U.S. patent number RE39,586 [Application Number 10/465,633] was granted by the patent office on 2007-04-24 for human monoclonal antibody against hepatitis b virus surface antigen (hbvsag).
This patent grant is currently assigned to XTL Biopharmaceuticals Limited. Invention is credited to Shlomo Dagan.
United States Patent |
RE39,586 |
Dagan |
April 24, 2007 |
Human monoclonal antibody against Hepatitis B virus surface antigen
(HBVSAG)
Abstract
Disclosed is a hybridoma cell line which produces human
antibodies capable of binding to the hepatitis B virus surface
antigen (HBVsAg), as well as antibodies produced by the cell line.
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 activated in vitro with pokeweed mitogen and
then fused with heteromyeloma cells to generate hybridomas
secreting human antibodies having a high affinity and specificity
to HBVsAg.
Inventors: |
Dagan; Shlomo (Rehovot,
IL) |
Assignee: |
XTL Biopharmaceuticals Limited
(Rehovot, IL)
|
Family
ID: |
11068956 |
Appl.
No.: |
10/465,633 |
Filed: |
June 10, 1997 |
PCT
Filed: |
June 10, 1997 |
PCT No.: |
PCT/IL97/00183 |
371(c)(1),(2),(4) Date: |
January 22, 1998 |
PCT
Pub. No.: |
WO97/47653 |
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: |
09000088 |
Jan 22, 1998 |
06146629 |
Nov 14, 2000 |
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Foreign Application Priority Data
Current U.S.
Class: |
424/149.1;
424/141.1; 424/133.1; 424/142.1; 435/326; 435/70.21; 435/339;
424/161.1; 424/130.1 |
Current CPC
Class: |
A61P
31/20 (20180101); A61P 31/12 (20180101); A61P
43/00 (20180101); C07K 16/082 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
A61K
39/42 (20060101); A61K 39/395 (20060101) |
Field of
Search: |
;424/149.1,130.1,133.1,142.1,161.1 ;435/70.21,326,339 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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179 483 |
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Apr 1985 |
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EP |
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0168234 |
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Jan 1988 |
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EP |
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0468866 |
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Jan 1992 |
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EP |
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0179483 |
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Apr 1986 |
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JP |
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WO 93/16192 |
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Aug 1993 |
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WO |
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9411495 |
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May 1994 |
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WO |
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WO9411495 |
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May 1994 |
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WO |
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94/26784 |
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Nov 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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WO 97/40164 |
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Oct 1997 |
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WO |
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WO 94/47654 |
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Dec 1997 |
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WO |
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Other References
Ehrlich et al. Human Antibodies and Hybridomas (Jan., 1992) vol. 3,
p 2-7. cited by examiner .
Ehrlich et al. Hum. Antibod. Hybridomas 3: 2-7, 1992. cited by
examiner .
Colucci et al., Preparation and characterization of human
monoclonal antibodies directed against the hepatitis B virus
surface antigen, Liver, 6:145-152 (1986). cited by other .
Ogata et al., Markedly prolonged incubation period of hepatitis B
in a chimpanzee passively immunized with a human monoclonal
antibody to the .alpha. determinant of hepatitis B surface antigen,
Proc. Natl. Acad. Sci. USA, 90:3014-3018 (Apr. 1993). 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(suppl. 1)80 (Aug. 1996). cited
by other .
Inchimori et al., Establishment of hybridoma secreting human
monoclonal antibodies against tetanus toxin and hepatitis B virus
surface antigen, Biochemical and Biophysical Research
Communications, 129(1)26-33 (May 31, 1985). cited by other .
Ichimori et al., Establishment of hybridoma secreting human
monoclonal antibody against hepatitis B virus surface antigen,
Biochemical and Biophysical Research Communications, 142(3)805-812
(Feb. 13, 1987). cited by other .
Wright et al., Clinical aspects of hepatitis B virus infection, The
Lancet, 342:1340-1344 (1993). cited by other .
Ehrlich et al. 1992 Human antibodies and Hybridomas, vol. 3 pp.
2-7, Jan. 1992. cited by examiner.
|
Primary Examiner: Zeman; Mary K.
Attorney, Agent or Firm: Browdy and Neimark, PLLC
Claims
What is claimed is:
1. A human monoclonal antibody Ab17.141, which is secreted by the
European Collection of hybridoma cell line deposited in the Cell
Cultures (ECACC) under Accession No. 96052169, .Iadd.or a fragment
thereof which retains the antigen binding characteristics of
Ab17.1.41.Iaddend..
2. The hybridoma cell line deposited at the ECACC on May 22, 1996
under Accession No. 96052169.
3. A pharmaceutical composition for the treatment of Hepatitis B
Virus (HBV) infections comprising as an active ingredient an
antibody in accordance with claim 1 together with a
pharmaceutically acceptable carrier.
4. A method for the treatment of Hepatitis B Virus (HBV) infections
comprising administering to an individual in need a therapeutically
effective amount of antibodies according to claim 1.
5. A method for reducing the occurrence of Hepatitis B virus (HBV)
infections in a population of individuals, comprising administering
a human monoclonal antibody Ab 17.1.41 or a fragment thereof which
retains the antigen binding characteristics of Ab 17.1.41 in
accordance with claim 1 to a population of individuals to reduce
the occurrence of HBV infections in the population.
6. A pharmaceutical composition for the treatment of Hepatitis B
Virus infections comprising as an active ingredient an antibody in
accordance with claim 1 adopted for use in combination with at
least one other active ingredient being an anti viral agent.
7. A pharmaceutical composition according to claim 6 wherein the
anti viral agent is selected from the group consisting of:
interferons, anti-Hepatitis B (HB) monoclonal antibodies, anti HB
polyclonal antibodies, nucleoside analogues and inhibitors of DNA
polymerase.
8. A pharmaceutical composition according to claim 6 wherein the
anti viral agent is a nucleoside analogue.
9. A method for the treatment of HBV infections comprising
administering to an individual in need thereof a therapeutically
effective amount of a pharmaceutical composition according to claim
6.
10. A method for the treatment of HBV infections comprising
administering to an individual in need thereof a therapeutically
effective amount of a pharmaceutical composition according to claim
7.
11. A method for the treatment of HBV infections comprising
administering to an individual in need thereof a therapeutically
effective amount of a pharmaceutical composition according to claim
8.
.Iadd.12. A method for reducing the occurrence of HBV infections
according to claim 5, wherein the population of individuals include
liver transplantation patients..Iaddend.
Description
FIELD OF THE INVENTION
The present invention concerns a hybridoma cell line producing
human antibodies capable of binding to the hepatitis B virus
surface antigen, 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. (Progressive
Hepatitis Research: Hepatitis B virus (HBV), Hepatitis C virus
(HCV) and Hepatitis Delta virus (HDV) Ed. O. Crivelli, Sorina
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 showing 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 infections
concerns the use of monoclonal antibodies (MoAb).
PCT patent application PCT/NL94/00102 discloses human monoclonal
antibodies directed against Hepatitis B surface antigen 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 and 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.
SUMMARY OF THE INVENTION
In accordance with the present invention, a hybridoma cell line is
provided which secretes human antibodies capable of binding to the
Hepatitis B surface antigen (HBVsAg).
In accordance with the invention, peripheral blood lymphocytes
(PBL) were obtained from human individuals having a high titer of
anti HBVsAg antibodies. Such individuals may either have been
previously infected with HBV, actively immunized with HBV antigens
or spontaneously showing a high level of such antibodies. A most
preferred human donor is an individual which tested negative for
the presence of HBV but shows a high titer of antibodies against
HBVsAg. PBLs from the human donor may be obtained either by whole
blood donation or by leukophoresis.
The human PBLs are then activated in vitro by their incubation with
pokeweed mitogen (PWM). After activation the PBLs are fused in
vitro 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). The generated 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 immunosorbent assay (ELISA) or radioimmuno assay
(RIA). Hybridomas testing positive for human IgG are then further
screened for production of anti HBVsAg antibodies by their
capability to bind to HBVsAg.
In accordance with the preferred embodiment of the present
invention, a hybridoma cell line designated herein as "17.1.41"
which was deposited on May 22, 1996, at the European Collection of
Cell Cultures (ECACC, CAMR, Salisbury, Wiltshire, SP40JG, U.K.)
under the Accession No. 96052169 is provided. Anti HBVsAg human
monoclonal antibodies secreted by the above hybridoma cell line
designated herein as "Ab17.1.41" as well as fragments thereof
retaining the antigen binding characteristics of the antibodies and
antibodies capable of binding to the antigenic epitope bound by
Ab17.1.41 are also provided. 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.
The antibodies of the invention have a relatively high affinity to
HBVsAg being in the range of about 10.sup.-9M to about 10.sup.-10M
as determined by a competitive ELISA assay.
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 Ab 17.1.41 monoclonal
antibodies and the Ag bound by these antibodies. In accordance with
this aspect of the invention, pharmaceutical compositions
comprising the Ab17.1.41 antibodies may be used for the treatment
of chronic Hepatitis B patients by administering to such a patient
a therapeutically effective amount of the antibodies or fragments
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.
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 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.
Such pharmaceutical compositions may also be used, for example, for
immunization of new born babies against HBV infections or for
immunization .[.cf.]. .Iadd.of .Iaddend.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
anti 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 of HB infection in an
individual by contacting a body fluid sample from the tested
individual with the antigen as described above and determining the
formation of antigen Ab in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph showing Hepatitis B infected liver sections
stained with the anti HBVs antibodies of the invention. All
sections were stained with a "secondary" antibody, i.e. goat anti
human or anti mouse 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
HBVsAg Ab 17.1.41.
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. 2 is a schematic representation of the binding of Ab17.1.41 to
a set of well characterized HBsAg types. The y axis represents
optical density units. The x axis represents different HBsAg
types.
FIG. 3 is a graphic representation of hepatitis B viremia score, as
defined in example 3. Each dot in the graph represents one
animal.
FIG. 4 is a graphic representation of the percentage of HBV
infected animals at days 18 and 25 in the untreated group and
Ab17.1.41 treated group (in the treatment model).
FIG. 5 is a graphic representation of the percentage of HBV
infected animals at days 10 and 17 in the untreated group and
Ab17.1.41 treated group (in the combined prophylaxis/inhibition
model).
FIG. 6 is a graphic representation of the percentage of HBV
infected animals at days 11 and 18 in the untreated group and Ab
17.1.41 treated group (in the combined inhibition/treatment
model).
FIG. 7 is a graphic representation of the percentage of HBV
infected animals at days 21 and 27 in the untreated group
(control), the group treated with an anti viral drug, the group
treated with Ab17.1.41 and the group treated with both the anti
viral drug and Ab17.1.41 (Mab+Drug).
FIG. 8 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 Ab17.1.4 1.
FIG. 9 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 17.1.41.
EXAMPLES
Materials and Methods
In vitro Activation:
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. PBL were
separated from granulocytes and erythrocytes on a Ficoll-hypaque
gradient (UNI-SEP maxi; Eldan Tech., Jerusalem, Israel) and
subsequently stimulated for 3-4 days with pokeweed mitogen (PWM;
Gibco BRL, Life Technologies Inc., Grand Island, N.Y.) diluted
1:100 and with Antigen at 200 ng/ml in RPMI-1640 medium with 10%
(v/v) fetal calf serum (FCS) supplemented with 10 U/ml penicillin,
10 .mu.g/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate,
1% (v/v) non-essential amino acids (Biological Industries, Belt
Haemek, Israel) and 10.sup.-4 M 2-mercaptoethanol (Sigma, St.
Louis) (Complete Medium).
Cell Fusion:
Cells were mixed with the human-mouse heteromyeloma HMMA2.1 1TG/0
(Posner et al.) at 3:1 ratio. Fusion was performed with 50% (w/v)
PEG 1500 (Boehringer Manheim GmbH) in a standard procedure. Fused
cells were seeded at a concentration of 30000 cells/well in 96-well
U-bottom microtiter plates (Nunc, Denmark) in complete medium
containing HAT-supplement (1.times.) (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).sub.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 37C, 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).sub.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, Calif.) 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 (K.sub.D) 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 o.n. 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 microtitration 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, 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. Section 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.) using Histostain-SPTM kit (Zymed) according to
the manufacture's recommendation. Control slides without using the
1 st 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.sub.K5' 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
Human peripheral blood lymphocytes (PBL) from donors positive for
anti HBVs antibodies were obtained and activated in vitro with PWM
as described above. The cells were then fused with a human mouse
heteromyeloto form hybridoma cell lines. One stable hybridoma clone
secreting specific human anti HBVsAg designated 17.1.41 was
characterized. The antibodies secreted by the above clone were
purified on a protein A column as well as on an anti human
Ig-agarose column and were found to be of the IgG1 V.kappa. type.
The affinity constant of the antibodies to HBVsAg was
1.34.times.10.sup.-9. Specificity was tested by competitive
inhibition assay using HBV surface antigen of the ad-ay (1:1).
Example 2
The 17.1.41 antibodies were used for staining human liver fragments
as described above. As seen in FIG. 1, the 17.1.41 antibodies were
able to detect HBV particles present in the infected liver
fragments.
The gene encoding the variable region of Ab 17.1.41 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. 8
shows the nucleotide sequence of the cDNA encoding the light chain
of the variable region of Ab 17.1.41 and its corresponding amino
acid sequence (Sequence identification nos. I and 3). FIG. 9 shows
the nucleotide sequence of the cDNA encoding the heavy chain of the
variable region of Ab17.1.41 and its corresponding amino acid
sequence (Sequence identification nos. 2 and 4).
The sequencing data revealed that the variable region of Ab 17.1.41
consists of the subgroups V.sub.H3, J.sub.H6, V.sub.K2 and
J.sub.K2.
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 serotypes 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 four 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 17.1.41 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. 2. Ab 17.1.41 has a broad reactivity
towards all tested subtypes and genotypes, except for C adw2.
Example 3
The biological activity of Ab 17.1.41 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 scid (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: treatment mode, combined
prophylaxis/inhibition mode and combined inhibition/treatment. 1.
Treatment mode--This model demonstrates the ability to use the
antibody to treat chronic HBV infection. Mice were transplanted
with HBV infected human liver fragments. The mice were treated with
Ab 17.1.41 at days 16, and 17 post liver transplantation. HBV DNA
was tested on days 18 and 25. The number of HBV DNA copies (the
viral load) in mouse sera was determined using PCR. We use the term
"viremia score" as a mathematical representation of the viral load.
The viremia score was determined as follows:
TABLE-US-00001 Viremia score viral load = HBV DNA copies/ml serum 0
viral load < 5 .times. 10.sup.3 1 5 .times. 10.sup.3 < viral
load < 5 .times. 10.sup.4 2 5 .times. 10.sup.4 < viral load
< 5 .times. 10.sup.5 3 viral load > 5 .times. 10.sup.5
As can be seen in FIG. 3, there is a significant reduction in the
viremia score in the group treated with the antibody. In addition,
as can be seen in FIG. 4, the percentage of infected animals in the
treated group are significantly lower (very low p values) as
compared to the untreated group. 2. Combined prophylaxis/inhibition
mode--This model represents liver transplantation. In this model
mice were treated with Ab 17.1.41 (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 17.1.41 (100 I.U.). HBV DNA was tested in mice sera 10 and 17
days after transplantation. As can be seen in FIG. 5, 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 17.1.41 followed by standard ex vivo liver
infection. b) Mice were treated with Ab 17.1.41 at days 0 and 7
post transplantation. HBV DNA in mice sera was tested on days 11
and 18. As can be seen in FIG. 6, the percentage of infected
animals in the Ab 17.1.41 treated group was significantly reduced
but rebounded about two weeks after the treatment was stopped.
Example 4
In the following experiment we tested the possibility to use
17.1.41 in combination with another anti viral agent in the HBV
model described above. Mice were treated with the anti viral drug
(a nucleoside analogue, 0.5 mg/mouse/day) at days 17-20 post
transplantation. A group of mice was further treated with Ab
17.1.41 at days 19 and 20. The presence of HBV DNA in mice sera was
tested on days 21 and 27. As can be seen in FIG. 7, immediately
after treatment either with the anti viral drug or with our
monoclonal antibody there was a marked reduction in the number of
animals infected. However, viral load rebounded in each group that
was treated with one individual drug. Only the group that was
treated with the combination of the anti viral drug and Ab 17.1.41
did not show an increase in the number of animals infected.
SEQUENCE LISTINGS
1
41336DNAHUMANCDS(1)..(336) 1gat att gtg atg act cag tct cca ctc tcc
ctg tcc gtc acc cct gga 48Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Ser Val Thr Pro Gly 1 5 10 15gag ccg gcc tcc atc tcc tgc agg
tct agt cag agc ctc ctg cat agg 96Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Arg 20 25 30tct gga aac aac tat ttg gat
tgg tac ctg cag aag cca ggg cac tct 144Ser Gly Asn Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly His Ser 35 40 45cca cag ctc ctg atc tat
gtg ggt tct aat cgg gcc tcc ggg gtc cct 192Pro Gln Leu Leu Ile Tyr
Val Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60gac agg ttc agt ggc
agt gga tca ggc aca gag tat aca ctg aaa atc 240Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Lys Ile 65 70 75 80agt aga gtg
gag gct gag gat gtt ggg gta tat tac tgc atg caa gct 288Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95cta caa
act cct cgg act ttt ggc cag ggg acc aag ctg gag atc aaa 336Leu Gln
Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
1102112PRTHUMAN 2Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser
Val Thr Pro Gly 1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Arg 20 25 30Ser Gly Asn Asn Tyr Leu Asp Trp Tyr
Leu Gln Lys Pro Gly His Ser 35 40 45Pro Gln Leu Leu Ile Tyr Val Gly
Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Tyr Thr Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro
Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
1103387DNAHUMANCDS(1)..(387) 3cag gtg cag ctg gtg gag tca ggg gga
ggc gtg gtc cgg cct ggg agg 48Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Arg Pro Gly Arg 1 5 10 15tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc gcc ttc agt gac tat 96Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Ala Phe Ser Asp Tyr 20 25 30agt ata aac tgg gtc cgc
cag gct cca ggc aag gga ctg gag tgg gtg 144Ser Ile Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45gca att att tca tat
gat gga aga att aca tac tat aga gac tcc gtg 192Ala Ile Ile Ser Tyr
Asp Gly Arg Ile Thr Tyr Tyr Arg Asp Ser Val 50 55 60aag ggc cga ttc
acc atc tcc aga gac gac tcc aag aac acg ctg tat 240Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80ctg caa
atg aac agc ctg aga act gag gac acg gct gtg tat tac tgc 288Leu Gln
Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcg
aga cag tat tac gat ttt tgg agt ggt tct tcg gtt ggg cgt aac 336Ala
Arg Gln Tyr Tyr Asp Phe Trp Ser Gly Ser Ser Val Gly Arg Asn 100 105
110tac gac ggc atg gac gtc tgg ggc cta ggg acc acg gtc acc gtc tcc
384Tyr Asp Gly Met Asp Val Trp Gly Leu Gly Thr Thr Val Thr Val Ser
115 120 125tca 387Ser4129PRTHUMAN 4Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Arg Pro Gly Arg 1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ala Phe Ser Asp Tyr 20 25 30Ser Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Ser
Tyr Asp Gly Arg Ile Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu
Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gln Tyr Tyr Asp Phe Trp Ser Gly Ser Ser Val Gly Arg Asn
100 105 110Tyr Asp Gly Met Asp Val Trp Gly Leu Gly Thr Thr Val Thr
Val Ser 115 120 125Ser
* * * * *