U.S. patent number RE40,831 [Application Number 10/423,036] was granted by the patent office on 2009-07-07 for human monoclonal antibodies to the hepatitis b surface antigen.
This patent grant is currently assigned to Yeda Research and Development Co., Ltd.. Invention is credited to Shlomo Dagan, Yair Reisner.
United States Patent |
RE40,831 |
Reisner , et al. |
July 7, 2009 |
Human monoclonal antibodies to the hepatitis B surface antigen
Abstract
Disclosed is a process for obtaining hybridoma cell lines which
produce human antibodies capable of binding to the hepatitis B
virus surface antigen (HBVsAg), as well as the hybridoma cell
lines, and antibodies produced by the cell lines. Also disclosed
are various uses of said antibodies in the prevention and treatment
of HBV infection. Peripheral blood lymphocytes obtained from human
donors having a high titer of anti HBVsAg antibodies are engrafted
into normal strains of mice which were lethally irradiated and
radioprotected with SCID bone marrow. After immunization of such
chimeric mice with HBVsAg, human cells are obtained from the mice
spleens and fused in vitro with heteromyeloma cells to generate
hybridomas secreting human antibodies having a high affinity and
specificity to HBVsAg.
Inventors: |
Reisner; Yair (Old Jaffa,
IL), Dagan; Shlomo (Rehovot, IL) |
Assignee: |
Yeda Research and Development Co.,
Ltd. (Rehovot, IL)
|
Family
ID: |
11068955 |
Appl.
No.: |
10/423,036 |
Filed: |
June 10, 1997 |
PCT
Filed: |
June 10, 1997 |
PCT No.: |
PCT/IL97/00184 |
371(c)(1),(2),(4) Date: |
December 10, 1998 |
PCT
Pub. No.: |
WO97/47654 |
PCT
Pub. Date: |
December 18, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
09202181 |
Dec 10, 1998 |
06254867 |
Jul 3, 2001 |
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Foreign Application Priority Data
Current U.S.
Class: |
424/149.1;
424/133.1; 424/142.1; 435/339; 435/5; 530/388.1; 530/388.15;
530/388.3 |
Current CPC
Class: |
A01K
67/0271 (20130101); C07K 16/082 (20130101); A61K
38/00 (20130101); A61K 39/00 (20130101) |
Current International
Class: |
C07K
16/08 (20060101) |
Field of
Search: |
;434/133.1,142.1,149.1
;530/388.15,388.3,388.1 ;435/5,339 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0179483 |
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Apr 1986 |
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EP |
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0438053 |
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Jul 1991 |
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EP |
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WO9411495 |
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May 1994 |
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WO |
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WO9426784 |
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Nov 1994 |
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WO |
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Other References
Greenspan et al. (Nature Biotechnology 17: 936-937, 1999). cited by
examiner .
Bowie et al (Science, 1990, 257:1306-1310). cited by examiner .
Webster II, 1984, Houghton Mifflin Company, MA, p. 612. cited by
examiner .
Ichimori et al., Establishment of hybridomas secreting human
monoclonal antibody against tetanus toxin and hepatitis B virus
surface antigen, Biochemical and Biophysical Research
Communications, 129:26-33 (1985). cited by other .
Ichimori et al., Establishment of hybridomas secreting human
monoclonal antibody against tetanus toxin and hepatitis B virus
surface antigen, Biochemical and Biophysical Research
Communications, 142:805-812 (1987). cited by other .
Marcus et al., Human/Mouse radiation chimera are capable of
mounting a human primary humoral response, Blood, 86:398-406
(1995). cited by other .
Lubin et al., Engraftment of human peripheral blood lymphocytes in
normal strains of mice, Blood, 83:2368-2381 (1994). cited by other
.
Ehrlich et al., Characterization of human monoclonal antibodies
directed against hepatitis B surface antigen, Hum. Antibod.
Hybridoma, 3:2-7 (1992). cited by other .
Eren et al., Production of specific human monoclonal antibodies to
Hepatitis B virus by human lymphocytes engrafted in normal strains
of mice, Journal of Hepatology, 25:80 (1996). cited by other .
Denes et al., Natural antibodies do not inhibit xenogeneic
transplantation of human PBL in lethally irradiated mice,
Xenotransplantation, 2:8-18 (1995). cited by other .
XP0020442g4, Continuous cultures of fused cells secreting antibody
of predefined specificity, Nature, 256:495-497 (1975). cited by
other.
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Primary Examiner: Zeman; Robert A
Attorney, Agent or Firm: Browdy and Neimark, P.L.L.C.
Claims
What is claimed is:
1. A human monoclonal antibody being selected from the group
consisting of: (a) the monoclonal antibody .[.18.5.103.].
.Iadd.18.5.1013 .Iaddend.which is secreted by the hybridoma cell
line deposited in the European Collection of Cell Cultures (ECACC)
under Accession No. 96052170; and (b) fragments of the antibody of
(a) which retain the antigen binding .[.characteristics.].
.Iadd.specificity .Iaddend.of the .[.whole.]. antibody .Iadd.of
(a).Iaddend..
2. A pharmaceutical composition for reducing the occurrence of HBV
infections in a population of individuals by passive immunotherapy
and/or for treating HBV infections comprising as active ingredient
an antibody in accordance with claim 1 together with a
pharmaceutically acceptable carrier.
3. A method for the treatment of HBV infections comprising
administering to an individual in need a therapeutically effective
amount of a pharmaceutical composition according to claim 2 to
treat HBV infection.
4. A method for reducing the occurrence of HBV infections in a
population of individuals by passive immunotherapy, comprising
administering to a population of individuals a pharmaceutical
composition according to claim 2, to reduce the occurrence of HBV
infections in the population.
5. A method for the treatment of HBV infections comprising
administering to an individual in need a therapeutically effective
amount of the antibody of claim 1 to treat HBV infection.
6. A method for reducing the occurrence of HBV infections in a
population of individuals by passive immunotherapy, comprising
administering to a population of individuals an antibody of claim
1, to reduce the occurrence of HBV infections in the
population.
7. A pharmaceutical composition for reducing the occurrence of HBV
infections in a population of individuals by passive immunotherapy,
and/or for treating HBV infections comprising as an active
ingredient at least one antibody in accordance with claim 1 in
combination with at least one other active ingredient being an anti
viral agent.
8. A pharmaceutical composition according to claim 7 wherein the
anti viral agent is selected from the group consisting of:
interferons, anti HB polyclonal antibodies, nucleoside analogues
and inhibitors of DNA polymerase.
9. A method for the diagnosis of HBV infections in a body fluid
sample comprising: (a) contacting said sample with an antibody of
claim 1 under conditions enabling the formation of antibody-antigen
complexes; .Iadd.and .Iaddend. (b) determining the level of
antibody-antigen complexes formed, wherein a determination of the
presence of a level of antibody-antigen complexes significantly
higher than that formed in a control sample indicates an HBV
infection in the tested body fluid sample.
10. A human monoclonal antibody being selected from the group
consisting of: (a) the monoclonal antibody 19.79.5 which is
secreted by the hybridoma cell line deposited in the European
Collection of Cell Cultures (ECACC) under Accession No. 96052168;
and (b) fragments of the antibody of (a) which retain the antigen
binding .[.characteristics.]. of the .[.whole.]. antibody .Iadd.of
(a).Iaddend..
11. The hybridoma cell line deposited at the ECACC on May 22, 1996
under Accession No. 96052170.
12. The hybridoma cell line deposited at the ECACC on May 22, 1996
under Accession No. 96052168.
.Iadd.13. A pharmaceutical composition for reducing the occurrence
of HBV infections in a population of individuals by passive
immunotherapy and/or for treating HBV infections comprising as
active ingredient an antibody in accordance with claim 10 together
with a pharmaceutically acceptable carrier..Iaddend.
.Iadd.14. A method for the treatment of HBV infections comprising
administering to an individual in need a therapeutically effective
amount of a pharmaceutical composition according to claim 13 to
treat HBV infection..Iaddend.
.Iadd.15. A method for reducing the occurrence of HBV infections in
a population of individuals by passive immunotherapy, comprising
administering to a population of individuals a pharmaceutical
composition according to claim 13, to reduce the occurrence of HBV
infections in the population..Iaddend.
.Iadd.16. A method for the treatment of HBV infections comprising
administering to an individual in need a therapeutically effective
amount of the antibody of claim 10 to treat HBV
infection..Iaddend.
.Iadd.17. A method for reducing the occurrence of HBV infections in
a population of individuals by passive immunotherapy, comprising
administering to a population of individuals an antibody of claim
10, to reduce the occurrence of HBV infections in the
population..Iaddend.
.Iadd.18. A pharmaceutical composition for reducing the occurrence
of HBV infections in a population of individuals by passive
immunotherapy, and/or for treating HBV infections comprising as an
active ingredient at least one antibody in accordance with claim 10
in combination with at least one other active ingredient being an
anti viral agent..Iaddend.
.Iadd.19. A pharmaceutical composition according to claim 18
wherein the anti viral agent is selected from the group consisting
of interferons, anti HB polyclonal antibodies, nucleoside analogues
and inhibitors of DNA polymerase..Iaddend.
.Iadd.20. A method for the diagnosis of HBV infections in a body
fluid sample comprising: (a) contacting said sample with an
antibody of claim 10 under conditions enabling the formation of
antibody-antigen complexes; and (b) determining the level of
antibody-antigen complexes formed, wherein a determination of the
presence of a level of antibody-antigen complexes significantly
higher than that formed in a control sample indicates an HBV
infection in the tested body fluid sample..Iaddend.
Description
FIELD OF THE INVENTION
The present invention concerns a process for obtaining hybridoma
cell lines which produce human antibodies capable of binding to the
hepatitis B virus surface antigen, the hybridoma cell lines,
antibodies produced by the cell lines, and various uses
thereof.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infection is a major worldwide health
problem. Approximately 5% of the world population is infected by
HBV and chronically infected patients carry a high risk of
developing cirrhosis and hepatocellular carcinoma. (Progress in
Hepatitis Research: Hepatitis B virus (HBV), Hepatitis C virus
(HCV) and Hepatitis Delta virus (HDV) Ed. O. Crivelli, Sorin
Biomedica, 1991).
The immune response to HBV-encoded antigens includes both a
cellular immune response which is active in the elimination of HBV
infected cells, as well as a humoral antibody response to viral
envelope antigens which contributes to the clearance of circulating
virus particles. The dominant cause of viral persistence during HBV
infection is the development of a weak antiviral immune
response.
Recombinant HBV vaccines provide a safe and effective means for
active immunization against HBV, however, they do not always induce
a sufficient and rapid antibody response.
Interferon-.alpha. has been used in the therapy of Hepatitis B
infection shown an efficacy of only 30-40% in highly selected
patients.
In addition, passive immunization with human polyclonal anti
Hepatitis B antisera has been shown to be effective in delaying and
even preventing recurrent HBV infection (Wright, T. L. and Lau, J.
Y. N. The Lancet 342:1340-1344, (1993)). Such human polyclonal
antisera are prepared from pooled plasma of immunized donors. These
preparations are very expensive and available in relatively small
amounts. Furthermore, pooled plasma may contain contaminated blood
samples and thus treatment with such antisera increases the
patient's risk to contract other viral infections such as hepatitis
C or HIV.
An alternative approach for the treatment of HBV infection is the
use of monoclonal antibodies (MoAb).
PCT patent application PCT/NL94/00102 discloses human monoclonal
antibodies directed against Hepatitis B surface antigen HBVsAg
which are secreted by the hybridoma cell lines Mab 4-7B and Mab
9H9. The monoclonal antibody secreted by the cell line Mab 4-7B
recognizes a linear epitope of HBVsAg and is different from the Mab
9H9 monoclonal antibody which recognizes a conformational epitope.
The antibodies are claimed for simultaneous use in the treatment of
chronic Hepatitis B infections.
PCT patent application PCT/US92/09749 discloses human monoclonal
antibodies against HBVsAg which are secreted by the hybridoma cell
lines PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3. The antibodies bind to
different HBV epitopes and are used for reducing the level of
circulating HBVsAg.
Japanese Patent Application JP 93066104 discloses a hybridoma of a
human lymphocyte cell strain TAW-925 and a human lymphocyte
transformed by Epstein-Barr virus. The hybridoma produces a human
monoclonal antibody against HBVsAg.
U.S. patent application Ser. No. 4,883,752 discloses preparation of
human-derived monoclonal antibody to HBVsAg, by administration of
HBVsAg vaccine to humans, recovering their lymphocytes, stimulating
the lymphocytes in vitro by a non specific stimulator, fusing said
cells with a myeloma cell, and selecting for hybridomas with
secrete anti HBVsAg antibodies.
Ichimori et al, Biochem. and Biophysic. Research Communications
129(1):26-33, 1985 discloses a hybridoma secreting human anti
HBVsAg monoclonal antibodies which recognize the a-determinant of
HBVsAg. Later, Ichimori, et al., supra 142(3):805-812, 1987
disclosed another hybridoma which stably secretes human monoclonal
antibody against HbsAg.
The abovementioned antibodies were all developer by in vitro
immortalization of antibody-producing cells from individuals
positive for anti-HBV antibodies.
A new approach enabling adaptive transfer of human peripheral blood
mononuclear cells (PBMC) into lethally irradiated normal strains of
mice radioprotected with severe combined immune deficiency (SCID)
bone marrow was recently described (Lubin I., et al., Blood,
83:2368, 1994). Secondary humoral responses to various recall
antigens as well as a primary humoral response to other antigens
were shown to be generated effectively in such human/mouse chimeras
(Marcus H., et al, Blood 86:398-406, 1995).
SUMMARY OF THE INVENTION
In accordance with the present invention, it was found that
hybridoma cell lines secreting human antibodies capable of binding
to the Hepatitis B surface antigen (HBVsAg) may be obtained using
the above mentioned human/mouse chimeras. In accordance with the
present invention, human peripheral blood lymphocytes (PBL) from
human donors positive for anti HBVsAg antibodies are engrafted into
normal strains of mice which were lethally irritated and
radioprotected wits SCID bone marrow. After immunization of such
chimeric mice with HBVsAg, human cells are obtained from the mice
spleens and fused in vitro with heteromyeloma cells to generate
hybridomas secreting human antibodies having a high affinity and
specificity to HBVsAg.
The present invention thus provides a process for obtaining human
monoclonal antibodies (hMoAb) capable of binding to Hepatitis B
virus surface antigen (HBVsAg) comprising: (a) immunizing a
chimeric rodent M4 having xenogeneic hematopoietic cells with
Hepatitis B surface antigen (HBVsAg) such that xenogeneic
antibody-producing cells are produced in said rodent, wherein said
rodent M4 is a rodent M1, the hematopoietic cells of which have
been substantially destroyed, said rodent M1 having transplanted
therein hematopoietic cells derived from a mouse M2 having a
hematopoietic deficiency, and xenogeneic hematopoietic cells
derived from human M3; (b) removing and immortalizing said
antibody-producing cells; (c) selecting and cloning the
immortalized antibody producing cells producing the antibodies
capable of binding to HBVsAg and; (d) isolating the antibodies
produced by the selected, cloned immortalized antibody producing
cells.
In accordance with the invention, spleens of the immunized chimeric
rodent M4 are removed between 12 and 20 days after human PBL
transplantation, preferably at day 14 after transplantation
thereof. Cell suspensions are prepared prepared from the spleens
and the antibody producing cells obtained from the immunized [a
heteromyeloma by techniques well known in the art (e.g. Kohler
& Milstein,] chimeric rodent M4 are fused preferably with a
human-mouse fusion partner such as a heteromyeloma by techniques
well known in the art (e.g., Kohler & Milstein, Nature,
256:495-497, 1975). In order to isolate the antibodies produced by
the selected hybridoma cell lines in accordance with the invention,
the hybridoma cell lines are either cultured in vitro in a suitable
medium wherein the desired monoclonal antibody is recovered from
the supernatant or, alternatively, the hybridoma cell lines may be
injected intraperitoneally into mice and the antibodies harvested
from the malignant ascitis or serum of these mice. The supernatant
of the hybridoma cell lines are first screened for production of
human IgG antibodies by any of the methods known in the art such as
enzyme linked (RIA). Hybridomas testing positive for human IgG are
then further screened for production of anti HBVsAg antibodies by
their capability to bind to HBVsAg.
The M1 rodent in accordance with the invention is preferably a
rodent conventionally used as a laboratory animal, most preferably
a rat or a mouse.
The mouse M2 may have any hematopoietic deficiency including
genetic hematopoietic deficiencies as well as induced hematopoietic
deficiencies. Non limiting examples of hematopoietic deficiencies
include SCID, Bg, Nu, Xid or mice having any combination of the
abovementioned hematopoietic deficiencies. In addition, the
hematopoietic deficiency may also be a result of gene deletion or
transgenic mice may be used.
The hematopoietic cells derived from the donor mouse M2 are
preferably bone marrow cells either untreated or depleted of T
cells. Other suitable sources of hematopoietic cells which may also
be used include, for example, spleen cells, fetal liver cells or
peripheral blood cells.
The xenogeneic hematopoietic cells derived from the human M3 are
preferably PBL cells but may also be derived from any suitable
source of human hematopoietic cells such as bone marrow cells, cord
blood cells, thymus spleen or lymphnode cells, etc.
By a most preferred embodiment, the rodent M1 is a mouse or rat,
the mouse M2 is a SCID mouse and the xenogeneic hematopoietic cells
derived from the human M3 are PBLs from a human M3 which has
already been exposed to the HBVsAg either spontaneously as a result
of a prior infection or induced following vaccinations. Such humans
will have a relatively high titer of anti HBVsAg antibodies as
compared to individuals which have never been infected with HBV
and, therefore, when PBLs from such donors are used as M3 donor
cells in accordance with the present invention, the immunization of
the M4 chimeric mouse with HBVsAg will elicit a secondary immune
response of the transplanted human PBLs in the M4 chimeric mouse. A
most preferred human donor M3 is such which tested negative for the
HB virus but shows a high titer of antibodies against HBVsAg. Such
PBLs from the human M3 donor may be obtained either by whole blood
donation or by leukophoresis.
The HBVsAg used for immunizing the chimeric rodent M4 in accordance
with the invention is preferably a Hepatitis B virus vaccine
containing the purified major surface antigen of the virus prepared
by recombinant DNA technology and formulated as a suspension of the
major surface antigen adsorbed on aluminum hydroxide (ENGERIX B,
SIB Biological (Rixensart, Belgium)).
The present invention is also directed to hybridoma cell lines
producing human monoclonal antibodies capable of binding to HBVsAg,
as well as to human monoclonal antibodies capable of binding to
HBVsAg and fragments thereof substantially maintaining the antigen
binding characteristics of the whole antibody. Such fragments may
be, for example, Fab or F(ab).sub.2 fragments obtained by digestion
of the whole antibody with various enzymes as known and described
extensively in the art. The antigenic characteristics of an
antibody are determined by testing the binding of an antibody to a
certain antigenic determinant using standard assays such as RIA,
ELISA or FACS analysis.
Typically, the human monoclonal antibodies obtained by the method
of the present invention have a relatively high affinity to HBVsAg
being in the range of about 10.sup.-9 M to about 10.sup.-10 M as
determined in a competative ELISA assay.
In accordance with a specific embodiment of the present invention
there are provided hybridoma cell lines designated herein as
"18.5.1013" and "19.79.5" which were deposited on May 22, 1996, in
the European Collection of Cell Cultures (ECACC, CAMR, Salisbury,
Wiltshire, SP40JG, U.K.) under Accession Nos. 96052170 and
96052168, respectively. Anti HBVsAg human monoclonal antibodies
secreted by the above hybridoma cell lines and designated herein as
"Ab18.5.1013" and "Ab19.79.5", respectively, are also provided as
well as fragments thereof retaining the antigen binding
characteristics of the antibodies, and antibodies capable of
binding to the antigenic epitope bound by "Ab18.5.1013" and
"Ab19.79.5".
The antigen bound by the antibodies defined above also constitutes
an aspect of the invention.
Further aspects of the present invention are various diagnostic,
prophylactic and therapeutic uses of the human anti HBVsAg
monoclonal antibodies and the Ag bound by them. In accordance with
this aspect of the invention, pharmaceutical compositions
comprising the human anti HBVsAg monoclonal antibodies may be used
for the treatment of chronic Hepatitis B patients by administering
to such a patient a therapeutically effective amount of the
monoclonal antibody or portion thereof capable of binding to the
HBVsAg being an amount effective in alleviating the symptoms of the
HBV infection or reducing the number of circulating viral particles
in an individual.
Such pharmaceutical compositions may comprise one or more
antibodies of the invention. In addition to the antibodies of the
invention the pharmaceutical Compositions may optionally also
comprise a carrier selected from any of the carriers known in the
art. One example of such a carrier is a liposome. The
pharmaceutical compositions of the invention may also comprise
various diluents and adjuvants known per se.
The compositions of the invention may be administered by a variety
of administration modes including parenterally, orally etc.
Compositions comprising the antibodies of the invention, as
described above, may be administered in combination with other anti
viral agents. Such agents may include, as a non limiting example:
Interferons, anti [Hepatitus HB] Hepatitis B (HB) monoclonal
antibodies, anti HB polyclonal antibodies, nucleoside analogs, and
inhibitors of DNA polymerase. In the case of such a combination
therapy the antibodies may be given simultaneously with the anti
viral agent or sequentially either before or after treatment with
the anti viral agent.
The pharmaceutical compositions of the invention may also be used,
for example, for immunization of new born babies against HBV
infections or for immunization of liver transplantation patients to
eliminate possible recurrent HBV infections in such patients.
By a further embodiment, the antibodies of the invention may also
be used in a method for the diagnosis of HBV infections in an
individual by obtaining a body fluid sample from the tested
individual which may be a blood sample, a lymph sample or any other
body fluid sample and contacting the body fluid sample with a human
and HBVsAG antibody of the invention under conditions enabling the
formation of antibody-antigen complexes. The level of such
complexes is then determined by methods known in the art, a level
significantly higher than that formed in a control sample
indicating an HV infection in the tested individual. In the same
manner, the specific antigen bound by the antibodies of the
invention may also be used for diagnosis. In the same manner, the
specific antigen of the invention may also be used for diagnosis of
HBV infection in an individual by contacting a body fluid sample
with the Ag and determining the presence of Ag-Ab complexes in the
sample as described above. In addition, the Ag of the invention may
be used for immunizing an individual to elicit a humoral response
against HBV.
The present invention further provides a kit for use in the therapy
of HB infections or diagnosis of such infections comprising the
antibodies of the invention, the antigen bound by the antibodies of
the invention and any further reagents necessary for detecting such
antibodies or antigens in a tested sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation showing the amount of total
human Ig (mg/ml) and the amount of specific anti HBs antibodies
(mU/ml) in the sera of irradiated mice which were radioprotected
with SCID bone marrow (chimeric mice). PBL+ENGERIX: the chimeric
mice were further transplanted with human PBL from donors positive
for anti HBs antibodies, and vaccinated with ENGERIX B in an
aluminum hydroxide adjuvant (alum).
PBL+Alum: the chimeric mice were further transplanted with human
PBL from donors positive for anti HBs antibodies, and vaccinated
with Alum alone (no ENGERIX B).
SCID-BM+ENGERIX: the chimeric mice were vaccinated with ENGERIX B
(no transplantation of human PBL).
SCID-BM+Alum: the chimeric mice were vaccinated with Alum (no human
PBL and no ENGERIX B).
The black line represents the initial level of anti HBs antibodies
in the serum of the human PBL donor.
FIG. 2 is a graphic representation showing the specific activity,
i.e. the levels of anti HBVs antibodies per mg of human Ig in the
sera of human donors (A-D, black columns) and the specific activity
in the sera of chimeric mice transplanted respectively with human
PBL of said donors (A-D, striped columns).
FIG. 3 is a graphic representation showing time response curve of
anti HBs antibodies specific activity (mU/mg) in sera of chimeric
mice (dotted line). The black columns represent the level of total
human Ig (mg/ml), and the striped columns represent the level of
specific anti HBs antibodies (mU/mi).
FIG. 4 is a graphic representation showing competitive inhibition
of binding of anti HBs antibodies to HBs particles. The extent of
binding was measured by ELISA using a horseradish peroxidase
labeled anti human IgG secondary antibody. The anti HBs antibodies
were diluted as indicated in the graph in medium (empty squares) or
in 0.5 .mu.g/ml HBs particles (black squares).
FIG. 5 is a photograph showing Hepatitis B infected liver sections
stained with anti HBVs antibodies. All sections were stained with a
"secondary" antibody, i.e. goat anti human Ig conjugated to biotin.
A--negative control. No first antibody. B--positive control. First
antibody-mouse anti HB antibody and a secondary anti-mouse Ig.
C--staining with anti HBs antibody No. 19.79.5. D--staining with
anti HBs antibody No. 18.5.1013.
Reference will now be made to the following Examples which are
provided by way of illustration and are not intended to be limiting
to the present invention.
FIG. 6 is a schematic representation of the binding of Ab 19.79.5
to a set of 15 well characterized HBsAg types. The y axis
represents optical density units. The x axis represents different
HBsAg types.
FIG. 7 is a graphic representation of the percentage of HBV
infected animals at days 11 and 18 in the untreated group and Ab
18.5.1013 treated group (in the inhibition model).
FIG. 8 is a graphic representation of the percentage of HBV
infected animals at days 10 and 17 in the untreated group and Ab
19.79.5 treated group (in the combined prophylaxis/inhibition
model).
FIG. 9 is a graphic representation of the percentage of HBV
infected animals at days 11 and 19 in the untreated group and Ab
19.79.5 treated group (in the combined inhibition/treatment
model).
FIG. 10 Nucleic acid sequence (SEQ ID NO:1) and corresponding amino
acid sequence (SEQ ID NO:2) of the light chain of the variable
domain of Ab 19.79.5.
FIG. 11 Nucleic acid sequence (SEQ ID NO:3) and corresponding amino
acid sequence (SEQ ID NO:4) of the heavy chain of the variable
domain of Ab 19.79.5.
EXAMPLES
Materials and Methods
Mice:
Animals used were 6-10 weeks old. BALB/c mice were obtained from
Harlan (Weizmann Institute Animal Breeding Center (Rehovot,
Israel)), SCID/NOD mice from the Weizmann Institute Animal Breeding
Center (Rehovot, Israel). All mice were fed sterile food and acid
water containing cyprofloxacin (20 .mu.g/ml) (Bayer, Leverkusen,
Germany). Whenever necessary, mice were injected daily with 1 mg
Fortum i.p. for five days post BMT (Glaxo Operations UK, Greenford,
England).
Conditioning Regimens:
BALB/c mice were exposed to total body irradiation (TBI), from a
gamma beam 150-A 60Co source (produced by the Atomic Energy of
Canada, Kanata, Ontario) with F.S.D of 75 cm and a dose rate of 0.7
Gy/min, with 4 Gy followed 3 days later by 10-11 Gy (split
dose).
Preparation and Transplantation of Bone Marrow Cells:
The femoral and tibial bones were removed from mice and homogenized
in a sterilized 50 ml Omni-Mixer stainless steel chamber
(Omni-Mixer Hmogenizer, Model No. 17106, OMNI International,
Waterbury, Conn. USA).
Recipient mice were injected i.v. with 4-6.times.10.sup.6 of
SCID/NOD bone marrow cells (in 0.2 ml PBS) immediately after
irradiation.
Transplantation of Peripheral Blood Lymphocytes:
Peripheral blood lymphocytes (PBL) were obtained after informed
consent by leukophoresis from donors positive for HBs antibodies
and negative for HBV. PBLs were washed twice, counted and
resuspended in PBS to the desired cell concentration.
100.times.10.sup.6 human PBL were injected intraperitoneally (i.p.)
into recipient mice, conditioned as described above. Control mice
did not receive human PBL.
Immunization of the Chimeric Animals:
Mice were immunized once with hepatitis B vaccine (ENGERIX-B; SB
Biologicals Rixensart, Belgium) administered i.p. together with the
PBL.
Cell and Plasma Collection from Human Mouse Chimera:
Animals were bled from the retro-orbital vein using heparin-coated
glass capillaries. Plasma was kept for human-Ig determination.
Spleens were removed after the animals were sacrificed by cervical
dislocation, cut into pieces and pressed through stainless steel
sieves to make a cell suspension in PBS.
Cell Fusion:
Cells were mixed with the human-mouse heteromyeloma HMMA2.11TG/0
(Posner et al. Hybridoma, 6:611-625, 1987) at 3:1 ratio. Fusion was
performed with 50% (w/v) PEG 1500 (Boehringer Manheim GmbH) in a
concentration of 3000 cells/well in 96-well U-bottom microtiter
plates (Nunc, Denmark) in complete medium containing HAT-supplement
(1x) (Biological Industries, Beit Haemek, Israel). Cells were fed
with fresh HAT-medium a week latter. Two weeks after fusion
supernatants were harvested for ELISA and medium was replaced with
fresh HT-medium.
Hybridoma cultures secreting specific anti-HBs Ig were cloned at
0.5 cell/well in 96-well U-bottom microtiter plates.
Determination of Human Immunoglobulin:
Sera were tested for antigen specific and total human Ig. Total
human Ig was quantified by sandwich ELISA using goat
F(ab)2-purified anti-human IgG+IgM+IgA (Zymed Laboratories, San
Francisco, Calif.) as the capture agent and peroxidase-conjugated
purified goat anti-human (Zymed Laboratories) as the detection
reagent. Human serum of known immunoglobulin concentration was used
as the standard (Sigma, Rehovot,Israel). Microplates (Nunc,
Roskilde, Denmark) pre-coated with the capture reagent (2.5 ug/ml,
50 ul/well) and blocked with 1% BSA were incubated overnight at 4C
with dilutions of plasma from 1:20000 to 1:640000, or the standard
from 0.2 to 0.06 ug/ml, then washed 5 times with PBS-Tween
solution. The detection reagent was added and the plates were
incubated for 1 h at 37 C, then washed again 3 times. Fresh
substrate solution (TMB, Sigma) was added and, after
peroxidase-catalyzed color development, the reaction was stopped by
addition of 10% sulfuric acid. Absorbance at 450 nm was quantified
on an ELISA reader (Dynatech, Port Guernsey, Channel Islands,
UK).
Concentration of antigen-specific human antibodies in mice sera was
determined by HBsAb EIA kit (ZER, Jerusalem, Israel).
Human antibodies in hybridoma supernatants were determined by
overnight incubation of supernatants on goat anti-human IgG+A+M
(Zymed) coated plates, with goat anti-human IgG-peroxidase
conjugated as the secondary reagent.
Antigen-specific antibodies in hybridoma supernatants were
determined as above using Hbs antigen coated plates.
Determination of Human IgG Subclasses:
Human IgG subclasses were determined by sandwich ELISA using goat
F(ab)2-purified anti-human IgG+IgM+IgA (Zymed Laboratories, San
Francisco, Calif.) coated plates and Hbs antigen coated plates.
Mouse anti-human IgG subclasses (Sigma) were used as second
antibody and peroxidase-conjugated purified goat anti-human (Zymed
Laboratories) as the detection reagent.
Statistic Analysis:
Statistical analysis was performed using the STAT VIEW II program
(Abacus Concepts, Inc., Berkeley, CA) on a Mackintosh Quadra 605 or
Microsoft EXCEL 5.0 (Microsoft) on a 486 DX2 PC compatible. Student
t-test, Anova correlation and regression analysis were utilized to
calculate probability (p) and correlation coefficient (r) values.
Results are presented as mean.+-.standard error.
Affinity Constant Measurements:
Determination of affinity constants (KD) of the different anti-HBs
antibodies to ad antigen (Chemicon Cat. No. AG 850) in solution
were performed according to Friguet et al. (Journal of
Immunological Methods, 77:305-319, 1985). The antigen at various
concentrations (3.5.times.10.sup.-10M to 1.4.times.10.sup.-9M) was
first incubated in solution with a constant amount of antibody
(3.4.times.10.sup.-11M), in 0.1 M sodium phosphate buffer
containing 2 mM EDTA and 10 mg/ml BSA, pH 7.8 (medium buffer).
After overnight incubation at 20 C the concentration of free
antibody was determined by an indirect ELISA. A volume of 300 ul of
each mixture were transferred and incubated for 2 h at 20 C into
the wells of a microtiteration plate (Nunc) previously coated with
Ad (50 .mu.l/well at 1 .mu.g/ml in 0.1 M NaHCO.sub.3 buffer, pH 9.6
for 2 h at 37.degree. C.). After washing with PBS containing 0.04%
TWEEN 20, (polyoxythylene sorbitan monolaurate) the bound
antibodies were detected by adding HRP-F(ab').sub.2 Goat anti human
IgG (Zymed) diluted 1:3000 with medium buffer, 50 .mu.l/well 2 h at
20.degree. C. The plate was developed with TMB chromogen (Sigma
T-3405 tablets) 50 .mu.l/well, the reaction stopped with 10%
H.sub.2SO.sub.4 50 .mu.l/well and the plate read in an ELISA reader
at 450 nm. The conditions were chosen so that the resulting f
values (see Friguet et al.) were around 0.1. The antibody
concentration used was deduced from an ELISA calibration done on
the same plate. The affinity constant KD was calculated from the
relevant Scatchard plot.
Inhibition Assays:
The inhibition assay was performed in microtiter plates coated with
HBs particles (2 .mu.g/ml in PBS). The plate was blocked with 3%
BSA in PBS.
Hybridoma supernatants containing anti HBs antibodies were serially
diluted. 50 .mu.l of each dilution were added to the coated
microtiter wells. Subsequently, 50 .mu.l of HBs particles (ad/ay,
0.5 .mu.l/ml in PBS) or PBS alone were added to each well. The
plates were incubated overnight at room temperature in a humid
chamber and washed 5 times with PBS-Tween. Next, 50 .mu.l of goat
anti human IgG conjugated to HRP (diluted 1:5000 in PBS) were added
to each well. After a 4 hour incubation at room temperature in a
humid chamber the plates were washed 5 times with PBS-Tween, and
TMB was added to each well. Results were read using an ELISA
reader, in a wavelength of 450 nm.
Immunohistostaining:
HBV positive liver fragment was fixed in 4% neutral buffered
formaldehyde for 24 h and then embedded in paraffin using routine
procedures. Sections of 4 .mu.m thickness were cut from paraffin
blocks and mounted on polylysine-coated slides. After
deparaffinization and peroxidase quenching staining was performed
using our monoclonal Human anti-HBs Protein A-purified antibodies
followed by biotinylated Goat anti-Human IgG (H+L) (Zymed, San
Francisco, Calif.) usig Histostain-SPTM kit (Zymed) according to
the manufacture's recommendation. Control slides without using the
1st Human anti-HBs antibody were stained in parallel.
Sequence analysis:
Total RNA was isolated from 10.times.10.sup.6 hybridoma cells with
RNAsol B reagent (TEL-TEX, Inc. Friendswood, Tex.). cDNA was
prepared from 10 .mu.g of total RNA with reverse transcriptase and
oligo dT (Promega, Madison, Wis.) according to standard procedures.
PCR was performed on 1/50 of the RT reaction mixture with V.sub.H,
V.sub..lamda., or [V.tau.5'] V.kappa.5'leader primers and 3'
primers corresponding to human constant region. The PCR fragments
were cloned into pGEM-T vector (Promega). The inserts were
sequenced using an ABI 377 sequencing machine. Sequences were
analyzed by comparison to Genbank and by alignment to Kabat
sequences (Kabat et al. 1991, Sequences of proteins of
immunological interest (5.sup.th Ed.) U.S. Dept. of Health and
Human Services, National Institutes of Health, Bethesda, Md.).
Example 1
Production of Human anti HBs Antibodies in Chimeric Mice
Human peripheral blood lymphocytes (PBL) from donors positive for
anti HBs antibodies were implanted intraperitoneally into
irradiated BALB/C mice which were radioprotected by transplantation
of bone marrow from SCID mice. These chimeric mice were immunized
with Hepatitis B vaccine (Engerix B) to include a secondary immune
response. The production of specific anti HBs antibodies along with
total human Ig secretion was measured in mice sera. FIG. 1 shows
levels of total human Ig and specific anti HBs antibodies in mice
sera 14 days after transplantation of human PBL. Although the
levels of human Ig secreted are similar in immunized and control
mice, a strong specific immune response develops in mice vaccinated
with hepatitis B vaccine as compared to the control group.
Comparison of the levels of specific human antibodies produced in
response to the antigen in immunized mice to their levels in the
donors sera, indicates a 5-10 fold increase in the mice. Moreover,
the specific activity measured in mice sera, i.e. the levels of
anti HBs specific antibodies per mg of human Ig secreted, is
102-104 fold higher than the specific activity observed in the
donor. This increase demonstrates a very high amplification of anti
HBs antibody production in response to the antigen in the chimeric
mice (FIG. 2). Production of human antibodies is detectable 10 days
after immunization and reaches a plateau after three weeks. The
specific activity is high at day 13 after immunization and
decreases thereafter (due to increase in total human Ig secretion)
(FIG. 3).
Example 2
Preparation and Characterization of Human Monoclonal Antibodies
Against HBs
Human B cells harvested from mice spleens two weeks after
immunization were fused to human-mouse heteromyeloma cells (Posner
et al. Supra). Hybridoma cells were tested for their growth rate,
total Ig secretion and specific antibody production. Control fusion
experiments were performed on the donor PBL that were activated in
vitro with PWM and HBVsAg. Fusion frequencies in different
experiments range from 0.9-5.times.10.sup.-5. Most of the growing
hybridoma clones secrete human Ig of which 0.1-4% produce specific
human anti HBs antibodies. Anti-HBs secreting hybridoma cells
derived from chimeric mice spleens were compared to those obtained
from fusion of the donors in vitro activated PBL in terms of Ig
type and stability as seen in Table 1 below. The majority of the
hybridomas from chimeric mice were found to be IgG type and all
were stable for more than 12 months. In contrast, hybridomas
derived from donor PBL were mostly unstable, only one clone has
been stable for more than 12 months. Two stable hybridoma clones
that secrete specific human anti HBs monoclonal antibodies were
characterized. As seen in Table 2 below, these antibodies were
purified on a protein A column as well as on an anti human
Ig-agarose column and were both found to be of IgG1 subclass.
Affinity constants range from 1.3.times.10.sup.-9 M to
6.times.10.sup.-9 M as tested by competitive ELISA. Specificity was
tested by competitive inhibition assay using HB surface antigen of
the ad-ay (1:1) subtype (FIG. 4). FIG. 5 shows specific binding of
the human MoAbs of the invention to HBV by staining human liver
fragments infected with HBV.
The gene encoding the variable region of Ab 19.79.5 was isolated,
fully sequenced, and its subgroups and CDRs were determined.
The antibody has a fully human Ig gene sequence as determined by
alignment to Genebank sequences and Kabat protein sequences. FIG.
10 shows the nucleotide sequence of the cDNA encoding the light
chain of the variable region of Ab 19.79.5 and its corresponding
amino acid sequence (Sequence identification nos. 1 and 3). FIG. 11
shows the nucleotide sequence of the cDNA encoding the heavy chain
of the variable region of Ab 19.79.5 and its corresponding amino
acid sequence (Sequence identification nos. 2 and 4).
The sequencing data reveled that the variable region of Ab 19.79.5
consists of the subgroups V.sub.H3, J.sub.H2, V.sub..lamda.3 and
J.sub..lamda.3.
HBV genomes are classified into six groups A to F, based on the
degree of similarity in their nucleotide sequences. The genetic
variability of HBV is further reflected in the occurrence of
different scrotypes of HBsAg. The common determinant `a` and two
pairs of mutually exclusive determinants `d/y` and `w/r` enable the
distinction of four major subtypes of HBsAg: adw, adr, ayw and ayr.
Additional determinants designated subdeterminants of w (w1 to w4)
have allowed the definition of our serotypes of ayw (ayw1-4) and
two serotypes of adw, i.e. adw2 and adw4. Additional subtype
variation is added by the q determinant, which is present on almost
all subtypes. Its absence is marked by a `q-` sign.
The kind of HBV serotypes recognized by Ab 19.79.5 was examined
using a set of 15 different HBsAg types (Norder et al., 1992,
Journal of General Virology, 73, 3141; Magnius and Norder, 1995,
Intervirology, 38, 24-34). As can be seen in FIG. 6, Ab 19.79.5 has
a complex recognition pattern of the different HBsAg serotypes.
Example 3
Biological Activity of Human Monoclonal Antibodies Against HBs
The biological activity of Ab 19.79.5 and Ab 18.5.1013 was
characterized using the following HBV animal model: a mouse was
treated so as to allow the stable engraftment of human liver
fragments. The treatment included intensive irradiation followed by
transplantation of acid (severe combined immunodeficient) mice bone
marrow. Viral infection of human liver fragments was performed
ex-vivo using HBV positive human serum (EP 699 235).
The animal model was used in three different modes representing
various potential uses of the antibodies: inhibition of infection
mode, combined prophylaxis/inhibition mode and combined
inhibition/treatment.
1. Inhibition Mode
This model demonstrates the ability to use the antibody to inhibit
liver infection by HBV. HBV positive human serum was preincubated
with Ab 18.5.1013, followed by standard ex-vivo liver infection.
HBV-DNA in mice sera was tested 11 and 18 days after
transplantation. As seen in FIG. 7 there was a significant
reduction in the percentage of infected animals in the antibody
treated group as compared to the untreated group.
2. Combined Prophylaxis/Inhibition Mode
This model represents liver transplantation. In this model mice
were treated with Ab 19.79.5(10 I.U./mouse) three days before liver
transplantation followed by transplantation of human liver
fragments which were ex vivo infected with HBV in the presence of
Ab 19.79.5 (100 I.U.). HBV DNA was tested in mice sera 10 and 17
days after transplantation. As can be seen in FIG. 8, there was a
significant reduction in the percentage of infected animals in the
treated group compared to the control group.
3. Combined Inhibition/Treatment Mode
a) HBV positive human serum was preincubated with Ab 19.79.5
followed by standard ex vivo liver infection. b) Mice were treated
with Ab 19.79.5 at days 0 and 7 past transplantation. HBV DNA in
mice sera was tested on days 11 and 19. As can be seen in FIG. 9,
the percentage of infected animals in the Ab 19.79.5 treated group
was significantly reduced but rebounded about two weeks after the
treatment was stopped.
Example 4
Combination Therapy of Human Monoclonal Antibodies Against HBs and
an Anti Viral Agent
Using the HBV model described above, mice are treated with an anti
viral drug (a nucleoside analogue, 0.5 mg/mouse/day) at days 17-20
post transplantation. A group of mice is further treated with the
human monoclonal antibodies of the invention at days 19 and 20. The
presence of HBV DNA in mice sera is tested on days 21 and 27.
TABLE-US-00001 TABLE 1 Anti-HBs Secretors Source of Hybridoma
Stability IgM IgG Cells 1 stable for >10 months 25 (52%) 23
(48%) In Vitro Activated 47 unstable PBL 6 stable for >10 months
3 (33%) 6 (67%) Chimeric Mouse 3 unstable Splenocytes
TABLE-US-00002 TABLE 2 Production Kd (M) .mu.g/10.sup.3 cells/day
Type Clone 6.1 .times. 10.sup.-3 10.3 IgG1 V.lamda. 18.5.1013 1.62
.times. 10.sup.-3 5.8 IgG1 V.lamda. 19.79.5
SEQUENCE LISTINGS
1
41327DNAhumanCDS (1)..(327) 1 tcc tat gtg ctg act cag cca ccc tcg
gtg t ca gtg gcc cca gga aag 48 Ser Tyr Val Leu Thr Gln Pro Pro Ser
Val S er Val Ala Pro Gly Lys 1 5 10 15 acg gcc agg att tcc tgt ggg
gga aac aac a tt gga act aaa aat gtg 96 Thr Ala Arg Ile Ser Cys Gly
Gly Asn Asn I le Gly Thr Lys Asn Val 20 25 30 cac tgg tac cag cag
aag cca ggc cag gcc c ct gtg ctg gtc gtc tat 144 His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala P ro Val Leu Val Val Tyr 35 40 45 gct gat
agc gac cgg ccc tca ggg atc cct g ag cga ttc tct ggc tcc 192 Ala
Asp Ser Asp Arg Pro Ser Gly Ile Pro G lu Arg Phe Ser Gly Ser 50 55
60 aac tct ggg aac acg gcc acc ctg acc atc a gc agg gtc gaa gtc ggg
240 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile S er Arg Val Glu Val
Gly 65 70 75 80 gat gag gcc gac tat tac tgt cag gtg tgg g at agt
gtt agt tat cat 288 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp A sp
Ser Val Ser Tyr His 85 90 95 gtg gta ttt ggc gga ggg acc acg ctg
acc g tc cta ggt 327 Val Val Phe Gly Gly Gly Thr Thr Leu Thr V al
Leu Gly 100 105 2109PRThuman 2 Ser Tyr Val Leu Thr Gln Pro Pro Ser
Val S er Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Ser Cys Gly
Gly Asn Asn I le Gly Thr Lys Asn Val 20 25 30 His Trp Tyr Gln Gln
Lys Pro Gly Gln Ala P ro Val Leu Val Val Tyr 35 40 45 Ala Asp Ser
Asp Arg Pro Ser Gly Ile Pro G lu Arg Phe Ser Gly Ser 50 55 60 Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile S er Arg Val Glu Val Gly 65 70
75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp A sp Ser Val Ser Tyr
His 85 90 95 Val Val Phe Gly Gly Gly Thr Thr Leu Thr V al Leu Gly
100 105 3363DNAhumanCDS (1)..(363) 3 cag gtg cag ctg gtg gag tct
ggg gga ggc g tg gtc cag cct ggg ggg 48 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly V al Val Gln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc
tgt gca ccg tct gga t tc gtc ttc agg agt tat 96 Ser Leu Arg Leu Ser
Cys Ala Pro Ser Gly P he Val Phe Arg Ser Tyr 20 25 30 ggc atg cac
tgg gtc cgc cag act cca ggc a ag ggg ctt gag tgg gtg 144 Gly Met
His Trp Val Arg Gln Thr Pro Gly L ys Gly Leu Glu Trp Val 35 40 45
tca ctt ata tgg cat gat gga agt aat aga t tc tat gca gac tcc gtg
192 Ser Leu Ile Trp His Asp Gly Ser Asn Arg P he Tyr Ala Asp Ser
Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat t cc aag aac
aca ttg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn S er Lys
Asn Thr Leu Tyr 65 70 75 80 ttg caa atg aac agc ctg aga gcc gaa gac
a cg gct atg tac ttc tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp T hr Ala Met Tyr Phe Cys 85 90 95 gcg aga gag agg ctg att gca
gca cct gct g cc ttt gac ctc tgg ggc 336 Ala Arg Glu Arg Leu Ile
Ala Ala Pro Ala A la Phe Asp Leu Trp Gly 100 105 110 cag gga acc
ctg gtc acc gtc tcc tcg 363 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 4121PRThuman 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly V al Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly P
he Val Phe Arg Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Thr Pro
Gly L ys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Trp His Asp Gly
Ser Asn Arg P he Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn S er Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp T hr Ala Met Tyr Phe Cys 85 90 95
Ala Arg Glu Arg Leu Ile Ala Ala Pro Ala A la Phe Asp Leu Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
* * * * *