or reduce a serum level of HBV DNA and/or
HBsAg in a subject.
13. An isolated nucleic acid molecule, which encodes the antibody
or antigen-binding fragment thereof according to any one of claims
1 to 12, or its heavy chain variable region and/or light chain
variable region.
14. A vector, which comprises the nucleic acid molecule according
to claim 13; preferably, the vector is a cloning vector or an
expression vector.
15. A host cell, which comprises the nucleic acid molecule
according to claim 13 or the vector according to claim 14.
16. A method for preparing the antibody or antigen-binding fragment
thereof according to any one of claims 1 to 12, which comprises
culturing the host cell according to claim 15 under a condition
that allows the expression of the antibody or antigen-binding
fragment thereof, and recovering the antibody or antigen-binding
fragment thereof from the cultured host cell culture.
17. A pharmaceutical composition, which comprises the antibody or
antigen-binding fragment thereof according to any one of claims 1
to 12, and a pharmaceutically acceptable carrier and/or
excipient.
18. Use of the antibody or antigen-binding fragment thereof
according to any one of claims 1 to 12 or the pharmaceutical
composition according to claim 17 in the manufacture of a
medicament for the prevention and/or treatment of an HBV infection
or HBV infection-associated disease (for example, hepatitis B) in a
subject (for example, a human), for neutralizing a virulence of HBV
in vitro or in a subject (for example, a human), for reducing a
serum level of HBV DNA and/or HBsAg in a subject (for example, a
human), and/or for activating a humoral immune response against HBV
in a subject (for example, a person with chronic HBV infection or a
patient with chronic hepatitis B).
19. The antibody or antigen-binding fragment thereof according to
any one of claims 1 to 12 or the pharmaceutical composition
according to claim 17, for use in the prevention and/or treatment
of an HBV infection or HBV infection-associated disease (for
example, hepatitis B) in a subject (for example, a human), for use
in neutralizing a virulence of HBV in vitro or in a subject (for
example, a human), for use in reducing a serum level of HBV DNA
and/or HBsAg in a subject (for example, a human), and/or for use in
activating a humoral immune response against HBV in a subject (for
example, a person with chronic HBV infection or a patient with
chronic hepatitis B).
20. A method, which is used for the prevention and/or treatment of
an HBV infection or HBV infection-associated disease (for example,
hepatitis B) in a subject, for neutralizing a virulence of HBV in a
subject (for example, a human), for reducing a serum level of HBV
DNA and/or HBsAg in a subject (for example, a human), and/or for
activating a humoral immune response against HBV in a subject (for
example, a person with chronic HBV infection or a patient with
chronic hepatitis B), the method comprises administering an
effective amount of the antibody or antigen-binding fragment
thereof according to any one of claims 1 to 12, or the
pharmaceutical composition according to claim 17 to a subject in
need thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of molecular
virology and immunology, especially the field of treatment of
hepatitis B virus (HBV) infection. Specifically, the present
invention relates to an antibody against hepatitis B virus surface
antigen (HBsAg) and a nucleic acid encoding the antibody, and a use
thereof. The anti-HBsAg antibody of the present invention has a
higher binding affinity for HBsAg at neutral pH than at acidic pH.
The novel antibody can be used for the prevention and/or treatment
of an HBV infection or a disease associated with HBV infection (for
example, hepatitis B), for neutralizing a virulence of HBV in a
subject (for example, a human), or for reducing a serum level of
HBV DNA and/or HBsAg in a subject. Therefore, the present invention
further relates to a use of the antibody and variant thereof in the
manufacture of a pharmaceutical composition for the prevention
and/or treatment of an HBV infection or a disease related to an HBV
infection (for example, hepatitis B), for neutralizing a virulence
of HBV in a subject (for example, a human), for reducing a serum
level of HBV DNA and/or HBsAg in a subject, or for activating a
humoral immune response to HBV in a subject (for example, a person
with chronic HBV infection or a patient with chronic hepatitis
B).
BACKGROUND ART
[0002] Hepatitis B virus infection, especially chronic HBV
infection, is one of the most important public health problems in
the world (Dienstag J L. 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), liver
cirrhosis (LC) and primary hepatocellular carcinoma (HCC) (Liaw Y
F, Chu C M. Hepatitis B virus infection. Lancet 2009 Feb. 14;
373(9663): 582-592). According to reports, there are currently
about 2 billion people in the world who have been infected with
HBV, there are now about 350 million persons with chronic hepatitis
B virus infections, the risk of these infected persons eventually
dying from HBV infection-associated liver diseases can reach 15% to
25%, and more than one million people die from such diseases each
year worldwide (Dienstag J L., ibid; and Liaw Y F, et al.,
ibid).
[0003] The current treatment drugs for chronic HBV infection can be
divided into interferons (IFNs) and nucleoside or nucleotide
analogs (NAs) (Dienstag J L., ibid.; Kwon H, Lok A S. Hepatitis B
therapy. Nat Rev Gastroenterol Hepatol 2011 May; 8(5): 275-284; and
Liaw Y F et al., ibid.). For HBV-infected patients (such as CHB
patients), the above-mentioned drugs alone or in combination can
effectively inhibit viral replication in the body and greatly
reduce HBV DNA levels; in particular, after 52 weeks or more of
such treatments, the response rate where the HBV DNA level in the
body is below the lower limit of detection (virological response)
can reach 40-80% (Kwon H et al., ibid.). However, the treatment
with the above-mentioned drugs alone or in combination cannot
completely eliminate the HBV virus in the infected persons, and the
response rate of HBsAg negative conversion or HBsAg seroconversion
(a sign of complete HBV virus clearance in the infected person)
caused thereby is usually less than 5% (Kwon H et al., ibid.).
[0004] The development of new drugs for the treatment of chronic
HBV infection based on immunological means is one of the important
research directions in this field. Immunotherapy for chronic HBV
infection is usually carried out in two ways: active immunotherapy
(its corresponding drug forms including vaccines, etc.) and passive
immunotherapy (its corresponding drug forms including antibodies,
etc.). Active immunotherapy refers to administration of a
therapeutic vaccine (including protein vaccine, peptide vaccine,
nucleic acid vaccine, etc.) in order to stimulate the body of
chronic HBV infected person to actively produce a cellular immune
response (CTL effect, etc.) or/and humoral immune response against
HBV (antibodies, etc.), so as to achieve the purpose of inhibiting
or eliminating HBV. Currently, there is no definitely significant
and effective active immunotherapy drug/vaccine that can be used to
treat chronic HBV infection. Passive immunotherapy (taking antibody
as an example) refers to administration of an antibody with
therapeutic properties to a HBV infected person, and a therapeutic
effect can be achieved by the antibody-mediated virus
neutralization to block HBV from infecting newborn hepatocytes, or
by the antibody-mediated immune clearance to remove viruses and
infected liver cells from the body. At present, the anti-HBs
polyclonal antibody purified from the serum/plasma of those who had
a response to a prophylactic hepatitis B vaccine or those who have
recovered from HBV infection, namely high-potency hepatitis B
immunoglobulin (HBIG), has been widely used to block
mother-to-child vertical transmission of HBV, prevent HBV
reinfection after liver transplantation in patients with chronic
HBV infection, and prevent people accidentally exposed to HBV from
being infected. However, the direct application of HBIG in the
treatment of HBV-infected patients (for example, CHB patients) has
no obvious effect, and it has many limitations such as fewer
sources for high-potency plasma, high price, unstable nature, and
potential safety issues.
[0005] Therefore, it is urgent and necessary to develop innovative
treatment methods and drugs for HBV infected persons that can more
effectively remove HBV virus, especially HBsAg.
Contents of the Present Invention
[0006] The present inventors have previously developed an
anti-HBsAg humanized antibody with excellent properties, which can
neutralize the virulence of HBV in vivo and reduce the serum levels
of HBV DNA and/or HBsAg. On the basis of the previous research, the
present inventors have paid a lot of creative work to conduct
in-depth research and engineering of the humanized antibody,
thereby developing an anti-HBsAg antibody with pH-dependent antigen
binding ability. The anti-HBsAg antibody of the present invention
has a higher binding affinity for HBsAg at neutral pH than at
acidic pH, so that the reuse of antibody is realized, the antibody
half-life is significantly extended, and the efficiency of HBV
clearance is enhanced. Furthermore, the present inventors obtain a
scavenger antibody and further extend the antibody half-life by
introducing a mutation into the Fc region of the above-mentioned
antibody to enhance its affinity to hFcRn or mFc.gamma.RII under
neutral condition.
[0007] The antibody of the present invention is extremely
advantageous, since it not only retains the activity of reducing
the serum level of HBV DNA and/or HBsAg, but also has a longer time
of antigen suppression, thereby greatly reducing the injection
dosage and administration frequency of treatment, and having
significant clinical value.
Antibody of the Present Invention
[0008] Therefore, in one aspect, the present invention provides an
antibody or antigen-binding fragment thereof capable of
specifically binding to HBsAg, in which the antibody or
antigen-binding fragment thereof binds to HBsAg with higher
affinity at neutral pH than at acidic pH.
[0009] In certain embodiments, the neutral pH is pH 6.7 to pH 7.5,
such as pH 7.4.
[0010] In certain embodiments, the acidic pH is pH 4.0 to pH 6.5,
such as pH 6.0.
[0011] In certain embodiments, a ratio of K.sub.D of binding to
HBsAg at an acidic pH (for example, pH 6.0) to K.sub.D of binding
to HBsAg at neutral pH (for example, pH 7.4) (i.e., value of
K.sub.D (acidic pH)/K.sub.D (neutral pH)), of the antibody or
antigen-binding fragment thereof, is greater than 1, for example
not less than 1.5, not less than 2, not less than 3, not less than
4, not less than 5, not less than 6, not less than 7, not less than
8, not less than 9, not less than 10, not less than 15, not less
than 20, not less than 30, not less than 40, not less than 50, not
less than 60, not less than 70, not less than 80, not less than 90,
not less than 100, not less than 300, not less than 500, not less
than 800, not less than 1000, not less than 2000, not less than
5000, or not less than 10,000. In some embodiments, the value of
K.sub.D (acidic pH)/K.sub.D (neutral pH) is greater than 1 and not
greater than 10000, for example, not greater than 5000, not greater
than 2000, not greater than 1000, not Greater than 900, not greater
than 800, not greater than 700, not greater than 600, not greater
than 500, not greater than 400, not greater than 300, not greater
than 200, not greater than 100, not greater than 90, not greater
than 80, not greater than 70, not greater than 60, not greater than
50, not greater than 40, not greater than 30, not greater than 20,
or not greater than 10. The K.sub.D can be measured by a technique
known in the art, for example, by SPR technique (for example,
Biacore).
[0012] In some embodiments, a ratio of K.sub.D of binding to HBsAg
at pH 6.0 to K.sub.D of binding to HBsAg at pH 7.4 of the antibody
or antigen-binding fragment thereof, is greater than 1, for example
not less than 1.5, not less than 2. In certain embodiments, the
K.sub.D value of the antibody of the invention at neutral pH may be
10.sup.-7M, 10.sup.-8M, 10.sup.-9M, 10.sup.-10M, 10.sup.-11M,
10.sup.-12M or less. In some embodiments, the K.sub.D value of the
antibody of the present invention at acidic pH may be 10.sup.-9M,
10.sup.-8M, 10.sup.-7M, 10.sup.-6M or more.
[0013] In certain embodiments, a ratio of EC50 of binding to HBsAg
at an acidic pH (for example, pH 6.0) to EC50 of binding to HBsAg
at neutral pH (for example, pH 7.4) (i.e., value of EC50 (acidic
pH)/EC50 (neutral pH)), of the antibody or antigen-binding fragment
thereof, is greater than 1, for example not less than 1.5, not less
than 2, not less than 3, not less than 4, not less than 5, not less
than 6, not less than 7, not less than 8, not less than 9, not less
than 10, not less than 15, not less than 20, not less than 30, not
less than 40, not less than 50, not less than 60, not less than 70,
not less than 80, not less than 90, not less than 100, not less
than 300, not less than 500, not less than 800, not less than 1000,
not less than 2000, not less than 5000, or not less than 10,000. In
some embodiments, the value of EC50 (acidic pH)/EC50 (neutral pH)
is greater than 1 and not greater than 10000, for example, not
greater than 5000, not greater than 2000, not greater than 1000,
not greater than 900, and not greater than 800, not greater than
700, not greater than 600, not greater than 500, not greater than
400, not greater than 300, not greater than 200, not greater than
100, not greater than 90, not greater than 80, not greater than 70,
not greater than 60, not greater than 50, not greater than 40, not
greater than 30, not greater than 20, or not greater than 10. In
some embodiments, the EC50 is measured by ELISA method, for
example, calculated by the regression analysis of a dose-response
curve generated by the ELISA method.
[0014] In certain embodiments, a ratio of EC50 of binding to HBsAg
at pH 6.0 to EC50 of binding to HBsAg at pH 7.4 of the antibody or
antigen-binding fragment thereof, is greater than 1, for example,
not less than 1.5, or not less than 2.
[0015] In certain embodiments, the antibody or antigen-binding
fragment thereof of the present invention is derived from the
anti-HBV humanized antibody 162 (which is described in detail in
Chinese Patent Application 201610879693.5).
[0016] In certain embodiments, the antibody or antigen-binding
fragment thereof of the present invention binds to aa121-124 of
HBsAg with higher affinity at neutral pH than at acidic pH.
[0017] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises a heavy chain variable region (VH)
comprising HCDR1, HCDR2 and HCDR3, which has one or more of the
following characteristics:
[0018] (i) HCDR1 has at least one amino acid (for example, 1, 2, 3,
4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 11;
[0019] (ii) HCDR2 has at least one amino acid (for example, 1, 2,
3, 4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 12; and/or,
[0020] (iii) HCDR3 has at least one amino acid (for example, 1, 2,
3, 4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 13.
[0021] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises a heavy chain variable region (VL)
comprising LCDR1, LCDR2 and LCDR3, which has one or more of the
following characteristics:
[0022] (i) LCDR1 has at least one amino acid (for example, 1, 2, 3,
4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 14;
[0023] (ii) LCDR2 has at least one amino acid (for example, 1, 2,
3, 4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 15; and/or,
[0024] (iii) LCDR3 has at least one amino acid (for example, 1, 2,
3, 4 or 5 amino acids) replaced with histidine as compared with a
sequence shown in SEQ ID NO: 16.
[0025] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0026] (a) a heavy chain variable region (VH) comprising the
following 3 CDRs:
[0027] (i) HCDR1 with a sequence of X.sub.1X.sub.2YHX.sub.3N (SEQ
ID NO: 26), wherein X.sub.1 is selected from Y or H, X.sub.2 is
selected from G or R, X.sub.3 is selected from W or Y;
[0028] (ii) HCDR2 with a sequence of
YIX.sub.4X.sub.5DGSVX.sub.6YNPSLEN (SEQ ID NO: 27), wherein X.sub.4
is selected from S, N or H, X.sub.5 is selected from Y or H,
X.sub.6 is selected from L, H or Q; and
[0029] (iii) HCDR3 with a sequence of GFDH (SEQ ID NO: 13);
and/or,
[0030] (b) a light chain variable region (VL) comprising the
following 3 CDRs:
[0031] (iv) LCDR1 with a sequence of RSSQSLVHSYGDX.sub.7YLH (SEQ ID
NO: 28), wherein X.sub.7 is selected from T or N;
[0032] (v) LCDR2 with a sequence of KVSNRFS (SEQ ID NO: 15);
and
[0033] (vi) LCDR3 with a sequence of SQNTHX.sub.8PYT (SEQ ID NO:
29), wherein X.sub.8 is selected from V, L or H.
[0034] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0035] (a) a heavy chain variable region (VH) comprising the
following 3 CDRs:
[0036] (i) HCDR1, which is composed of a sequence selected from the
following: SEQ ID NOs: 17, 21, 24;
[0037] (ii) HCDR2, which is composed of a sequence selected from:
SEQ ID NOs: 18, 20, 22, 12; and
[0038] (iii) HCDR3, which is composed of a sequence shown in SEQ ID
NO: 13; and/or,
[0039] (b) a light chain variable region (VL) comprising the
following 3 CDRs:
[0040] (iv) LCDR1, which is composed of a sequence selected from
the following: SEQ ID NOs: 14, 25;
[0041] (v) LCDR2, which is composed of a sequence shown in SEQ ID
NO: 15; and
[0042] (vi) LCDR3, which is composed of a sequence selected from
the following: SEQ ID NOs: 19, 16, 23.
[0043] In certain embodiments, X.sub.1 is selected from H, X.sub.2
is selected from G, X.sub.3 is selected from W or Y, X.sub.4 is
selected from S or H, X.sub.5 is selected from Y, X.sub.6 is
selected from L or H, X.sub.7 is selected from T or N, X.sub.8 is
selected from V, L or H.
[0044] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0045] (a) a heavy chain variable region (VH) comprising the
following 3 CDRs:
[0046] (i) HCDR1, which is composed of a sequence selected from the
following: SEQ ID NOs: 17, 24;
[0047] (ii) HCDR2, which is composed of a sequence selected from:
SEQ ID NOs: 18, 12; and
[0048] (iii) HCDR3, which is composed of a sequence shown in SEQ ID
NO: 13; and/or,
[0049] (b) a light chain variable region (VL) comprising the
following 3 CDRs:
[0050] (iv) LCDR1, which is composed of a sequence selected from
the following: SEQ ID NOs: 14, 25;
[0051] (v) LCDR2, which is composed of a sequence shown in SEQ ID
NO: 15; and
[0052] (vi) LCDR3, which is composed of a sequence selected from
the following: SEQ ID NOs: 19, 16, 23.
[0053] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0054] (1) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 21, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
23;
[0055] (2) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
19;
[0056] (3) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 17, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
16;
[0057] (4) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 24, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
16;
[0058] (5) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
23; or
[0059] (6) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ
ID NO: 17, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO:
13; and, a VL comprising the following 3 CDRs: LCDR1 shown in SEQ
ID NO: 25, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
16.
[0060] In certain embodiments, the antibody or antigen-binding
fragment thereof further comprises a framework region of a human
immunoglobulin (for example, 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 (for example,
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues
to murine residues.
[0061] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises: a heavy chain framework region
contained in an amino acid sequence encoded by a human heavy chain
germline gene, and/or a light chain framework region contained in
an amino acid sequence encoded by a human light chain germline
gene.
[0062] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises: a heavy chain framework region
contained in an amino acid sequence encoded by human heavy chain
germline gene 4-28-02 (SEQ ID NO: 38), and a light chain framework
region contained in an amino acid sequence encoded by human light
chain germline gene 2D-28-01 (SEQ ID NO: 39), and the heavy chain
framework region and/or the light chain framework region optionally
comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or
10) back mutations from human residues to murine residues.
[0063] In certain embodiments, the VH of the antibody or
antigen-binding fragment thereof comprises: VH FR1 as shown in SEQ
ID NO: 30, VH FR2 as shown in SEQ ID NO: 31, VH FR3 as shown in SEQ
ID NO: 32, and VH FR4 shown in SEQ ID NO: 33.
[0064] In some embodiments, the VL of the antibody or
antigen-binding fragment thereof comprises: VL FR1 as shown in SEQ
ID NO: 34, VL FR2 as shown in SEQ ID NO: 35, VL FR3 as shown in SEQ
ID NO: 36, and VL FR4 shown in SEQ ID NO: 37.
[0065] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0066] (a) a heavy chain variable region (VH), which comprises an
amino acid sequence selected from the following:
[0067] (i) a sequence shown in any one of SEQ ID NOs: 3, 5, 6,
8;
[0068] (ii) a sequence with substitution, deletion or addition of
one or several amino acids (for example, substitution, deletion or
addition of 1, 2, 3, 4 or 5 amino acids) as compared with a
sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; or
[0069] (iii) a sequence with a sequence identity of at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% as compared with a sequence shown
in any one of SEQ ID NOs: 3, 5, 6, 8;
[0070] and
[0071] (b) a light chain variable region (VL), which comprises an
amino acid sequence selected from the following:
[0072] (iv) a sequence shown in any one of SEQ ID NOs: 4, 2, 7, 9,
10;
[0073] (v) a sequence with substitution, deletion or addition of
one or several amino acids (for example, substitution, deletion or
addition of 1, 2, 3, 4 or 5 amino acids) as compared with a
sequence shown in any one of SEQ ID NOs: 4, 2, 7, 9, 10; or
[0074] (vi) a sequence with a sequence identity of at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% as compared with a sequence shown in any
one of SEQ ID NOs: 4, 2, 7, 9, 10.
[0075] Preferably, the substitution described in (ii) or (v) is a
conservative substitution.
[0076] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0077] (1) a VH with a sequence shown in SEQ ID NO: 3 and a VL with
a sequence shown in SEQ ID NO: 4;
[0078] (2) a VH with a sequence shown in SEQ ID NO: 5 and a VL with
a sequence shown in SEQ ID NO: 2;
[0079] (3) a VH with a sequence shown in SEQ ID NO: 6 and a VL with
a sequence shown in SEQ ID NO: 7;
[0080] (4) a VH with a sequence shown in SEQ ID NO: 8 and a VL with
a sequence shown in SEQ ID NO: 9;
[0081] (5) a VH with a sequence shown in SEQ ID NO: 3 and a VL with
a sequence shown in SEQ ID NO: 10; or
[0082] (6) a VH with a sequence shown in SEQ ID NO: 3 and a VL with
a sequence shown in SEQ ID NO: 9.
[0083] In certain embodiments, the antibody or antigen-binding
fragment thereof further comprises a constant region derived from a
human immunoglobulin.
[0084] In certain embodiments, the heavy chain of the antibody or
antigen-binding fragment thereof comprises a heavy chain constant
region derived from a human immunoglobulin (for example, IgG1,
IgG2, IgG3, or IgG4), and the light chain of the antibody or
antigen-binding fragment thereof comprises a light chain constant
region derived from a human immunoglobulin (for example, .kappa. or
.lamda.).
[0085] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0086] (a) a heavy chain constant region (CH) of a human
immunoglobulin or a variant thereof, wherein the variant has
substitution, deletion or addition of one or more amino acids or
any combination thereof (for example, substitution, deletion or
addition of at most 20, at most 15, at most 10, or at most 5 amino
acids or any combination thereof; for example, substitution,
deletion or addition of 1, 2, 3, 4 or 5 amino acids or any
combination thereof) as compared with a wild-type sequence from
which it is derived; and/or
[0087] (b) a light chain constant region (CL) of a human
immunoglobulin or a variant thereof, wherein the variant has
substitution, deletion or addition of one or more amino acids or
any combination thereof (for example, substitution, deletion or
addition of at most 20, at most 15, at most 10, or at most 5 amino
acids or any combination thereof; for example, substitution,
deletion or addition of 1, 2, 3, 4 or 5 amino acids or any
combination thereof) as compared with a wild-type sequence from
which it is derived.
[0088] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises a human IgG1 or IgG4 heavy chain
constant region. In certain embodiments, the antibody or
antigen-binding fragment thereof comprises a heavy chain constant
region (CH) as shown in SEQ ID NO: 40.
[0089] In certain embodiments, the antibody or antigen-binding
fragment thereof of the present invention comprises a variant of a
heavy chain constant region (CH) of a human immunoglobulin, in
which the variant has an enhanced affinity to hFcRn or
mFc.gamma.RII at neutral pH (for example, pH 7.4) as compared with
a wild-type sequence from which it is derived. In such embodiments,
the variant generally has substitution of at least one amino acid
as compared with a wild-type sequence from which it is derived.
[0090] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises a variant of a human IgG1 heavy chain
constant region, in which the variant has the following
substitutions as compared to a wild-type sequence from which it is
derived: (i) M252Y, N286E, N434Y; or, (ii) K326D, L328Y; wherein
the above-mentioned amino acid positions are positions according to
the Kabat numbering system. In certain embodiments, the antibody or
antigen-binding fragment thereof comprises a heavy chain constant
region (CH) as shown in SEQ ID NO: 42 or 43.
[0091] In certain embodiments, the light chain constant region is a
.kappa. light chain constant region. In certain embodiments, the
antibody or antigen-binding fragment thereof comprises a light
chain constant region (CL) as shown in SEQ ID NO: 41.
[0092] In certain embodiments, the antibody or antigen-binding
fragment thereof comprises:
[0093] (1) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41;
[0094] (2) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41;
[0095] (3) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41;
[0096] (4) a heavy chain comprising a VH shown in SEQ ID NO: 5 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41;
[0097] (5) a heavy chain comprising a VH shown in SEQ ID NO: 5 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41;
[0098] (6) a heavy chain comprising a VH shown in SEQ ID NO: 5 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41;
[0099] (7) a heavy chain comprising a VH shown in SEQ ID NO: 6 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41;
[0100] (8) a heavy chain comprising a VH shown in SEQ ID NO: 6 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41;
[0101] (9) a heavy chain comprising a VH shown in SEQ ID NO: 6 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41;
[0102] (10) a heavy chain comprising a VH shown in SEQ ID NO: 8 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41;
[0103] (11) a heavy chain comprising a VH shown in SEQ ID NO: 8 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41;
[0104] (12) a heavy chain comprising a VH shown in SEQ ID NO: 8 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41;
[0105] (13) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41;
[0106] (14) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41;
[0107] (15) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41;
[0108] (16) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 40, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41;
[0109] (17) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 47, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; or
[0110] (18) a heavy chain comprising a VH shown in SEQ ID NO: 3 and
a CH shown in SEQ ID NO: 48, and a light chain comprising a VL
shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41.
Preparation of Antibody
[0111] The antibody of the present invention can be prepared by
various methods known in the art, for example, obtained by genetic
engineering recombination technology. For example, DNA molecules
encoding the heavy chain and light chain genes of the antibody of
the present invention are obtained by chemical synthesis or PCR
amplification. The resulting DNA molecule is inserted into an
expression vector and then transfected into a host cell. Then, the
transfected host cell is cultured under specific conditions, and
the antibody of the present invention is expressed.
[0112] The antigen-binding fragment of the present invention can be
obtained by hydrolyzing a complete antibody molecule (see Morimoto
et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan
et al., Science 229:81 (1985)). In addition, these antigen-binding
fragments can also be directly produced by recombinant host cells
(reviewed in 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 host cells; Fab'
fragments can be chemically coupled to form F(ab').sub.2 fragments
(Carter et al., Bio/Technology, 10: 163-167 (1992)). In addition,
Fv, Fab or F(ab').sub.2 fragments can also be directly isolated
from a recombinant host cell culture medium. Those of ordinary
skill in the art are fully aware of other techniques for preparing
these antigen-binding fragments.
[0113] Therefore, in another aspect, the present invention provides
an isolated nucleic acid molecule comprising a nucleotide sequence
encoding the antibody or antigen-binding fragment thereof of the
present invention, or heavy chain variable region and/or light
chain variable region thereof. In certain preferred embodiments,
the isolated nucleic acid molecule encodes the antibody or
antigen-binding fragment thereof of the present invention, or heavy
chain variable region and/or light chain variable region
thereof.
[0114] In another aspect, the present invention provides a vector
(for example, a cloning vector or an expression vector) comprising
the isolated nucleic acid molecule of the present invention. In
certain preferred embodiments, the vector of the present invention
is, for example, plasmid, cosmid, bacteriophage and the like.
[0115] In another aspect, the present invention provides a host
cell comprising the isolated nucleic acid molecule of the present
invention or the vector of the present invention. Such host cell
includes, but is not limited to, prokaryotic cell such as E. coli
cell, and eukaryotic cell such as yeast cell, insect cell, plant
cell and animal cell (for example, mammalian cell, such as mouse
cell, human cell, etc.). In certain preferred embodiments, the host
cell of the present invention is a mammalian cell, such as CHO (for
example, CHO-K1, CHO-S, CHO DG44).
[0116] In another aspect, a method for preparing the antibody or
antigen-binding fragment thereof of the present invention is
provided, which comprises culturing the host cell of the present
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 cultured host cell
culture.
Derived Antibody
[0117] The antibody or antigen-binding fragment thereof of the
present invention can be derivatized, for example linked to another
molecule (for example, another polypeptide or protein). Generally,
the derivatization (for example, labeling) of the antibody or
antigen-binding fragment thereof will not adversely affect its
binding to HBsAg. Therefore, the antibody or antigen-binding
fragment thereof of the present invention is also intended to
include such derivatized forms. For example, the antibody or
antigen-binding fragment of the present invention can be
functionally linked (by chemical coupling, gene fusion,
non-covalent linkage or other means) to one or more other molecular
groups, such as another antibody (for example, to form a bispecific
antibody), detection reagent, pharmaceutical reagent, and/or
protein or polypeptide capable of mediating the antibody or
antigen-binding fragment to bind to another molecule (for example,
avidin or polyhistidine tag).
[0118] Therefore, in certain embodiments, the antibody of the
present invention or antigen-binding fragment thereof is labeled.
In some embodiments, the antibody or antigen-binding fragment
thereof of the present invention bears a detectable label, such as
enzyme, radionuclide, fluorescent dye, luminescent substance (for
example, chemiluminescent substance), or biotin. The detectable
label of the present invention can be any substance that can be
detected by fluorescence, spectroscopy, photochemistry,
biochemistry, immunology, electrical, optical or chemical means.
Such labels are well known in the art, examples of which include,
but are not limited to, enzyme (for example, horseradish
peroxidase, alkaline phosphatase, (.beta.-galactosidase, urease,
glucose oxidase, etc.), radioactive nuclide (for example, .sup.3H,
.sup.125I, .sup.35S, .sup.14C or .sup.32P), fluorescent dye (for
example, fluorescein isothiocyanate (FITC), fluorescein,
tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE),
Texas red, rhodamine, quantum dots or cyanine dye derivatives (for
example, Cy7, Alexa 750)), luminescent substance (for example,
chemiluminescent substance, such as acridine ester compound),
magnetic beads (for example, Dynabeads.RTM.), calorimetric marker
such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads, and biotin used to
ligate avidin (for example, streptavidin) modified by the
above-mentioned marker. In certain embodiments, such label can be
suitable for immunological detection (for example, enzyme-linked
immunoassay, radioimmunoassay, fluorescent immunoassay,
chemiluminescence immunoassay, etc.). In certain embodiments, the
detectable label as described above can be ligated to the antibody
or antigen-binding fragment thereof of the present invention
through a linker of different length to reduce potential steric
hindrance.
Pharmaceutical Composition and Therapeutic Use
[0119] The antibody or antigen-binding fragment thereof of the
present invention can be used for the prevention or treatment of an
HBV infection in a subject (for example, a human) or a disease
associated with HBV infection (for example, hepatitis B), for
neutralizing in vitro or in a subject (for example, a human) a
virulence of HBV, for reducing a serum level of HBV DNA and/or
HBsAg in a subject (for example, a human), and for activating a
humoral immune response to HBV in a subject (for example, a patient
with chronic HBV infection or chronic hepatitis B).
[0120] Therefore, in another aspect, the present invention provides
a pharmaceutical composition, which comprises the antibody or
antigen-binding fragment thereof of the present invention, and a
pharmaceutically acceptable carrier and/or excipient. The
pharmaceutical composition of the present invention may also
comprises an additional pharmaceutically active agent. In certain
embodiments, the additional pharmaceutically active agent is a drug
used to prevent or treat an HBV infection or a disease associated
with HBV infection (for example, hepatitis B), for example,
interferon drug, such as interferon or pegylated interferon.
[0121] In another aspect, the present inveiton provides a use of
the antibody or antigen-binding fragment thereof of the present
invention or the pharmaceutical composition of the present
invention in the manufacture of a medicament for the prevention
and/or treatment of an HBV infection (for example, a human) or a
disease associated with HBV infection (for example, hepatitis B) in
a subject, for neutralizing a virulence of HBV in vitro or in a
subject (for example, a human), for reducing a serum level of HBV
DNA and/or HBsAg in a subject (for example, a human), and/or for
activating a humoral immune response to HBV in a subject (for
example, a patient with chronic HBV infection or chronic hepatitis
B).
[0122] In another aspect, the present invention provides a method
for preventing or treating an HBV infection or a disease associated
with HBV infection (for example, hepatitis B) in a subject (for
example, a human), for neutralizing a virulence of HBV in vivo or
in a subject (for example, a human), for reducing a serum level of
HBV DNA and/or HBsAg in a subject (for example, a human), and/or
for activating a humoral immune response to HBV in a subject (for
example, a patient with chronic HBV infection or chronic hepatitis
B), the method comprises administering an effective amount of the
antibody or antigen-binding fragment thereof according to the
present invention or the pharmaceutical composition according to
the present invention to a subject in need thereof.
[0123] The drugs and pharmaceutical compositions provided by the
present invention can be used alone or in combination, and can also
be used in combination with other pharmaceutically active agents
(for example, other antiviral agents, such as interferon drugs,
such as interferon or pegylated interferon).
[0124] The antibody or antigen-binding fragment thereof of the
present invention or the pharmaceutical composition of the present
invention can be administered by a traditional route of
administration, including but not limited to oral, buccal,
sublingual, ocular, topical, parenteral, rectal, intrathecal,
intracytoplasmic reticulum, inguinal, intravesical, topical (e.g.,
powder, ointment or drops), or nasal route. The antibody or
antigen-binding fragment thereof of the present invention can be
administered by various methods known in the art. However, for many
therapeutic applications, the preferred route/mode of
administration is parenteral administration (for example,
intravenous injection, subcutaneous injection, intraperitoneal
injection, intramuscular injection). The skilled person should
understand that the route and/or mode of administration will vary
according to the intended purpose. In a preferred embodiment, the
antibody or antigen-binding fragment thereof of the present
invention is administered by intravenous infusion or injection.
[0125] The antibody or antigen-binding fragment thereof of the
present invention or the pharmaceutical composition of the present
invention can be formulated into a variety of dosage forms, such as
liquid, semisolid, and solid forms, for example, solution (e.g.
injection), dispersion or suspension, tablet, powder, granule,
emulsion, pill, syrup, powder, liposome, capsule and suppository.
The preferred dosage form depends on the intended mode of
administration and therapeutic use.
[0126] For example, one preferred dosage form is an injection. Such
an injection may be a sterile injectable solution. For example, a
sterile injectable solution can be prepared by the following
method: a necessary dose of the antibody or an antigen binding
fragment thereof according to the invention is incorporated into a
suitable solvent, and optionally, other expected ingredients
(including, but not limited to, a pH regulator, a surfactant, an
adjuvant, an ionic strength enhancer, an isotonic agent, a
preservative, a diluent, or any combination thereof) are
incorporated simultaneously, and then filtered sterilization is
carried out. In addition, the sterile injectable solution can be
prepared into a sterile powder (for example, by vacuum drying or
freeze drying) for the convenience of storage and use. Such sterile
powder can be dispersed in a suitable vehicle before use, such as
sterile pyrogen-free water.
[0127] Another preferred dosage form is a dispersion. A dispersion
can be prepared by the following method: the antibody or an antigen
binding fragment thereof according to the invention is incorporated
in a sterile vehicle comprising a basic dispersion medium and
optionally, other expected ingredients (including, but not limited
to, a pH regulator, a surfactant, an adjuvant, an ionic strength
enhancer, an isotonic agent, a preservative, a diluent, or any
combination thereof). In addition, an absorption delaying agent can
also be incorporated in a dispersion, such as monostearate salt and
gelatin, in order to obtain an expected pharmacokinetic
property.
[0128] Another preferred dosage form is an oral solid dosage form,
including capsule, tablet, powder, granule, and the like. Such a
solid dosage form generally comprises at least one of: (a) inert
drug excipient (or vehicle), such as sodium citrate and calcium
phosphate; (b) filler, such as starch, lactose, sucrose, mannose
and silicic acid; (c) binder, such as carboxymethyl cellulose,
alginate, gelatin, polyvinylpyrrolidone, sucrose and arabic gum;
(d) wetting agent, such as glycerol; (e) disintegrating agent, such
as agar, calcium carbonate, potato or tapioca starch; (f) retarder,
such as olefin; (g) absorption enhancer, such as quaternary
ammonium compound; (h) humectant, such as cetyl alcohol and
glyceryl monostearate; (i) adsorbent, such as kaolin and bentonite;
(j) lubricant, such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycol, sodium dodecyl sulfate, or any
combination thereof. In the case of tablet and capsule dosage
forms, a buffer can also be comprised.
[0129] In addition, a release rate modifier (i.e. an agent capable
of changing drug release rate) may also be added to an oral solid
dosage form, in order to obtain a modified release or pulsed
release dosage form. Such a release rate modifier includes, but is
not limited to carboxypropyl methylcellulose, methylcellulose,
carboxymethyl cellulose sodium, ethyl cellulose, cellulose acetate,
polyethylene oxide, xanthan gum, isoacrylic amino copolymer,
hydrogenated flavoring oil, carnauba wax, paraffin, cellulose
acetate phthalate, carboxypropyl methylcellulose phthalate,
methacrylic acid copolymer, or any combination thereof. A modified
release or pulsed release dosage form may comprise one or a group
of release rate modifiers.
[0130] Another preferred dosage form is an oral liquid dosage form,
including emulsion, solution, suspension, syrup, and the like. In
addition to active ingredients, such an oral liquid dosage form may
further comprise inert solvents commonly used in the art, for
example water or other solvents, such as ethyl alcohol,
isopropanol, propylene glycol, 1,3-butylene glycol, oil (such as
cotton seed oil, peanut oil, corn oil, olive oil, flavoring oil and
sesame oil), glycerol, polyethylene glycol and sorbitan fatty acid
ester, and any combination thereof. In addition to these inert
solvents, such an oral liquid dosage form may further comprise
humectant, emulsifying agent, suspending agent, sweetening agent,
flavoring agent, fragrant agent, and the like.
[0131] In addition, the antibody or an antigen binding fragment
thereof according to the invention may be present in a unit dosage
form in a pharmaceutical composition, for the convenience of
administration. The pharmaceutical composition according to the
invention should be sterile, and stable under the conditions of
manufacture and storage conditions.
[0132] The medicament and pharmaceutical composition provided in
the invention may be used alone or in combination, or may be used
in combination with an additional pharmaceutically active agent
(for example, other antiviral agents, e.g. interferon-type agents,
such as interferon or pegylated interferon). In some preferred
embodiments, the antibody or an antigen binding fragment thereof
according to the invention is used in combination with other
antiviral agent(s), in order to prevent and/or treat a disease
associated with HBV infection. The antibody or an 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 agents, ribavirin, adamantane, hydroxyurea,
IL-2, L-12 and pentacarboxy cytosolic acid, etc.
[0133] The pharmaceutical composition according to the invention
may comprise "a therapeutically effective amount" or "a
prophylactically effective amount" of the antibody or an antigen
binding fragment thereof according to the invention. "A
prophylactically effective amount" refers to an amount that is
sufficient to prevent, suppress or delay the development of a
disease (such as HBV infection or a disease associated with HBV
infection). "A therapeutically effective amount" refers to an
amount that is sufficient to cure or at least partially suppress a
disease and its complications in a patient with the disease. The
therapeutically effective amount of the antibody or an antigen
binding fragment thereof according to the invention may vary
depending on the following factors: the severity of a disease to be
treated, general state of the immune system in a patient, general
conditions of a patient such as age, weight and gender,
administration modes of drugs, additional therapies used
simultaneously, and the like.
[0134] A dosage regimen can be adjusted to provide an optimal
desired effect (for example, a therapeutic or prophylactic effect).
For example, a single dose may be administered, or multiple doses
may be administered within a period of time, or the dose can be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation.
[0135] For the antibody or antigen binding fragment thereof
according to the invention, an exemplary and 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 0.1-25 mg/kg, 0.1-10 mg/kg. It should be noticed that a
dose can vary depending on the type and severity of a disease to be
treated. In addition, a person skilled in the art understands that
for any specific patient, specific dosage regimen should be
adjusted over time depending on the patient's need and the
professional evaluation made by a doctor; the dose range provided
here is only provided for the purpose of exemplification, rather
than defining the use or scope of the pharmaceutical composition
according to the invention.
Kit and Detection Use
[0136] The antibody or antigen-binding fragment thereof of the
present invention can specifically bind to HBsAg, so that it can be
used to detect the presence or level of HBsAg in a sample.
[0137] Therefore, in another aspect, the present invention provides
a kit comprising the antibody or antigen-binding fragment thereof
of the present invention. In some embodiments, the antibody or
antigen-binding fragment thereof of the present invention bears a
detectable label. In other embodiments, the kit further comprises a
second antibody, which specifically recognizes the antibody or
antigen-binding fragment thereof of the present invention.
Preferably, the second antibody further comprises a detectable
label. Such detectable labels are well known to those skilled in
the art, and include, but are not limited to, radioisotope,
fluorescent substance, luminescent substance, colored substance and
enzyme (for example, horseradish peroxidase) and the like.
[0138] In another aspect, the present invention provides a method
for detecting the presence or level of HBsAg protein in a sample,
which comprises: using the antibody or antigen-binding fragment
thereof of the present invention. In some embodiments, the antibody
or antigen-binding fragment thereof of the present invention
further comprises a detectable label. In other embodiments, the
method further comprises using a second antibody carrying a
detectable label to detect the antibody or antigen-binding fragment
thereof of the present invention. The method can be used for
diagnostic purposes, or for non-diagnostic purposes (for example,
the sample is a cell sample, not a sample from a patient).
[0139] In some embodiments, the method comprises: (1) contacting
the sample with the antibody or antigen-binding fragment thereof of
the present invention; (2) detecting the formation of a complex
between the antibody or antigen-binding fragment thereof and HBsAg
protein or detecting an amount of the complex. The formation of the
complex indicates the presence of HBsAg protein and/or HBV.
[0140] In another aspect, the present invention provides a method
for diagnosing whether a subject is infected with HBV, which
comprises: using the antibody or antigen-binding fragment thereof
of the present invention to detect the presence of HBsAg protein in
a sample from the subject. In some embodiments, the antibody or
antigen-binding fragment thereof of the present invention further
comprises a detectable label. In other embodiments, the method
further comprises using a second antibody carrying a detectable
label to detect the antibody or antigen-binding fragment thereof of
the present invention.
[0141] In another aspect, there is provided a use of the antibody
or antigen-binding fragment thereof of the present invention in the
manufacture of a kit for detecting the presence or level of HBsAg
protein in a sample, or for diagnosing whether a subject is
infected with HBV.
Definition of Terms
[0142] In the present invention, unless otherwise specified, the
scientific and technical terms used herein have the meanings
commonly understood by those skilled in the art. Moreover, the cell
culture, biochemistry, nucleic acid chemistry, immunology
laboratory and other operating steps used in this article are all
routine steps widely used in the corresponding fields. At the same
time, in order to better understand the present invention,
definitions and explanations of related terms are provided
below.
[0143] As used herein, the term "antibody" refers to an
immunoglobulin molecule typically composed of two pairs of
polypeptide chains, each pair having a light chain (LC) and a heavy
chain (HC). Antibody light chains can be classified into .kappa.
(kappa) and .lamda. (lambda) light chains. Heavy chains can be
classified as .mu., .delta., .gamma., .alpha., or .epsilon., and
the isotypes of antibody are defined as IgM, IgD, IgG, IgA, and
IgE, respectively. Within the light and heavy chains, the variable
and constant regions are connected by a "J" region of about 12 or
more amino acids, and the heavy chain also comprises a "D" region
of about 3 or more amino acids. Each heavy chain is composed of a
heavy chain variable region (VH) and a heavy chain constant region
(CH). The heavy chain constant region is composed of 3 domains
(CH1, CH2, and CH3). Each light chain is composed of a light chain
variable region (VL) and a light chain constant region (CL). The
light chain constant region is composed of a domain CL. The
constant domain does not directly participate in the binding of
antibody and antigen, but exhibits a variety of effector functions,
such as mediating the binding of immunoglobulin to a host tissue or
factor, including various cells of immune system (for example,
effector cells) and the first component of classical complement
system (C1q). The VH and VL regions can also be subdivided into
hypervariable regions (called complementarity determining regions
(CDRs)), interspersed with relatively conservative regions called
framework regions (FRs). Each VH and VL is composed of 3 CDRs and 4
FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4 from the amino terminus to the carboxy terminus. The
variable regions (VH and VL) of each heavy chain/light chain pair
form antigen binding site respectively. The assignment of amino
acids in each region or domain can follow the definitions of 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.
[0144] As used herein, the term "complementarity determining
region" or "CDR" refers to amino acid residues in a variable region
of an antibody that are responsible for antigen binding. Each of
the variable regions of the heavy chain and the light chain
contains three CDRs, named CDR1, CDR2, and CDR3. The precise
boundaries of these CDRs can be defined according to various
numbering systems known in the art, for example, according to the
Kabat numbering system (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991), Chothia numbering
system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917;
Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system
(Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003). For a
given antibody, those skilled in the art will easily identify the
CDRs defined by each numbering system. Moreover, the correspondence
between different numbering systems is well known to those skilled
in the art (for example, see Lefranc et al., Dev. Comparat.
Immunol. 27:55-77, 2003).
[0145] In the present invention, the CDRs contained in the antibody
or antigen-binding fragment thereof of the present invention can be
determined according to various numbering systems known in the art.
In certain embodiments, the CDRs contained in the antibody or
antigen-binding fragment thereof of the present invention are
preferably determined by the Kabat, Chothia or IMGT numbering
system. In certain embodiments, the CDRs contained in the antibody
or antigen-binding fragment thereof of the present invention are
preferably determined by the Kabat numbering system.
[0146] As used herein, the term "framework region" or "FR" residues
refers to those amino acid residues in a variable region of an
antibody other than the CDR residues as defined above.
[0147] The term "antibody" is not limited by any specific method
for producing the antibody. For example, it comprises recombinant
antibody, monoclonal antibody, and polyclonal antibody. The
antibody may be an antibody of different isotype, for example, IgG
(for example, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD,
IgE or IgM antibody.
[0148] As used herein, the term "antigen-binding fragment" of
antibody refers to a polypeptide comprising a fragment of a
full-length antibody that retains the ability to specifically bind
to the same antigen to which the full-length antibody binds, and/or
competes with the full-length antibody to specifically bind to the
antigen, which is also called "antigen binding portion". See
generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd
edition, Raven Press, NY (1989), which is incorporated herein by
reference in its entirety for all purposes. Antigen-binding
fragment of antibody can be produced by recombinant DNA technology
or by the enzymatic or chemical cleavage of the intact antibody.
Non-limiting examples of antigen-binding fragment include Fab,
Fab', F(ab').sub.2, Fd, Fv, complementarity determining region
(CDR) fragments, scFv, diabody, single domain antibody, chimeric
antibody, linear antibody, nanobody (technology from Domantis),
probody and such polypeptides which comprise at least a portion of
the antibody that is enough to confer a specific antigen-binding
capacity to the polypeptides. Engineered antibody variants are
reviewed in Holliger et al., 2005; Nat Biotechnol, 23:
1126-1136.
[0149] As used herein, the term "full-length antibody" refers to an
antibody composed of two "full-length heavy chains" and two
"full-length light chains." "full-length heavy chain" refers to a
polypeptide composed of a heavy chain variable region (VH), a heavy
chain constant region CH1 domain, a hinge region (HR), a heavy
chain constant region CH2 domain and a heavy chain constant region
CH3 domain in the N-terminal to C-terminal direction; and, when the
full-length antibody is of the IgE isotype, it optionally also
comprises a heavy chain constant region CH4 domain. Preferably, the
"full-length heavy chain" is a polypeptide chain composed of VH,
CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
The "full-length light chain" is a polypeptide chain composed of a
light chain variable region (VL) and a light chain constant region
(CL) in the N-terminal to C-terminal direction. The two pairs of
full-length antibody chains are connected by a disulfide bond
between CL and CH1 and a disulfide bond between HRs of the two
full-length heavy chains. The full-length antibody of the present
invention can be derived from a single species, such as human; it
can also be a chimeric antibody or a humanized antibody. The
full-length antibody of the present invention comprises two antigen
binding sites formed by VH and VL pairs respectively, and the two
antigen binding sites specifically recognize/bind the same
antigen.
[0150] As used herein, the term "Fd" refers to an antibody fragment
composed of VH and CH1 domains; the term "dAb fragment" refers to
an antibody fragment composed of VH domain (Ward et al., Nature
341:544 546 (1989)); the term "Fab fragment" refers to an antibody
fragment composed of VL, VH, CL and CH1 domains; the term
"F(ab').sub.2 fragment" refers to an antibody fragment composed of
two Fab fragments connected by a disulfide bridge on the hinge
region; the term "Fab' fragment" refers to a fragment obtained by
reducing the disulfide bond connecting the two heavy chain
fragments in the F(ab').sub.2 fragment, and is composed of an
intact light chain and a Fd fragment (consisting of VH and CH1
domains) of heavy chain.
[0151] As used herein, the term "Fv" refers to an antibody fragment
composed of a single-arm VL and VH domains of an antibody. Fv
fragment is generally considered to be the smallest antibody
fragment that can form a complete antigen-binding site. It is
generally believed that six CDRs confer antigen-binding specificity
to an antibody. However, even one variable region (e.g., Fd
fragment, which contains only three antigen-specific CDRs) can
recognize and bind to antigen, although its affinity may be lower
than the complete binding site.
[0152] As used herein, the term "Fc" refers to an antibody fragment
that is formed by linking the second, third constant region of a
first heavy chain of an antibody and the second, third constant
region of a second heavy chain via disulfide bonding. The Fc
fragment of an antibody has many different functions, but does not
participate in antigen binding.
[0153] As used herein, the term "scFv" refers to a single
polypeptide chain comprising VL and VH domains, wherein the VL and
VH are connected by a linker (see, for example, Bird et al.,
Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci.
USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of
Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds,
Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules
may have the general structure: NH.sub.2-VL-linker-VH-COOH or
NH.sub.2-VH-linker-VL-COOH. Suitable prior art linkers consist of
repeated GGGGS amino acid sequences or variants thereof. For
example, a linker having the amino acid sequence (GGGGS).sub.4 can
be used, but variants thereof can also be used (Holliger et al.
(1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers
that can be used in the present invention are described by 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. In some cases, there may also be
disulfide bonds between the VH and VL of the scFv.
[0154] As used herein, the term "diabody" refers to that its VH and
VL domains are expressed on a single polypeptide chain, but the
used linker is too short to allow pairing between the two domains
of the same chain, thereby forcing one domain to pair with the
complementary domain of another chain and generating two
antigen-binding sites (see, for example, Holliger P. et al., Proc.
Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al.,
Structure 2:1121-1123 (1994)).
[0155] Each of the aforementioned antibody fragments maintains the
ability to specifically bind to the same antigen to which the
full-length antibody binds, and/or competes with the full-length
antibody to specifically bind to the antigen.
[0156] Conventional techniques known to those skilled in the art
(for example, recombinant DNA technology or enzymatic or chemical
fragmentation) can be used to obtain from a given antibody (for
example, the antibody provided by the present invention) the
antigen-binding fragments of the antibody (for example, the
above-mentioned antibody fragments), and can be screened for
specificity in the same manner by which intact antibodies are
screened.
[0157] Herein, unless the context clearly dictates otherwise, when
the term "antibody" is referred to, it includes not only intact
antibody but also antigen-binding fragments of the antibody.
[0158] As used herein, the term "monoclonal antibody", "McAb" and
"mAb" have the same meaning and can be used interchangeably. It
refers to an antibody or a fragment of an antibody from a
population of highly homologous antibody molecules, i.e. a
population of completely identical antibody molecules except for
natural mutation that may occur spontaneously. A monoclonal
antibody has a high specificity for a single epitope of an antigen.
Polyclonal antibody, relative to monoclonal antibody, generally
comprises at least two or more different antibodies which generally
recognize different epitopes on an antigen. In addition, the
modifier "monoclonal" merely indicates the character of the
antibody as being obtained from a highly homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method.
[0159] As used herein, the term "chimeric antibody" refers to an
antibody that a part of its light chain or/and heavy chain is
derived from an antibody (which may be derived from a specific
species or belong to a specific antibody class or subclass), and
another part of its light chain or/and heavy chain is derived from
another antibody (which may be derived from the same or different
species or belong to the same or different antibody class or
subclass), but in any case, it still retains the binding activity
to the target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.;
Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
For example, the term "chimeric antibody" may include such an
antibody (e.g., human-mouse chimeric antibody), in which the heavy
and light chain variable regions of the antibody are derived from a
first antibody (e.g., mouse antibody), while the heavy chain and
light chain constant regions of the antibody are derived from a
second antibody (e.g., human antibody). In order to prepare a
chimeric antibody, the methods known in the art can be used to link
immunoglobulin variable regions of an immunized animal to human
immunoglobulin constant regions (see, for example, U.S. Pat. No.
4,816,567 to Cabilly et al.). For example, a DNA encoding VH is
operably linked to another DNA molecule encoding the heavy chain
constant region to obtain a full-length heavy chain gene. The
sequence of the human heavy chain constant region gene is known in
the art (see, for example, Kabat, E A et al. (1991), Sequences of
Proteins of Immunological Interest, Fifth Edition, US Department of
Health and Human Services, NIH Publication No. 91-3242), the DNA
fragments comprising these regions can be obtained by standard PCR
amplification. The heavy chain constant region may be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is
generally preferably an IgG1 or IgG4 constant region. For example,
the DNA encoding VL is operably linked to another DNA molecule
encoding the light chain constant region CL to obtain a full-length
light chain gene (and a Fab light chain gene). The sequence of the
human light chain constant region gene is known in the art (see,
for example, Kabat, EA et al. (1991), Sequences of Proteins of
Immunological Interest, Fifth Edition, US Department of Health and
Human Services, NTH Publication No. 91-3242), and DNA fragments
comprising these regions can be obtained by standard PCR
amplification. The light chain constant region can be a or .lamda.,
constant region, but is generally preferably a .kappa. constant
region.
[0160] As used herein, the term "humanized antibody" refers to a
genetically engineered non-human antibody, whose amino acid
sequence has been modified to increase homology with the sequence
of a human antibody. Generally speaking, all or part of the CDR
regions of a humanized antibody are derived from a non-human
antibody (donor antibody), and all or part of the non-CDR regions
(for example, variable region FR and/or constant region) are
derived from a human immunoglobulin (receptor antibody). In some
embodiments, the CDR regions of the humanized antibody are derived
from a non-human antibody (donor antibodies), and all or part of
the non-CDR regions (for example, variable region FR and/or
constant regions) are derived from a human immunoglobulin (receptor
antibody). The humanized antibody generally retains the expected
properties of the donor antibody, including, but not limited to,
antigen specificity, affinity, reactivity, etc. The donor antibody
may be a mouse, rat, rabbit, or non-human primate (for example,
cynomolgus monkey) antibody with desired properties (for example,
antigen specificity, affinity, reactivity, etc.). In order to
prepare the humanized antibody, the methods known in the art can be
used to insert the CDR regions of the immunized animal into the
human framework sequences (see U.S. Pat. No. 5,225,539 to Winter;
U.S. Pat. No. 5,530,101 to Queen et al.; U.S. PAt. Nos. 5,585,089;
5,693,762 and 6,180,370; and Lo, Benny, K C, editor, in Antibody
Engineering: Methods and Protocols, volume 248, Humana Press, New
Jersey, 2004).
[0161] As used herein, the term "germline antibody gene" or
"germline antibody gene segment" refers to a sequence present in
the genome of an organism encoding immunoglobulin, which has not
undergone a maturation process that can lead to genetic
rearrangements and mutations for expression of a particular
immunoglobulin. In the present invention, the expression "heavy
chain germline gene" refers to an germline antibody gene or gene
fragment encoding an immunoglobulin heavy chain, which includes V
gene (variable), D gene (diversity), J gene (joining) and C gene
(constant); similarly, the expression "light chain germline gene"
refers to an germline antibody gene or gene fragment encoding an
immunoglobulin light chain, which includes V gene (variable), J
gene (joining), and C gene (constant). In the present invention,
the amino acid sequence encoded by the germline antibody gene or
the germline antibody gene fragment is also referred to as
"germline sequence". The germline antibody gene or germline
antibody gene fragment and their corresponding germline sequences
are well known to those skilled in the art and can be obtained or
queried from professional databases (e.g., IMGT, unswag, NCBI or
VBASE2).
[0162] As used herein, the term "specific binding" refers to a
non-random binding reaction between two molecules, such as the
reaction between an antibody and an antigen to which it is
directed. The strength or affinity of a specific binding
interaction can be expressed by an equilibrium dissociation
constant (K.sub.D) of the interaction. In the present invention,
the term "K.sub.D" refers to a dissociation equilibrium constant of
a specific antibody-antigen interaction, which is used to describe
the binding affinity between the antibody and the antigen. The
smaller the equilibrium dissociation constant, the tighter the
antibody-antigen binding, and the higher the affinity between the
antibody and the antigen. The specific binding properties between
two molecules can be measured using methods known in the art, for
example, using surface plasmon resonance (SPR) of BIACORE
instrument.
[0163] As used herein, the expression "binding at a neutral pH with
an affinity higher than that at an acidic pH" or the equivalent
expression "pH-dependent binding" refers to that the antibody of
the present invention has a K.sub.D value or EC50 value for binding
HBsAg at an acidic pH that is higher than its K.sub.D value or EC50
value for binding HBsAg at a neutral pH. The K.sub.D can be
measured by a technique known in the art, for example, by SPR
technique (for example, Biacore). In the present invention, the
term "EC50" refers to an antibody-antigen half maximum effect
concentration, that is, an antibody concentration required to reach
50% of the maximum binding effect between a specific
antibody-antigen, and it is used to describe the binding capacity
between the antibody and the antigen. The smaller the EC50, the
higher the binding capacity between the antibody and the antigen.
The antibody-antigen half maximum effect concentration (EC50) can
be determined using methods known in the art, for example, using an
enzyme-linked immunosorbent assay (ELISA) in which an antigen is
bound to a solid phase carrier, and the antibody specifically binds
to the antigen.
[0164] As used herein, "neutralizing antibody" refers to an
antibody or antigen-binding fragment thereof that can significantly
reduce or completely inhibit the virulence (for example, the
ability to infect cells) of the target virus. Generally speaking,
neutralizing antibodies can recognize and bind the target virus,
and prevent the target virus from entering/infecting the subject's
cells. The antibody of the present invention is a neutralizing
antibody.
[0165] However, it should be understood that in the present
application, the virus-neutralizing ability of an antibody is not
directly equivalent to the virus-clearing ability of an antibody.
As used herein, "neutralizing virus" means that the virulence of a
target virus is neutralized (i.e. the virulence of a target virus
is significantly reduced or completely inhibited) by inhibiting the
target virus from entering/infecting the cell of a subject. As used
herein, "clearing virus" means that a target virus (no matter it
infects a cell or not) is eliminated from an organism, and
therefore the organism turns toward the state before infection by
the virus (e.g. the serological test result of virus turns
negative). Therefore, in general, neutralizing antibodies do not
necessarily have virus-clearing ability. However, in the present
application, the inventor surprisingly found that the antibodies
according to the invention can not only neutralize HBV, but also
clear virus (i.e. can clear HBV DNA and/or HBsAg in vivo, clear HBV
and HBV-infected cells in vivo), and therefore have important
clinical value.
[0166] As used herein, the term "isolated" refers to a state
obtained from natural state by artificial means. If a certain
"isolated" substance or component is present in nature, it is
possible because its natural environment changes, or the substance
is isolated from natural environment, or both. For example, a
certain un-isolated polynucleotide or polypeptide naturally exists
in a certain living animal body, and the same polynucleotide or
polypeptide with a high purity isolated from such a natural state
is called isolated polynucleotide or polypeptide. The term
"isolated" excludes neither the mixed artificial or synthesized
substance nor other impure substances that do not affect the
activity of the isolated substance.
[0167] As used herein, the term "vector" refers to a nucleic acid
vehicle into which a polynucleotide can be inserted. When a vector
enables the expression of a protein encoded by an inserted
polynucleotide, the vector is referred to as an expression vector.
A vector can be introduced into a host cell by transformation,
transduction or transfection, so that the genetic material elements
carried by the vector can be expressed in the host cell. Vectors
are well known to those skilled in the art and include, but are not
limited to: plasmids; phagemids; cosmids; artificial chromosomes,
such as yeast artificial chromosomes (YAC), bacterial artificial
chromosomes (BAC) or P1-derived artificial chromosomes (PAC);
bacteriophages such as .lamda. phage or M13 phage and animal
viruses. Animal viruses that can be used as vectors include, but
are not limited to, retroviruses (including lentiviruses),
adenoviruses, adeno-associated viruses, herpes viruses (e.g.,
herpes simplex virus), poxviruses, baculoviruses, papillomaviruses,
papovavirus (e.g., SV40). A vector may comprise a variety of
elements that control expression, including, but not limited to,
promoter sequence, transcription initiation sequence, enhancer
sequence, selection element, and reporter gene. In addition, the
vector may comprise a replication initiation site.
[0168] As used herein, the term "host cell" refers to a cell into
which a vector can be introduced, which includes, but is not
limited to, prokaryotic cell such as Escherichia coli or Bacillus
subtilis, fungal cell such as yeast cell or Aspergillus, insect
cell such as S2 Drosophila cell or Sf9, or animal cell such as
fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK
293 cell or human cell.
[0169] As used herein, the term "identity" refers to the match
degree between two polypeptides or between two nucleic acids. When
two sequences for comparison have the same monomer sub-unit of base
or amino acid at a certain site (e.g., each of two DNA molecules
has an adenine at a certain site, or each of two polypeptides has a
lysine at a certain site), the two molecules are identical at the
site. The percent identity between two sequences is a function of
the number of identical sites shared by the two sequences over the
total number of sites for comparison.times.100. For example, if 6
of 10 sites of two sequences are matched, these two sequences have
an identity of 60%. For example, DNA sequences: CTGACT and CAGGTT
share an identity of 50% (3 of 6 sites are matched). Generally, the
comparison of two sequences is conducted in a manner to produce
maximum identity. Such alignment can be conducted by using a
computer program such as Align program (DNAstar, Inc.) which is
based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453,
1970). The percent identity between two amino acid sequences can
also be determined using the algorithm of E. Meyers and W. Miller
(Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated
into the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4. In
addition, the percentage of identity between two amino acid
sequences can be determined by the algorithm of Needleman and
Wunsch (J. Mol. Biol. 48:444-453 (1970)) which has been
incorporated into the GAP program in the GCG software package
(available at http://www.gcg.com), using either a Blossum 62 matrix
or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4
and a length weight of 1, 2, 3, 4, 5, or 6.
[0170] The twenty conventional amino acids involved herein are
expressed in routine manners. See, for example, Immunology-A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland, Mass. (1991)), which is incorporated herein
by reference. In the present disclosure, the terms "polypeptide"
and "protein" have the same meaning and are used interchangeably.
Also in the present disclosure, amino acids are generally
represented by single letter and three letter abbreviations as
known in the art. For example, alanine can be represented by A or
Ala. In addition, as used herein, the terms "monoclonal antibody"
and "McAb" have the same meaning and can be used interchangeably;
the terms "polyclonal antibody" and "PcAb" have the same meaning
and can be used interchangeably.
[0171] As used herein, the term "a pharmaceutically acceptable
carrier and/or excipient" refers to a carrier and/or excipient
pharmacologically and/or physiologically compatible with a subject
and an active agent, which is well known in the art (see, e.g.,
Remington's Pharmaceutical Sciences. Edited by Gennaro A R, 19th
ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but
is not limited to a pH adjuster, a surfactant, an adjuvant, an
ionic strength enhancer, a diluent, an osmotic pressure-controlling
agent, an absorption delaying agent, and a preservative. For
example, the pH adjuster includes, but is not limited to, phosphate
buffer. The surfactant includes, but is not limited to, cationic,
anionic, or non-ionic surfactant, e.g. Tween-80. The ionic strength
enhancer includes, but is not limited to, sodium chloride. The
preservative includes, but is not limited to a variety of
antibacterial agents and antifungal agents, such as paraben,
chlorobutanol, phenol, and sorbic acid. The osmotic
pressure-controlling agent includes, but is not limited to sugar,
NaCl and analogs thereof. The absorption delaying agent includes,
but is not limited to monostearate and gelatin.
[0172] As used herein, the term "prevention/preventing" refers to a
method that is carried out in order to suppress or delay the
occurrence of a disease, a disorder or a symptom (such as HBV
infection or a disease associated with HBV infection) in a subject.
As used herein, the term "treatment/treating" refers to a method
that is carried out in order to obtain a beneficial or desired
clinical outcome. For the purpose of the invention, the beneficial
or desired clinical outcome includes, but is not limited to, easing
symptom, narrowing the scope of disease, stabilizing (i.e. not
aggravating) the state of disease, delaying or slowing the progress
of disease, and alleviating symptoms (either partially or
completely), no matter detectable or not detectable. In addition,
"treatment" also refers to a prolonged survival period compared to
the expected survival period (if no treatment is accepted). In the
present application, the antibody according to the invention has
the ability of neutralizing HBV, and therefore can be used to
prevent/protect an unaffected subject or a cell thereof from
infection by HBV. In addition, the antibody according to the
invention has the ability of clearing HBV (i.e. able to clear HBV
DNA and/or HBsAg in vivo, clear HBV and cells infected by HBV in
vivo), and therefore can be used to treat HBV infection or a
disease associated with HBV infection in an infected subject.
[0173] As used herein, the term "subject" refers to a mammal, such
as a primate mammal, such as a human.
[0174] As used herein, the term "an effective amount" refers to an
amount that is sufficient to achieve or at least partially achieve
the expected effect. For example, an amount effective for
preventing a disease (such as HBV infection or diseases associated
with HBV infection) refers to an amount effective for preventing,
suppressing, or delaying the occurrence of a disease (such as HBV
infection or diseases associated with HBV infection). An effective
amount for treating a disease refers to an amount effective for
curing or at least partially blocking a disease and its
complication in a patient having the disease. The determination of
such an effective amount is within the ability of a person skilled
in the art. For example, an amount effective for a therapeutic use
depends on severity of a disease to be treated, general state of
the immune system in a patient, general conditions of a patient,
such as age, weight and gender, administration means of drugs,
additional therapies used simultaneously, and the like.
Beneficial Effects of the Present Invention
[0175] The antibody of the present invention not only can
specifically recognize/bind HBsAg, can neutralize the virulence of
HBV, can reduce the serum level of HBV DNA and/or HBsAg in the
subject, and can effectively eliminate HBV and HBV-infected cells
in the body, but also has a significantly enhanced antigen
clearance effect and antigen suppression time. It is particularly
surprising that it is known in the art that patients with chronic
hepatitis B tend to produce immune depletion (tolerance) against
HBV due to high levels of HBsAg in the body, thereby prolonging the
infection, but the antibody of the present invention can activate
the subject (for example patients with chronic HBV infection, or
patients with chronic hepatitis B) to regenerate a humoral immune
response against HBV, thereby increasing the clinical cure rate.
Therefore, the antibody of the present invention is particularly
suitable for preventing and treating HBV infection and diseases
associated with HBV infection (for example, hepatitis B). In
addition, the antibody of the present invention has pH-dependent
antigen binding properties, and a single molecule of antibody can
bind to multiple molecules of antigens, so that it can also reduce
the frequency and dosage of administration, and has great clinical
value.
[0176] The embodiments of the present invention will be described
in detail below in conjunction with the accompanying drawings and
examples. However, those skilled in the art will understand that
the following drawings and examples are only used to illustrate the
present invention, but not to limit the scope of the present
invention. According to the accompanying drawings and the following
detailed description of the preferred embodiments, various objects
and advantageous aspects of the present invention will become
apparent to those skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0177] FIG. 1 shows a schematic diagram of the working principle of
an antibody with pH-dependent antigen-binding activity. Human
plasma is neutral, with a pH of about 7.4, while the intracellular
environment is acidic, with a pH of about 6.0. An antibody with
pH-dependent antigen-binding activity can bind to an antigen in the
plasma, the antigen-antibody complex is then internalized into the
cell. The pH-dependent antibody will dissociate from the antigen in
the acidic environment of the endosome. The antibody dissociated
from the antigen will be captured by FcRn and circulated to the
outside of the cell. In the extracelluar neutral environment, the
FcRn releases the antibody, and the antibody returned to the plasma
can bind to other antigen again, thereby realizing the cycle use of
the antibody.
[0178] FIG. 2 shows the results of docking of Fab crystal structure
based on the structural analysis of 162 to a short antigen mimic
peptide, in which the blue structure is the short antigen peptide,
and the red structure is part of the binding region of 162
antibody.
[0179] FIG. 3 shows a schematic diagram of the recombinant vector
(pCGMT-scFv) encoding the scFv antibody, in which the scFv antibody
has a structure of: NH.sub.2-VH-linker-VL-COOH.
[0180] FIGS. 4A to 4D show the ELISA results of the phage library
displaying the pH-dependent scFv antibody derived from 162 and the
antigen HBsAg. FIG. 4A: the detection results of binding to HBsAg
at pH 7.4 and pH 6.0 for the phage library derived from 162 after
the third round of screening, the abscissa represents the phage
antibody number, and the ordinate represents the OD value. The
results show that these single clones all have strong antigen
binding activity and have a significant decrease in binding
activity at pH 6.0. FIG. 4B: the detection results of pH-dependent
binding to HBsAg for the 13 single clones with high
OD.sub.(450/630) value at pH 7.4 in the third round and showing the
largest difference between OD.sub.(450/630) values at pH7.4 and pH
6.0, with 8 gradients and 3-fold dilution, in which the abscissa
represents the dilution factor, and the ordinate represents the OD
value. The results show that the pH-dependent antigen binding
effect is better presented after the gradient dilution, in which
the C32, C27, C26 and C19 show the better performance and C27
molecule has the best effect (the remaining 9 molecules are not
shown). FIG. 4C: the detection results of binding to HBsAg at pH
7.4 and pH 6.0 for the phage library derived from 162 after the
fourth round of screening, the abscissa represents the phage
antibody number, and the ordinate represents the OD value. FIG. 4D:
the detection results of pH-dependent binding to HB sAg for the 8
single clones with high OD.sub.(450/630) value at pH 7.4 in the
fourth round and showing the largest difference between
OD.sub.(450/630) values at pH 7.4 and pH 6.0, with 8 gradients and
3-fold dilution, in which the abscissa represents the dilution
factor, and the ordinate represents the OD value. The results show
that the pH-dependent antigen binding effect is better presented
after the gradient dilution, in which D3, D4 and D5 show the better
performance, and D5 molecule has the best effect (the remaining 5
molecules are not shown).
[0181] FIG. 5 shows a summary of the mutation sites of C26, C27,
C32, D3, D4 and D5.
[0182] FIG. 6A shows the detection results of binding to HBsAg at
pH 7.4 and pH 6.0 for the quantified cell supernatant obtained from
the small scale eukaryotic transfection of C32, C27 and C26 in
Example 3. FIG. 6B shows the detection results of binding to HBsAg
at pH 7.4 and pH 6.0 for the quantified cell supernatant obtained
from the small scale eukaryotic transfection of D3, D4 and D5 in
Example 3. The abscissa represents the antibody concentration (Log
10 ng/ml), and the ordinate represents the OD value. The results
show that C32, C27, C26, D3, D4 and D5 all can maintain an
antigen-binding activity equivalent to that of the parent antibody
162 at neutral pH, and all have a significant decrease in binding
activity to antigen at pH 6.0.
[0183] FIG. 7 shows the working principle of scavenger antibody.
The pH-dependent antigen binding activity plays a role in cells.
Thus, if this first limiting factor of cell entry is not broken,
the pH-dependent antigen-binding properties will not be applied
subsequently, and the benefit of modification will be greatly
reduced. A scavenger antibody obtained by further mutation of amino
acids in the Fc region can enhance the binding to hFcRn receptor at
neutral pH, or enhance the binding to Fc.gamma.Rs receptor. Tthe
scavenger antibody is located outside the cell and acts as a
"transport helper" for reciprocally transporting antigens into the
cell, the antibody half-life can thus be extremely prolonged, and
it can bind to antigen again, thereby improving the cell entry
efficiency of antigens, and greatly improving the clearance
efficiency.
[0184] FIGS. 8A to 8B show the protein gel results of pH-dependent
antibodies and antibodies with DY modification. FIG. 8A: the
picture of protein gel of pH-dependent antibodies, in which 162 is
a positive control, and the results show that the expressed C26,
D3, D4 and D5 antibodies are single-component. FIG. 8B: the picture
of protein gel of antibodies with DY modification, in which 162 is
a positive control, and the results show that the expressed
antibodies C26 DY, D3 DY, D4 DY and D5 DY are single-component.
[0185] FIGS. 9A to 9D show the detection results of pH-dependent
antibodies and antibodies with DY modification binding to HBsAg at
pH 7.4 and pH 6.0 in Example 4, in which the abscissa represents
the antibody concentration (Log 10 ng/ml) and the ordinate
represents the OD value. The results show that D26, D3, D4 and D5
can maintain an antigen-binding activity equivalent to that of the
parent (162) at the neutral pH, and have a significant decrease in
antigen-binding activity under the condition of pH 6.0, and the
corresponding antibodies with DY modification also can maintain an
antigen-binding activity at the neutral pH and a pH-dependent
antigen-binding activity comparable to those of the parent.
[0186] FIG. 10A shows the immunofluorescence experiment of mouse
primary macrophages for the pH-dependent antibodies and antibodies
with DY modification in Example 4, in which the green fluorescence
represents hFcRn, the blue fluorescence represents nucleus, and the
red fluorescence represents HBsAg. The results show that the DY
modification enhances the phagocytosis of murine macrophages to the
antigen-antibody complexes. FIG. 10B shows phagocytosis experiment
based on human THP-1 phagocytic cells of the pH-dependent
antibodies and the antibodies with DY modification in Example 4.
The results show that the DY modification enhances the phagocytosis
of human THP-1 phagocytic cells to the antigen-antibody
complexes.
[0187] FIGS. 11A to 11B show the therapeutic effects of the C26 DY
scavenger antibody and 162 in HBV transgenic mice after injection
with a single dose of 5 mg/kg via tail vein in Example 4. FIG. 11C
shows the therapeutic effects of the D3 DY, D3 DY, D4 DY and D5 DY
in HBV transgenic mice after injection with a single dose of 5
mg/kg via tail vein in Example 4. FIG. 11A: the abscissa represents
the number of days (d) after the injection of antibody, and the
ordinate represents the HBsAg level in mouse serum after clearance
(log 10 IU/ml). FIG. 11B shows: the changes in the concentration of
antibody in mouse serum, in which the abscissa represents the
number of days (d) after the injection of antibody, and the
ordinate represents the antibody concentration (ng/ml). FIG. 11C:
the abscissa represents the number of days (d) after the injection
of antibody, and the ordinate represents the HBsAg level in mouse
serum after clearance (log 10 IU/ml). The results show that the
scavenger antibodies with DY modification C26 DY, D3 DY, D4 DY and
D5 DY are stronger in antigen clearance ability by more than one
order of magnitude than 162. This indicates that the scavenger
antibodies with DY modification C26 DY, D3 DY, D4 DY and D5 DY
could play the function of cyclically binding antigens and enhance
the effect of antigen clearance at alow injection dose of 5
mg/kg.
SEQUENCE INFORMATION
[0188] Information of partial sequences involved in the present
invention is provided in Table 1 below.
TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO
Description Sequence information 1 162 VH
EVQLQESGPGLVKPSQTLSLTCAVSGSSITYGYHWNWIRQFPGNKLE
WIGYISYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS
2 C27 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQS
PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HVPYTFGGGTKLEIK 3
C26 D4 D5 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLE
WIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS
4 C26 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQS
PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HHPYTFGGGTKLEIK 5
C27 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLE
WIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS
6 C32 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLE
WIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS
7 C32 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQS
PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HLPYTFGGGTKLEIK 8
D3 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEW
IGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYC ASGFDHWGQGTTLTVSS
9 D3 D5 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQ
SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THVPYTFGGGTKLEIK
10 D4 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQ
SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THLPYTFGGGTKLEIK
11 162 YGYHWN HCDR1 12 162 D3 D4 D5 YISYDGSVLYNPSLEN HCDR2 13 162
C26 C27 C32 GFDH D3 D4 D5 HCDR3 14 162 C26 C27 C32 RSSQSLVHSYGDTYLH
LCDR1 15 162 C26 C27 C32 KVSNRFS D3 D4 D5 LCDR2 16 162 C27 D3 D5
SQNTHVPYT LCDR3 17 C26 C27 D4 D5 HGYHWN HCDR1 18 C26
YIHYDGSVLYNPSLEN HCDR2 19 C26 SQNTHHPYT LCDR3 20 C27
YINHDGSVQYNPSLEN HCDR2 21 C32 YRYHWN HCDR1 22 C32 YINYDGSVHYNPSLEN
HCDR2 23 C32 D4 SQNTHLPYT LCDR3 24 D3 HGYHYN HCDR1 25 D3 D4 D5
RSSQSLVHSYGDNYLH LCDR1 26 General formula of
X.sub.1X.sub.1YHX.sub.1N HCDR1 27 General formula of
YIX.sub.4X.sub.5DGSVX.sub.6YNPSLEN HCDR2 28 General formula of
RSSQSLVHSYGDX.sub.7YLH LCDR1 29 General formula of SQNTHX.sub.8PYT
LCDR3 30 C26 C27 C32 D3 D4 EVQLQESGPGLVKPSQTLSLTCAVSGSSIT D5 HFR1
31 C26 C27 C32 D3 D4 WIRQFPGNKLEWIG D5 HFR2 32 C26 C27 C32 D3 D4
RVTITRDTSKNQFFLKLSSVTAEDTAKYYCAS D5 HFR3 33 C26 C27 C32 D3 D4
WGQGTTLTVSS D5 HFR4 34 C26 C27 C32 D3 D4 DVVMTQSPLSLPVTLGEPASISC D5
LFR1 35 C26 C27 C32 D3 D4 WYLQKPGQSPKLLIY D5 LFR2 36 C26 C27 C32 D3
D4 GVPDRFSGSGSGTDFTLKISRVETEDLGVYYC D5 LFR3 37 C26 C27 C32 D3 D4
FGGGTKLEIK D5 LFR4 38 4-28-02
QVQLQESGPGLVKPSQTLSLTCAVSGYSISSSNWWGW1RQPPGKGLE
WIGYIYYSGSIYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYY CAR 39 2D-28-01
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQS
PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 40 Human IgG1 heavy
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMTSRTPEVTCV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 Human .kappa.
light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDSAL
constant region QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC 42 Human IgG1 heavy
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region with V4
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCV mutation
VVDVSHEDPEVKFNWYVDGVEVHEAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK 43 Human IgG1 heavy
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constant
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region with DY
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV mutation
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNDAYPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 44
Primer 5'>GTTATTACTCGTGGCCCAGCCGGCCATGGCAGAGGTGCAGCTGC AGGAGTC
<3' 45 Primer 5'>CTCCAGCTTGTTCCCTGGGAACTGCCGGATCCAGTTSYRGTGGT
RGYSGTRGGTGATGGAGCTACCAGA <3' 46 Primer
5'>GTTCCCAGGGAACAAGCTGGAGTGGATTGGGYACMWCMRCYA
CSACGGCAGCSWYCWSYACAATCCATCTCTCG <3' 47 Primer
5'>GACTGTGAGAGTTGTGCCTTGGCCCCAGTGGTSGWRACCACTCG CACAGTA <3'
48 Primer 5'>CCAGATCCGCCACCTCCACTCCCGCCTCCACCTGAGGAGACTGT
GAGAGTTGTGCCTT <3' 49 Primer
5'>GTGGAGGTGGCGGATCTGGAGGGGGTGGTAGCGATGTTGTGAT GACCCAATC <3'
50 Primer 5'>CTTTGGAGACTGGCCTGGCTTCTGCAGGTACCAATGSWGGTRGK
KGTCTCCATAGYKGTGRWS <3' 51 Primer
5'>AGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAAC CGATTTTCTG
<3' 52 Primer 5'>TTTCCAGCTTGGTCCCCCCTCCGAAGKKGTRGKGRWSATGGKKG
TKSTGAGAGCAGTAATAAAC <3' 53 Primer
5'>TAGTCGACCAGGCCCCCGAGGCCTTTTATTTCCAGCTTGGTCCC CCCT <3' 54
Signal peptide MGWSCIILFLVATATGVHS 55 Primer 5'-
AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGA GTC 56 Primer 5'-
GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGACGGTGG 57 Primer 5'-
AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCA GTCTC 58 Primer 5'-
ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC
EXAMPLES
[0189] The present invention will now be described with reference
to the following examples which are intended to illustrate the
present invention rather than limit the present invention.
[0190] Unless otherwise specified, the molecular biology
experimental methods and immunoassay methods used in the present
invention basically refer to J. Sambrook et al., Molecular Cloning:
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory
Press, 1989, and F M Ausubel et al., Compiled Molecular Biology
Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995;
the restriction enzymes were used in accordance with the conditions
recommended by the product manufacturer. Those skilled in the art
know that the examples describe the present invention by way of
example, and are not intended to limit the scope of protection
sought to be protected by the present invention.
Example 1: Phage Screening of pH-Dependent Anti-HBsAg
Antibodies
1.1 Determination of Mutation Sites for pH-Dependent Antibody
Modification
[0191] The anti-HBV humanized antibody 162 (detailed in Chinese
patent application 201610879693.5) developed in the laboratory was
used as the parent antibody, and its variable region were modified
for pH-dependent antigen binding. As shown in FIG. 1, the modified
162 could maintain the antigen-binding activity under neutral
conditions, but its antigen-binding activity under acidic
conditions was greatly reduced. The dissociated modified 162 could
bind to intracellular FcRn so as to return to the plasma and bind
to the antigen again, so that one molecule of the modified 162 with
pH-dependent antigen binding ability could repeatedly bind and
neutralize a plurality of molecules of antigen. Histidine was
protonated under acidic conditions and was a key amino acid to
bring the pH-dependent antigen binding properties. The 162 Fab had
an analyzed crystal structure, the analyzed crystal structure was
docked by simulation with an antigen short peptide, and part of the
results was shown in FIG. 2, in which the blue structure
represented the antigen short peptide, and the red structure
represented part of the binding region of 162 antibody. According
to the docking results, a total of 14 key amino acids for antigen
and antibody binding were found. Considering that the simulated
docking results had greater reference value, the amino acids on the
interface and the amino acids on the both sides were selected for
mutation, and 26 sites were determined.
1.2 Construction of Phage Library of pH-Dependent scFv Antbodies
Derived from 162
[0192] Using the variable regions of the light and heavy chains of
the 162 antibody as a template, the determined sites in the
antibody variable region CDRs were mutated for pH-dependent
modification, and the target fragments were amplified according to
the primers in Table 2 to obtain the gene fragments coding the
pH-dependent scFv antibodies derived from 162. PCR conditions were:
95.degree. C., 5 min; 95.degree. C., 30 s; 57.degree. C., 30 s;
72.degree. C., 30 s; 72.degree. C., 10 min; for 25 amplification
cycles; SOE-PCR reaction conditions were: 95.degree. C., 5 min;
95.degree. C., 30 s; 57.degree. C., 30 s; 72.degree. C., 30 s;
72.degree. C., 10 min; for 5 amplification cycles. The amplified
products were analyzed by agarose gel electrophoresis, and the
amplification products were recovered/purified by using the DNA
purification and recovery kit (TianGen, DP214-03), thereby
obtaining the gene fragments H-K encoding the humanized scFv
antibodies derived from 162. The structure of scFv antibodies was:
NH.sub.2-VH-linker-VL-COOH, and the linker sequence could be
(G.sub.4S).sub.3. Each of the gene fragments H-K was digested with
SfiI, and then ligated to the vector pCGMT (from Scripps, Making
chemistry selectable by linking it to infectivity) at a molar ratio
of 10:1 (gene fragment:vector). The ligation products were
transformed into competent Escherichia coli ER2738 by
electroporation (electroporation conditions: 25 .mu.F, 2.5 KV, 200
.OMEGA.). The transformed Escherichia coli was recovered in SOC
medium for 45 min, and then 200 .mu.L of bacterial solution was
plated on LB plates (comprising 100 g/L ampicillin+tetracycline+2
g/mL glucose), and incubated by standing at 37.degree. C.
overnight. All colonies on the plates were the libraries that the
mutation sites determined in the variable regions were randomly
mutated into histidine, which were used for subsequent screening.
Monoclonal colonies were picked out from the plates and sequenced
to ensure the correctness of the sequences of recombinant vectors
encoding the scFv antibodies. The schematic diagram of the
recombinant vector (pCGMT-scFv) encoding the scFv antibody was
shown in FIG. 3.
TABLE-US-00002 TABLE 2 Mutation primers for pH-dependent scFv
antibodies derived from 162 Primer name Primer sequence VH-F SEQ ID
NO: 44 HCDR1-R SEQ ID NO: 45 HCDR2-F SEQ ID NO: 46 HCDR3-R SEQ ID
NO: 47 VH-R SEQ ID NO: 48 VK-F SEQ ID NO: 49 KCDR1-R SEQ ID NO: 50
KCDR2-F SEQ ID NO: 51 KCDR3-R SEQ ID NO: 52 VK-R SEQ ID NO: 53
1.3 Detection of Humanized scFv Antibodies
[0193] The library obtained in the previous step was screened for
multiple rounds, and the positive monoclonal colonies obtained in
the screening were cultured with 2.times.YT medium containing
ampicillin (100 g/L) and glucose (2 g/mL) to reach an OD value of
0.6, and then added with M13KO7 for auxiliary super-infection.
After 2 h, 100 g/L kanamycin was added and the super-infection was
performed at 37.degree. C. After 2 h, the culture was centrifuged
at 4000 rpm for 10 min, the supernatant was discarded, and the cell
pellet was collected. The cell pellet was resuspended in a medium
containing ampicillin and kanamycin (100 g/L), and cultured with
shaking at 30.degree. C. overnight. Subsequently, the culture was
centrifuged at 12000 rpm for 10 min, the cells and supernatant were
collected, and stored at 4.degree. C. for testing.
[0194] An ELISA plate coated with HBsAg (200 ng/mL) antigen was
used, and 100 .mu.L of the supernatant to be tested was added to
each well, and incubated at 37.degree. C. for 1 h (two wells for
each supernatant). Subsequently, the ELISA plate was washed once
with PBST, and then the two wells of each supernatant were added
with 120 .mu.L of PBS with pH 7.4 and pH 6.0 respectively and
incubated at 37.degree. C. for 30 min. After washing with PBST of
corresponding pH for 5 times, 100 .mu.L , of anti M13-HRP diluted
at 1:5000 was added, and incubated at 37.degree. C. for 30 min.
Subsequently, the ELISA plate was washed 5 times with PBST, and the
substrate TMB solution was added. After 15 minutes of color
development, the color reaction was terminated with
H.sub.2SO.sub.4, and the reading was measured at OD450/630. The
detection results of ELISA of the third round were shown in FIGS.
4A to 4D. The results showed that the phages displaying these scFv
antibodies all had reactivity in ELISA detection and weakly bound
to antigens at pH 6.0; six strains of pH-dependent phage antibodies
with good effects were initially obtained, named C-26, C-27, C-32,
D3, D4 and D5, respectively.
Example 2: Preparation of pH-Dependent Anti-HBsAg Antibodies
2.1 Construction of Recombinant Vector for Eukaryotic
Expression
[0195] In the present invention, a large amount of antibody
recombination needed to be carried out, so it was necessary to
construct a set of light and heavy chain vectors that can
efficiently recombine antibodies. In the present invention, the
existing eukaryotic expression vector pTT5 in the laboratory was
specially modified to construct a set of light and heavy chain
recombinant vectors for double plasmid co-transfection.
MGWSCIILFLVATATGVHS (SEQ ID NO: 54) was used as the signal peptide
for the light and heavy chains. The sequences encoding the constant
regions of the human antibody light and heavy chains were
separately ligated to the downstream of signal peptide to construct
a set of eukaryotic expression vectors pTT5-CH, pTT5-C.kappa. and
pTT5-C.lamda. that facilitated antibody recombination.
[0196] The six scFv antibodies obtained in 1.3 were used to amplify
the light and heavy chain variable region fragments with the
primers in Table 3. The specific amplification reaction conditions
were: 95.degree. C., 5 min; 95.degree. C., 30 s; 57.degree. C., 30
s; 72.degree. C., 30 s; 72.degree. C., 10 min; for 25 amplification
cycles. And the amplification products were recovered from the
gel.
[0197] The laboratory-made Gibson assembly solution was used to
ligate the above constructed eukaryotic expression vector with the
recovered PCR product of antibody variable region gene (the primer
carried a sequence homologous to the vector) to obtain the
recombinant vectors VH+pTT5-CH (comprising the CH shown in SEQ ID
NO: 40) and VH+pTT5-C.kappa. (comprising the CL shown in SEQ ID NO:
41). The recombinant vector was transformed into E. coli DH5.alpha.
strain, plated on LB plate, and cultivated overnight in a
37.degree. C. incubator. Monoclonal colonies were picked out from
the plate and sequenced, and the sequencing results were subjected
to sequence comparison using MEGA to confirm the correctness of its
genes, and exclude the genes with wrong information.
TABLE-US-00003 TABLE 3 Primers for construction of eukaryotic
expression vectors Primer name Primer sequence VH-F SEQ ID NO: 55
VH-R SEQ ID NO: 56 VK-F SEQ ID NO: 57 VK-R SEQ ID NO: 58
2.2 Small- and Large-Scale Expression of Antibody Genes
[0198] The constructed recombinant vectors VH+pTT5-CH and
VH+pTT5-C.kappa. were co-transfected into HEK293 cells, and double
plasmids for small-scale expression were co-transfected into a
24-well plate, 500 .mu.L per well; if the cell supernatant of
small-scale expression had antigenic activity, the transfection
system was enlarged to 100 mL (determined by the amount of antibody
used) of FreeStyle.TM. 293F suspension cells (the cell density was
about 2.times.10.sup.6 cells/ml). The transfected cells were
cultured in a shake flask in a 32.degree. C., 5% CO.sub.2
incubator, and the supernatant was collected after 7 days of
expression.
2.3 Antibody Purification
[0199] The cell expression supernatant was collected and purified
with a Protein A column according to the manufacturer's
instructions. The specific steps were as follows: the harvested
cell culture supernatant was centrifuged at 8000 rpm for 10 min,
the supernatant was retained, the pH value was adjusted to 8.4 with
dry powder Na.sub.2HPO.sub.4, and then filtered with a filter
membrane with 0.22 .mu.m pore diameter. 10 mL of Sepharose 4B
medium coupled with Protein A was loaded into column, it was
connected to AKTA Explorer100 system, the pump A was connected to
0.2 M disodium hydrogen phosphate solution, and the pump B was
connected to 0.2 M citric acid solution. Detection wavelength was
UV 280 nm, flow rate was 5 mL/min, and the sample injection
proportion of pumps A/B was adjusted. The column was first washed
with 100% B (pH 2.3) to remove protein impurities, the pH was
balanced with 10% B (pH 8.0), the signal at the detection
wavelength returned to zero after it was stable, then the sample
was loaded. After the flow through peak passed, 10% B was used for
balance until the signal at the detection wavelength was reduced to
zero and was stable, elution was performed using 70% B (pH 4.0),
and the elution peak was collected. The elution peak sample was
dialyzed into PBS buffer and subjected to assay of concentration
and SDS-PAGE and HPLC analysis to determine the purity of IgG
antibody.
Example 3: Property Analysis and Functional Evaluation of
pH-Dependent Anti-HB sAg Antibodies
[0200] Through the method of Example 1, six strains of pH-dependent
phage antibodies that bound to HBsAg were obtained by preliminary
screening, named C26, C27, C32, D3, D4 and D5, respectively.
Furthermore, the small-scale eukaryotic expression and purification
of the 6 strains of phage antibodies were carried out by the method
of Example 2. The VH and VL amino acid sequences of the 6
antibodies were shown in Table 4 below. In addition, the CDR
sequences of the 6 antibodies were determined, and the CDR amino
acid sequences of the heavy chain variable regions and the light
chain variable regions thereof were shown in Table 5. The mutation
sites that endowed C26, C27, C32, D3, D4 and D5 with pH-dependent
antigen binding properties to HBsAg were summarized in FIG. 5.
TABLE-US-00004 TABLE 4 Amino acid sequences of C26/C27/C32/D3/D4/D5
light and heavy chain variable regions Sequence SEQ name ID NO
Amino acid sequence C26 VH 3
EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL
EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK
YYCASGFDHWGQGTTLTVSS C26 VK 4
DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG
QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHHYTEGGGTKLEIK
C27 VH 5 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL
EWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK
YYCASGFDHWGQGTTLTVSS C27 VK 2
DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG
QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK
C32 VH 6 EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKL
EWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK
YYCASGFDHWGQGTTLTVSS C32 VK 7
DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPG
QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIK
D3 VH 8 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLE
WIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKY YCASGFDHWGQGTTLTVSS
D3 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG
QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK
D4 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL
EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK
YYCASGFDHWGQGTTLTVSS D4 VK 10
DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG
QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIK
D5 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKL
EWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK
YYCASGFDHWGQGTTLTVSS D5 VK 9
DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPG
QSPKLLIYKVSNRESGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC
SQNTHVPYTFGGGTKLEIK
TABLE-US-00005 TABLE 5 CDR sequences of C26/C27/C32/D3/D4/D5 light
and heavy chains C26 VH CDR1 HGYHWN SEQ ID NO: 17 VH CDR2
YIHYDGSVLYNPSLEN SEQ ID NO: 18 VH CDR3 GFDH SEQ ID NO: 13 VL CDR1
RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15 VL
CDR3 SQNTHHPYT SEQ ID NO: 19 C27 VH CDR1 HGYHWN SEQ ID NO: 17 VH
CDR2 YINHDGSVQYNPSLEN SEQ ID NO: 20 VH CDR3 GFDH SEQ ID NO: 13 VL
CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15
VL CDR3 SQNTHVPYT SEQ ID NO: 16 C32 VH CDR1 YRYHWN SEQ ID NO: 21 VH
CDR2 YINYDGSVHYNPSLEN SEQ ID NO: 22 VH CDR3 GFDH SEQ ID NO: 13 VL
CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14 VL CDR2 KVSNRFS SEQ ID NO: 15
VL CDR3 SQNTHLPYT SEQ ID NO: 23 D3 VH CDR1 HGYHYN SEQ ID NO:24 VH
CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL
CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO: 15 VL
CDR3 SQNTHVPYT SEQ ID NO:16 D4 VH CDR1 HGYHWN SEQ ID NO:17 VH CDR2
YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL CDR1
RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3
SQNTHLPYT SEQ ID NO:23 D5 VH CDR1 HGYHWN SEQ ID NO:17 VH CDR2
YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDH SEQ ID NO:13 VL CDR1
RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFS SEQ ID NO:15 VL CDR3
SQNTHVPYT SEQ ID NO:16
[0201] The inventors first performed small-scale transfection for
the six monoclonal antibodies; after quantifying the supernatant
after transfection, the pH-dependent antigen binding ability with
HBsAg was detected by ELISA method, and the antibody concentration
was uniformly diluted to 1111.11 ng/mL. Subsequently, 20% NBS was
used to carry out a 3-fold concentration gradient dilution of the
antibody concentration, for a total of 8 concentration gradients.
Subsequently, the diluted antibody was added to a commercial HBsAg
plate (purchased from Beijing Wantai), and incubated at 37.degree.
C. for 1 h (two wells per supernatant). Subsequently, the ELISA
plate was washed once with PBST and spin-dried. Then, the two wells
of each supernatant were added with 120 .mu.L, of pH 7.4 and pH 6.0
PBS respectively and incubated at 37.degree. C. for 30 min. It was
then washed 5 times with PBST of corresponding pH and spin-dried.
Subsequently, GAH-HRP-labeled secondary antibody was added,
incubated for 30 min, the plate was washed 5 times with PBST, and
spin-dried. The substrate TMB solution was added. After 15 minutes
of color development, the color reaction was terminated with
H.sub.2SO.sub.4, and the reading was measured at OD450/630.
[0202] The results were shown in FIGS. 6A to 6B. It could be seen
from the results that the candidate molecules all could maintain an
antigen-binding activity comparable to that of the parent antibody
162 at the neutral pH, and the antigen-binding activity was
significantly reduced at pH 6.0. The EC50 results were summarized
in Table 6.
TABLE-US-00006 TABLE 6 EC50 values of pH-dependent activity
detection for C26, C27, C32, D3, D4 and D5 EC50 in pH 6.0 EC50 in
pH 7.4 EC50(pH 6.0)/ antibody (ng/mL) (ng/mL) EC50(pH 7.4) C26
331.30 37.20 8.90 C27 949.60 491.30 1.93 C32 2255.00 1333 1.69 D3
473.10 35.43 13.35 D4 188.10 37.16 5.06 D5 60.04 21.04 2.85
Example 4: Construction and Functional Evaluation of Scavenger
Antibody
[0203] The pH-dependent antibody needs to enter the cell to exert
its pH-dependent antigen-binding activity. Therefore, if the first
limiting factor of cell entry is not broken, the subsequent
pH-dependent antigen-binding properties will have no chance to
"play". Therefore, in this example, the scavenger antibody was
obtained by further mutation of amino acids in the Fc region, which
could enhance the binding to hFcRn receptor at neutral pH, or
enhance the binding to Fc.gamma.Rs receptor. As shown in FIG. 7,
the scavenger antibody is located outside the cell and played the
role of a "transportation helper" that reciprocally transported
antigens into the cell, thereby extremely extending the antibody
half-life, and it could bind to antigen again, thereby improving
the efficiency of cell entry of antigen, and significantly
improving the clearance efficiency.
[0204] The C26, D3, D4 and D5 were selected as the antibodies for
subsequent evaluation, and subjected to Fc DY (K326D, L328Y)
mutations to enhance the affinity with mFc.gamma.RII under neutral
conditions (the modification of C26 Fc was commissioned to the
General Biologicals, order number G122413) to obtain scavenger
antibodies C26 DY, D3 DY, D4 DY and D5 DY that bound to
mFc.gamma.RII. The above antibodies were subjected to large-scale
eukaryotic expression and purification, and the specific steps were
same as Examples 1.2 and 1.3.
[0205] FIGS. 8A to 8B showed the protein gel results of the
original antibody and the modified antibodies. FIG. 8A: the picture
of protein gel of the original antibody, in which the 162 was a
positive control. The results showed that the expressed original
antibody is single-component. FIG. 8B: the picture of protein gel
of antibodies with DY modification, in which the 162 was a positive
control. The results showed that the expressed antibodies with DY
modification are single-component.
4.1 Evaluation of pH-Dependent Antigen-Binding Activity of
Scavenger Antibodies Binding to mFc.gamma.RII
[0206] For the original antibody and the antibodies with DY
modification after expression and purification, the inventors used
the ELISA method to detect their pH-dependent antigen binding
ability to HBsAg. First, a BCA protein quantification kit was used
to determine the concentrations of the purified antibodies, and the
antibodies were uniformly diluted to have a concentration of
1111.11 ng/mL. Subsequently, 20% NBS was used to carry out a 3-fold
concentration gradient dilution for the antibody concentrations,
for a total of 8 concentration gradients. Subsequently, the diluted
antibody was added to a commercial HBsAg plate (purchased from
Beijing Wantai) and incubated at 37.degree. C. for 1 h (two wells
per supernatant). Subsequently, the ELISA plate was washed once
with PBST and spin-dried. Then the two wells of each supernatant
were added with 120 .mu.L of pH 7.4 PBS and pH 6.0 PBS respectively
incubated at 37.degree. C. for 30 min, washed 5 times with PBST of
corresponding pH and spin-dried. Subsequently, GAH-HRP-labeled
secondary antibody was added, and incubated for 30 min, the plate
was washed 5 times with PBST, and spin-dried. And the substrate TMB
solution was added. After 15 minutes of color development, the
color reaction was terminated with H.sub.2SO.sub.4, and the reading
was measured at OD450/630.
[0207] The results were shown in FIGS. 9A to 9D, in which the C26,
D3, D4, D5 and their DY modification antibodies all had an
antigen-binding activity equivalent to that of antibody 162, but
showed a weak binding to antigen at pH 6.0, thereby exhibiting a
good pH-dependent antigen-binding activity.
4.2 Verification of Function at Cellular Level for Scavenger
Antibodies Binding mFc.gamma.RII
4.2.1 Labeling HBsAg with 488 Fluorescence
[0208] Take the labeling of 1 mg HBsAg as an example, the whole
process was protected from light.
[0209] (1) 1 mL of 1 mg/mL HBsAg was dialyzed into borate buffer
(PH 8.5, 500 mL), 4.degree. C., 4 h;
[0210] (2) the molar ratio of HBsAg to 488 label was 1:5, and
0.1988 mg of 488 fluorescence was required after calculation;
[0211] (3) 10 mg/mL of 488 fluorescence solution was prepared with
DMF and mixed well;
[0212] (4) 19.88 pL of 488 fluorescence was added to 1 mL of the
dialyzed HBsAg, mixed well, and incubated at room temperature for 1
h;
[0213] (5) the incubation mixture was dialyzed into PBS at
4.degree. C. overnight.
4.2.2 Immunofluorescence Experiment Based on Mouse Primary
Macrophages
[0214] (1) 4 days before the experiment, 1.5 mL of 3% sodium
thioglycolate solution was injected into the abdominal cavity of
each mouse, without injecting into the intestine;
[0215] (2) two mice were executed and soaked in 75% alcohol for 3
minutes;
[0216] (3) the mouse was horizontally fixed on a foam board to
expose the abdomen; the abdominal skin was cut with tissue
scissors, the peritoneum was disinfected and incised to expose the
abdominal cavity, the abdominal incision skin was pulled by two
toothed forceps hold in the left hand and fixed, 1640 culture
medium was pipetted by Pasteur pipette hold in the right hand for
peritoneal lavage with 4 mL/time, for a total of two times. The
pipette was used to gently and fully stir the abdominal cavity to
make the lavage more fully and thoroughly. After fully stirring for
about 2 minutes and standing for about 5 minutes to fully isolate
the macrophages, the lavage solution was pipetted and transferred
into a centrifuge tube;
[0217] (4) 4.degree. C., 1100 g, 5 min;
[0218] (5) the supernatant was carefully discard, the cells were
washed twice with 1640 medium, 4.degree. C., 1100 g, 5 min, the
supernatant was discarded, and the cells were resuspended in
RPM1640;
[0219] (6) After counting the cells, the cell density was adjusted
to 10.sup.6 cells/mL, the cells were cultured on a 24-well
glass-bottom cell imaging culture plate, 250 .mu.L/well, the medium
was replaced after 2 h, and washing was carried out once with
RPM1640, after the non-adherent cells were discarded, the cells
were incubated overnight in a 37.degree. C., CO.sub.2
incubator;
[0220] (7) the antibody and antigen labeled with the corresponding
fluorescence were diluted in serum-free medium to: 800 ng/mL for
antigen and 20 .mu.g/mL for antibody;
[0221] (8) 125 .mu.L of the antigen and 125 .mu.L of the antibody
were mixed uniformly, and then were allowed to stand for 1 hour in
a 37.degree. C., CO.sub.2 incubator;
[0222] (9) the cell supernatant in the cell imaging culture plate
was discarded, the antigen-antibody complex was added, shaken
evenly, and allowed to stand in a 37.degree. C., CO.sub.2 incubator
for 2 hours;
[0223] (10) the supernatant was discarded, and 1 mL of sterile PBS
incubated at 37.degree. C. in advance was used to "wash" the cell
surface 3 to 5 times, and then totally removed by a pipette;
[0224] (11) the 1:2000 diluted Dio was added in an amount that
immersed the cells, and allowed to stand at room temperature for 20
min;
[0225] (12) the supernatant was discarded, and 1 mL of sterile PBS
incubated at 37.degree. C. in advance was used to "wash" the cell
surface 3 to 5 times, and then totally removed by a pipette;
[0226] (13) a live cell nuclear dye was added (2 drops were added
to 1 mL of volume), allowed to stand at room temperature for 20
min, and placed in a high-content imager for imaging.
[0227] The results of the experiment were shown in FIG. 10A. It
could be seen from the results that the DY modification enhanced
the phagocytosis of mouse macrophages to the antigen-antibody
complexes, leading to more antigen degradation.
4.2.3 Validation by Chemiluminescence Method Based on human THP-1
Phagocytic Cells
[0228] (1) Adherent THP-1 cells were coated on a plate at
2.times.10.sup.5/well, added with 1640 medium containing 10% serum,
placed in a carbon dioxide incubator and cultured at 37.degree. C.
for 24 h;
[0229] (2) HBsAg was diluted with serum-free 1640 medium to 800
ng/mL, and the antibody to be tested with 20 ug/mL as the initial
concentration was subjected to 2-fold gradient dilution, for a
total of 11 gradients. 300 uL of the diluted HBsAg and 300 uL of
the antibody to be tested were mixed at ratio of 1:1, and allowed
to stand at 37.degree. C. for 1 h;
[0230] (3) the THP-1 cell supernatant was discarded, 250 uL of the
HBsAg-antibody mixture was added to the THP-1 cells, placed in a
carbon dioxide incubator and cultured at 37.degree. C. for 1
hour;
[0231] (4) the THP-1 cell supernatant was discarded, and washed 3
times with sterile PBS;
[0232] (5) 120 uL of DDM cell lysis solution was added to each well
of THP-1 cells and allowed to stand and react at 4.degree. C. for 1
hour;
[0233] (6) the supernatant of the lysate was subjected to detecting
the concentration of HBsAg by using hepatitis B surface antigen
quantitative detection kit (Beijing Wantai).
[0234] The results of the experiment were shown in FIG. 10B. It
could be seen from the results that the DY modification enhanced
the phagocytosis of human THP-1 phagocytic cells to the
antigen-antibody complexes.
4.3 Determination of Therapeutic Effect of Scavenger Antibody
Binding to mFc.gamma.RI in Animal Models
[0235] HBV transgenic mice were selected as animal models. The C26
DY, D3 DY, D4 DY and D5 DY scavenger antibodies and 162 were
injected at a single dose of 5 mg/kg via tail vein (4 mice in each
group) to the 6-8 weeks old HBV transgenic mice. By detecting the
concentrations of HBsAg, antibody and HBV DNA in serum, the antigen
clearance rates and antibody half-life of the scavenger antibodies
in vivo were analyzed.
Quantitative Detection of HBsAg
[0236] (1) Preparation of reaction plate: the mouse monoclonal
antibody HBs-45E9 was diluted with 20 mM PB buffer
(Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer, pH 7.4) to 2 .mu.g/mL,
and 100 .mu.L of coating solution was added to each well of a
chemiluminescence plate, and the coating was carried out at
2-8.degree. C. for 16-24 h, followed by another 2 hours at
37.degree. C., the plate was washed once with PBST washing
solution, and spin-dried. After washing, 200 .mu.L of blocking
solution was added to each well and the blocking was carried out at
37.degree. C. for 2 h. Subsequently, the blocking solution was
discarded, and the plate was placed in a drying room to dry, and
stored at 2-8.degree. C. for later use.
[0237] (2) Sample dilution: the collected mouse serum was diluted
with a PBS solution containing 20% NB S (newborn bovine serum) at
two gradients of 1:30 and 1:150 for subsequent quantitative
detection.
[0238] (3) Sample denaturation treatment: 15 .mu.L of the
above-diluted serum sample was mixed well with 7.5 .mu.L of
denaturation buffer (15% SDS, dissolved in 20 mM PB7.4), and
reacted at 37.degree. C. for 1 h. Then, 90 .mu.L of stop buffer (4%
CHAPS, dissolved in 20 mM PB7.4) was added, and mixed well.
[0239] (4) Sample reaction: 100 .mu.L of the above-mentioned
denatured serum sample was added to a reaction plate, and reacted
at 37.degree. C. for 1 hour. Subsequently, the reaction plate was
washed 5 times with PBST and spin-dried.
[0240] (5) Enzymatic label reaction: the HBs-A6A7-HRP reaction
solution was added at 100 .mu.L/well to a chemiluminescence plate,
and reacted at 37.degree. C. for 1 h. Then, the plate was washed 5
times with PBST and spin-dried.
[0241] (6) Luminescence reaction and measurement: a luminescence
solution (100 .mu.L/well) was added to the chemiluminescence plate,
and light intensity measurement was performed.
[0242] (7) Calculation of HBsAg concentration in mouse serum
sample: parallel experiments were performed using standard
products, and a standard curve was drawn based on the measurement
results of the standard products. Then, the light intensity
measurement value of the mouse serum sample was substituted into
the standard curve, and the concentration of HBsAg in the serum
sample to be tested was calculated.
[0243] The results of the detection of HBsAg in the serum were
shown in FIG. 11A and FIG. 11C. It could be seen from FIG. 11A that
the scavenger antibody with DY modification C26 DY had an antibody
clearance ability stronger more than one order of magnitude than
that of 162, which was consistent with the detection results of
antibody half-life in the serum (FIG. 11B). In the comparison of
the concentrations of antibodies in the serum, the half-life of C26
DY was longer than that of 162 by nearly 12 days, which indicated
that the scavenger antibody C26 DY had the function of circularly
and reciprocally binding antigen, thereby increasing the duration
time of antigen clearance. The experimental results in FIG. 11C
showed that the antigen clearance ability of D3 DY, D4 DY and D5 DY
was equivalent to that of C26 DY, and the duration time was longer
than that of C26 DY, indicating that at a low injection dose of 5
mg/kg, the scavenger antibodies with DY modification D3 DY, D4 DY
and D5 DY had a better function of circularly and reciprocally
binding antigen, thereby performing better antigen clearance.
Example 5: Affinity Determination of 162 and C26
[0244] HBsAg was dissolved in sodium acetate (pH 4.5) at 5
.mu.g/mL, and the chip coating program was run on the Biacore 3000
device to coat HBsAg on the CM5 chip. The coating volume of HBsAg
was 2400 RU. The analyte was diluted 2-fold from 100 nM to prepare
samples of 7 concentrations. The affinity determination program was
run on the Biacore 3000 device, the flow rate was set to 50
.mu.l/min, the binding time was set to 90 s, the dissociation time
was set to 600 s, the temperature of sample chamber was set to
10.degree. C., the regeneration solution was 50 mM NaOH, the
regeneration flow rate was set to 50 .mu.L/min, and the
regeneration time was set to 60 s. The results were summarized in
Table 7.
TABLE-US-00007 TABLE 7 Affinity determination of 162 and C26 KD(M)
in KD(M) in KD(pH 6.0)/ Antibody pH 7.4 pH 6.0 KD(pH 7.4) 162
9.34E-10 C26 3.45E-09 9.82E-09 2.85
[0245] Although the specific embodiments of the present invention
have been described in details, those skilled in the art will
understand that various modifications and changes can be made to
the details according to all the teachings that have been
published, and these changes are within the protection scope of the
present invention. All of the present invention is given by the
appended claims and any equivalents thereof.
Sequence CWU 1
1
581113PRTArtificial sequence162 VH 1Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly Ser Ser Ile Thr Tyr Gly 20 25 30Tyr His Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Ile Gly Tyr Ile Ser
Tyr Asp Gly Ser Val Leu Tyr Asn Pro Ser Leu 50 55 60Glu Asn Arg Val
Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Lys
Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Lys Tyr Tyr Cys 85 90 95Ala
Ser Gly Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105
110Ser2112PRTArtificial sequenceC27 VK 2Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Tyr Gly Asp Thr Tyr
Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Ser Gln Asn 85 90
95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 1103113PRTArtificial sequenceC26 D4 D5 VH 3Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Thr His Gly 20 25 30Tyr His
Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Ile
Gly Tyr Ile His Tyr Asp Gly Ser Val Leu Tyr Asn Pro Ser Leu 50 55
60Glu Asn Arg Val Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65
70 75 80Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Lys Tyr Tyr
Cys 85 90 95Ala Ser Gly Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser 100 105 110Ser4112PRTArtificial sequenceC26 VK 4Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Tyr
Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys
Ser Gln Asn 85 90 95Thr His His Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 1105113PRTArtificial sequenceC27 VH 5Glu
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Thr His Gly
20 25 30Tyr His Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu
Trp 35 40 45Ile Gly Tyr Ile Asn His Asp Gly Ser Val Leu Tyr Asn Pro
Ser Leu 50 55 60Glu Asn Arg Val Thr Ile Thr Arg Asp Thr Ser Lys Asn
Gln Phe Phe65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr
Ala Lys Tyr Tyr Cys 85 90 95Ala Ser Gly Phe Asp His Trp Gly Gln Gly
Thr Thr Leu Thr Val Ser 100 105 110Ser6113PRTArtificial sequenceC32
VH 6Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Thr
Tyr Arg 20 25 30Tyr His Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45Ile Gly Tyr Ile Asn Tyr Asp Gly Ser Val His Tyr
Asn Pro Ser Leu 50 55 60Glu Asn Arg Val Thr Ile Thr Arg Asp Thr Ser
Lys Asn Gln Phe Phe65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Glu
Asp Thr Ala Lys Tyr Tyr Cys 85 90 95Ala Ser Gly Phe Asp His Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser 100 105 110Ser7112PRTArtificial
sequenceC32 VK 7Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30Tyr Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln
Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Thr Glu Asp
Leu Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Leu Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
1108113PRTArtificial sequenceD3 VH 8Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly Ser Ser Ile Thr His Gly 20 25 30Tyr His Tyr Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Ile Gly Tyr Ile His
Tyr Asp Gly Ser Val Leu Tyr Asn Pro Ser Leu 50 55 60Glu Asn Arg Val
Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Lys
Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Lys Tyr Tyr Cys 85 90 95Ala
Ser Gly Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105
110Ser9112PRTArtificial sequenceD3 D5 VK 9Asp Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Tyr Gly Asp Asn
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 11010112PRTArtificial sequenceD4 VK 10Asp Val Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Tyr Gly
Asp Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Ser Gln
Asn 85 90 95Thr His Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110116PRTArtificial sequence162 HCDR1 11Tyr Gly Tyr
His Trp Asn1 51216PRTArtificial sequence162 D3 D4 D5 HCDR2 12Tyr
Ile Ser Tyr Asp Gly Ser Val Leu Tyr Asn Pro Ser Leu Glu Asn1 5 10
15134PRTArtificial sequence162 C26 C27 C32 D3 D4 D5 HCDR3 13Gly Phe
Asp His11416PRTArtificial sequence162 C26 C27 C32 LCDR1 14Arg Ser
Ser Gln Ser Leu Val His Ser Tyr Gly Asp Thr Tyr Leu His1 5 10
15157PRTArtificial sequence162 C26 C27 C32 D3 D4 D5 LCDR2 15Lys Val
Ser Asn Arg Phe Ser1 5169PRTArtificial sequence162 C27 D3 D5 LCDR3
16Ser Gln Asn Thr His Val Pro Tyr Thr1 5176PRTArtificial
sequenceC26 C27 D4 D5 HCDR1 17His Gly Tyr His Trp Asn1
51816PRTArtificial sequenceC26 HCDR2 18Tyr Ile His Tyr Asp Gly Ser
Val Leu Tyr Asn Pro Ser Leu Glu Asn1 5 10 15199PRTArtificial
sequenceC26 LCDR3 19Ser Gln Asn Thr His His Pro Tyr Thr1
52016PRTArtificial sequenceC27 HCDR2 20Tyr Ile Asn His Asp Gly Ser
Val Gln Tyr Asn Pro Ser Leu Glu Asn1 5 10 15216PRTArtificial
sequenceC32 HCDR1 21Tyr Arg Tyr His Trp Asn1 52216PRTArtificial
sequenceC32 HCDR2 22Tyr Ile Asn Tyr Asp Gly Ser Val His Tyr Asn Pro
Ser Leu Glu Asn1 5 10 15239PRTArtificial sequenceC32 D4 LCDR3 23Ser
Gln Asn Thr His Leu Pro Tyr Thr1 5246PRTArtificial sequenceD3 HCDR1
24His Gly Tyr His Tyr Asn1 52516PRTArtificial sequenceD3 D4 D5
LCDR1 25Arg Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asp Asn Tyr Leu
His1 5 10 15266PRTArtificial sequenceHCDR1 general
formulaMISC_FEATURE(1)..(1)X = Y or HMISC_FEATURE(2)..(2)X = G or
RMISC_FEATURE(5)..(5)X = W or Y 26Xaa Xaa Tyr His Xaa Asn1
52716PRTArtificial sequenceHCDR2 general
formulaMISC_FEATURE(3)..(3)X = S, N or HMISC_FEATURE(4)..(4)X = Y
or HMISC_FEATURE(9)..(9)X = L, H or Q 27Tyr Ile Xaa Xaa Asp Gly Ser
Val Xaa Tyr Asn Pro Ser Leu Glu Asn1 5 10 152816PRTArtificial
sequenceLCDR1 general formulaMISC_FEATURE(13)..(13)X = T or N 28Arg
Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asp Xaa Tyr Leu His1 5 10
15299PRTArtificial sequenceLCDR3 general
formulaMISC_FEATURE(6)..(6)X = V, L or H 29Ser Gln Asn Thr His Xaa
Pro Tyr Thr1 53030PRTArtificial sequenceC26 C27 C32 D3 D4 D5 HFR1
30Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Thr 20
25 303114PRTArtificial sequenceC26 C27 C32 D3 D4 D5 HFR2 31Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Ile Gly1 5
103232PRTArtificial sequenceC26 C27 C32 D3 D4 D5 HFR3 32Arg Val Thr
Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys1 5 10 15Leu Ser
Ser Val Thr Ala Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ser 20 25
303311PRTArtificial sequenceC26 C27 C32 D3 D4 D5 HFR4 33Trp Gly Gln
Gly Thr Thr Leu Thr Val Ser Ser1 5 103423PRTArtificial sequenceC26
C27 C32 D3 D4 D5 LFR1 34Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys
203515PRTArtificial sequenceC26 C27 C32 D3 D4 D5 LFR2 35Trp Tyr Leu
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr1 5 10
153632PRTArtificial sequenceC26 C27 C32 D3 D4 D5 LFR3 36Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Lys
Ile Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys 20 25
303710PRTArtificial sequenceC26 C27 C32 D3 D4 D5 LFR4 37Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys1 5 103898PRTArtificial sequence4-28-02
38Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser
Ser 20 25 30Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp 35 40 45Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ile Tyr Tyr Asn
Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Val Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg3993PRTArtificial
sequence2D-28-01 39Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys 85 9040330PRTArtificial sequenceHuman
IgG1 heavy chain constant region 40Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230
235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 33041107PRTArtificial sequenceHuman
kappa light chain constant region 41Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Ser Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10542330PRTArtificial
sequenceHuman IgG1 heavy chain constant region with V4 mutation
42Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Glu Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Tyr His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 33043330PRTArtificial sequenceHuman IgG1
heavy chain constant region with DY mutation 43Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Asp Ala Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 3304451DNAArtificial
sequencePrimer 44gttattactc gtggcccagc cggccatggc agaggtgcag
ctgcaggagt c 514569DNAArtificial sequencePrimer 45ctccagcttg
ttccctggga actgccggat ccagttsyrg tggtrgysgt rggtgatgga 60gctaccaga
694674DNAArtificial sequencePrimer 46gttcccaggg aacaagctgg
agtggattgg gyacmwcmrc yacsacggca gcswycwsya 60caatccatct ctcg
744751DNAArtificial sequencePrimer 47gactgtgaga gttgtgcctt
ggccccagtg gtsgwracca ctcgcacagt a 514858DNAArtificial
sequencePrimer 48ccagatccgc cacctccact cccgcctcca cctgaggaga
ctgtgagagt tgtgcctt 584952DNAArtificial sequencePrimer 49gtggaggtgg
cggatctgga gggggtggta gcgatgttgt gatgacccaa tc 525063DNAArtificial
sequencePrimer 50ctttggagac tggcctggct tctgcaggta ccaatgswgg
trgkkgtctc catagykgtg 60rws 635154DNAArtificial sequencePrimer
51agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt tctg
545264DNAArtificial sequencePrimer 52tttccagctt ggtcccccct
ccgaagkkgt rgkgrwsatg gkkgtkstga gagcagtaat 60aaac
645348DNAArtificial sequencePrimer 53tagtcgacca ggcccccgag
gccttttatt tccagcttgg tcccccct 485419PRTArtificial sequenceSignal
peptide 54Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser5548DNAArtificial sequencePrimer
55agtagcaact gcaaccggtg tacattctca ggtgcagctg caggagtc
485640DNAArtificial sequencePrimer 56gatgggccct tggtcgacgc
tgaagagacg gtgacggtgg 405750DNAArtificial sequencePrimer
57agtagcaact gcaaccggtg tacattctga catacagatg acgcagtctc
505839DNAArtificial sequencePrimer 58atggtgcagc caccgtacgt
ttgatttcca ccttggtcc 39
* * * * *
References