Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF HEPATITIS B VIRUS INFECTION WITH HUMAN
MONOCLONAL ANTIBODIES
FIELD OF THE INVENTION
The present invention concerns a pharmaceutical composition for the treatment
or
prevention of hepatitis B infection comprising a mixture of two human
monoclonal
antibodies.
BACKGROUND OF THE INVENTION
Despite introduction of universal vaccination against hepatitis B in over 100
countries,
persistent HBV infection is still a serious problem worldwide, causing an
estimated annual
death rate of one million (Kane, Lancet 1996; 348-696). It may take several
decades until
the effect of vaccination will be translated into reduced transmission and
morbidity.
Meanwhile, patients with persistent HBV infection require better anti-viral
therapeutic
modalities than are currently available. In the U.S., approximately 300,000
new cases of
acute HBV infection occur annually, 10% of whom will become HBV carriers, and
50% of
those will develop chronic liver disease with an increased risk for developing
hepatocellular
carcinoma (HCC) (El-Serag and Mason, N Eng J Med 1999; 340 745-750).
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Hepatitis B vaccines are effective in preventing primary infection but have
not shown
a significant effect in infected patients.
Two therapies are currently approved for treatment of chronic HBV infection:
interferon alfa-
2b (IFNa) (Wong et al., Ann Intern Med 1993; 119, 312-323) and lamivudine
(Dienstag et
al., N Eng JMed 1999; 341, 1256-1263). Both therapies provide only a partial
solution to the
disease due to a relatively low response rate, severe side effects of IFNa,
and development of
lamivudine resistant strains (Liaw et al., Hepatolo~ 1999; 30, 567-572).
Passive immunotherapy utilizing preparations of human hyperimmune
immunoglobulin from HBV-immune patients is commonly used as prophylaxis
against liver re-infection after liver transplantation. It is given
intramuscularly to
neonates to prevent vertical transmission of HBV from infected mothers. It is
not
used for treatment of chronic patients.
Overall, the use of plasma-derived polyclonal antibodies is limited because
these
preparations have variable activity, limited availability and there are
potential hazards
for the transmission of infectious agents.
In contrast, monoclonal antibodies (mAbs) can be consistently produced and do
not
carry the infectious risks associated with plasma-derived products.
Previous studies using a single human mAb for treating HBV-infected patients
undergoing liver transplantation resulted in emergence of escape mutants
(McMahon
et al., 1992 flepatology 1 S (5) 757-766). The same antibody was administered
for a
two-week period to chronic hepatitis B patients pre-treated with lamivudine
and was
shown to form complexes with HBsAg and to reduce its level in patients. Three
months after therapy HBsAg levels had returned to pre-treatment levels
(Heijtink et
al., 2001 J. Med Virol. 64 427-434).
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In another study, two fully human monoclonal antibodies were developed
directed
against different epitopes of hepatitis B surface antigen (HBsAg)
(PCT/IL97/00184
and PCT/IL97/00183). A single administration of a mixture of these antibodies
into
HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg
levels
followed by a recurrence to initial levels within a few days (Eren et al.,
2000
Hepatology 32, 588-596).
SUMMARY OF THE INVENTION
In accordance with the present invention a pharmaceutical composition is
provided
comprising a combination of two, fully human, high-affinity monoclonal
antibodies
directed against different epitopes of hepatitis B virus surface antigen
(HBsAg)
In accordance with one embodiment of the present invention, a pharmaceutical
composition is provided (designated HBV-AbxTL) comprising as an active
ingredient
a mixture of the human monoclonal antibody 19.79.5 as well as fragments
thereof
retaining the antigen binding characteristics of the antibodies, and the human
monoclonal antibody 17.1.41 as well as fragments thereof retaining the antigen
binding characteristics of the antibodies together with a pharmaceutically
acceptable
carrier. Antibody 19.79.5 is secreted by the hybridoma cell line deposited in
the
European Collection of Cell Cultures (ECACC) under Accession No. 96052168, and
antibody 17.1.41 secreted by the hybridoma cell line deposited in the ECACC
under
Accession No. 96052169. Antibodies 19.79.5 and 17.1.41 are further
characterized by
their sequence disclosed in PCT/IL97/00184 and PCT/IL97/00183. Fragments
retaining the antigen binding characteristics of the antibodies may be, for
example,
Fab or F(ab)2 fragments obtained by digestion of the whole antibody with
various
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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.
Further aspects of the present invention are various prophylactic and
therapeutic uses
of the antibody mixture. In accordance with this aspect of the invention, the
pharmaceutical composition comprising the antibody mixture may be used for the
treatment of chronic Hepatitis B patients by administering to such a patient a
therapeutically effective amount of the mixture of 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. Means to assess alleviation of symptoms of HBV infection may
include as a non limiting example measurement of liver functions by
determining
levels of the enzyme alanine aminotransferase (ALT) or by measuring sero
conversion
namely disappearance of the HBeAg or by examining liver biopsies and
determining
the level of tissue fibrosis by methods well known in the art. The number of
circulating viral particles can be determined for example by measuring HBV DNA
levels using PCR or by detecting HBsAg levels in the blood.
In one embodiment of the present invention the pharmaceutical composition is
given
in a dose ranging from 0.26 mg to 80 mg. Preferably 10 mg or 40 mg.
In a preferred embodiment of the present invention the pharmaceutical
composition
comprises an approximate ratio of 1:3 between antibodies 19.79.5 and 17.1.41
respectively.
In addition to the antibody mixture the pharmaceutical composition of the
invention
may optionally also comprise a carrier selected from any of the carriers known
in the
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art. One example of such a carrier is a liposome. The pharmaceutical
composition of
the invention may also comprise various diluents and adjuvants known per se.
The composition of the invention may be administered by a variety of
administration
modes including intra venous, intra muscular and subcutaneous administration.
The pharmaceutical composition of the invention may be administered in
combination
with other anti-viral agents. Such agents may include, as a non-limiting
example:
interferons, anti hepatitis B monoclonal antibodies, anti hepatitis B
polyclonal
antibodies, nucleoside analogues, inhibitors of DNA polymerase and therapeutic
vaccines. In 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 composition of the invention may also be used, for example
as a
prophylactic treatment of neonates born to HBV infected mothers or of
healthcare
workers exposed to the virus or of liver transplant recipients to eliminate
possible
recurrent HBV infection of the transplanted liver.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: HBsAg and HBV-DNA serum levels of two patients infused with a single
dose
the HBV-AbxT~ mixture. The HBV-AbxTL mixture was administered at time point 0.
The
time range is not to scale. A: patient no. 303, dose 0.26 mg, Ab:Ag molar
ratio = 1:14; B:
patient no. 310, dose 39 mg, Ab:Ag molar ratio = 1:2.
HBV-DNA ~ HBsAg _ _ _~ _ _ _ .
Figure 2: HBsAg and HBV-DNA serum levels in four patients administered with
multiple
infusions of the HBV-AbxTr. mixture. The HBV-AbxTL mixture was administered at
time
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points (days) 0, 8,15 and 22; arrows indicate administration time. A: patient
no. 303, dose 4
x 10 mg; B: patient no. 308, dose 4 x 20 mg; C: patient no. 105, dose 4 x 40
mg; D: patient
no. 301, dose 4 x 80 mg.
HBV-DNA T HBsAg _ _ _~ _ _ _ .
Figure 3: HBsAg and anti-HBsAg antibody serum levels in four patients
administered with
multiple infusions of the HBV-AbxTL mixture. The HBV-AbxTL mixture was
administered at
time points (days) 0, 8,15 and 22; arrows indicate administration time. A:
patient no. 303,
dose 4 x 10 mg; B: patient no. 308, dose 4 x 20 mg; C: patient no. 105, dose 4
x 40 mg; D:
patient no. 301, dose 4 x 80 mg.
HBsAg ,~ anti-HBsAg Ab --
Reference will now be made to the following Examples that are provided by way
of
illustration and are not intended to be limiting to the present invention.
EXAMPLES
Materials and Methods
Virological and immunological assays
Serum HBsAg levels. HBsAg levels were determined by a modified automated
immunoassay (IMX system, Abbott GmbH Diagnostika) using a purified HBsAg
preparation (Bio-Hep-B, Biotechnology General, Ness-Ziona, Israel) as
standard.
Serum anti-HBs levels. Anti-HBs levels were determined by AUSAB RIA and
compared to a WHO reference for anti-HBs. A reference serum for anti-HBs was
obtained from CLB, Red Cross Blood Transfusion Service, the Netherlands.
Serum HBV DNA levels. HBV-DNA levels in patients' serum were analyzed by
HBV-DNA PCR using the Amplicor HBV Monitor''" Test (Hoffman-La Roche Inc.,
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Roche Diagnostics, Branchburg, N.J., USA) according to the manufacturers'
instructions.
Preparation of HBV-AbxTL
Each dose of HBV-AbxTL is prepared by diluting the two antibodies 19.79.5 and
17.1.41 in 250 ml normal saline solution in an approximate ratio of 1:3
between the
antibodies respectively (i.e. for each mg of antibody 19.79.5 approximately 3
mg of
antibody 17.1.41 are added).
Example 1
HBV-AbxTL was first tested in a dose escalation (single-dose) phase IA study
in
patients with otherwise untreated chronic Hepatitis B infection (Galun et al.,
2000
Hepatology 32 (4 Pt.2): p221A). A total of 15 patients were enrolled in the
study and
each received a single dose of HBV-AbxTL. The doses ranging between 0.26 to 40
1 S mg. The dosing levels, were based on the molar ratio of antibody to
antigen (Ab:Ag)
(Table 1). HBV-AbxTL was administered as intravenous infusions over 2-8 hours.
Table 1: Pre-treatment clinical characterization of patients in phase 1 A
Dose Ab:Ag ALT HBsAg HBV-DNA
Patient Cohort
(mg) Molar ratio(U/L) (~g/ml) (copies/ml)
301 I 0.26 1:700 106 5.5 1.7 x 10'
302 I 0.26 1:600 10 3.7 3.5 x 10'
304 I 0.26 1:800 59 6.2 7.1 x 10
303 II 0.26 1:14 15 0.1 2.1 x 10'
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305 II 4.7 1:450 54 85 3.2 x 10
101 II 0.32 1:400 134 4.1 3.0 x 10'
306 III 8.9 1:70 61 18.2 1.8 x 10'
307 III 1.5 1:90 75 2.9 1.8 x 10'
102 III 0.26 1:30 27 0.2 7.0 x 10~
308 IV 30 1:30 19 29.7 6.5 x 10'
309 IV 0.47 1:20 186 0.4 5.6 x 106
103 IV 3.7 1:10 79 1.4 1.2 x 10'
310 V 39 1:2 46 2.8 8.5x10"
201 V 40 1:2 60 1.9 6.3 x 10'
311 V 40 1:3.5 102 4.8 3.1 x 10'
Reduction in HBsAg and HBV-DNA levels became detectable shortly after infusion
initiation but was only observed in patients receiving antibodies with a high
Ab:Ag
ratio. In the fifth group (Ab:Ag molar ratio of 1:2) HBsAg levels decreased to
undetectable levels and then started to increase 24 hr after initiation of the
infusion,
reaching pre-treatments levels only eight days after the infusion (Figure 1 ).
HBV-
DNA levels also decreased after the initiation of the HBV-AbxTL infusion and
reached
pre-treatment levels one day later. The reduction in HBV-DNA levels was
between
one to three orders of magnitude. The most common adverse event reported was
mild
myalgia observed in six patients (40%).
Example 2
In a subsequent, multiple-dose, dose escalation Phase IB study of patients
with
chronic Hepatitis B infection, 12 patients were enrolled, three patients in
each of 4
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sequential dose cohorts (Table 2). Each patient received 4 weekly infusions of
HBV-
AbxT~ at doses ranging from 10 to 80 mg per infusion. The intravenous
infusions
were given over 2 or 4 hours.
Table 2: Pre-treatment clinical characterization of patients in phase 1 B
Dose ALT HBsAg HBV-DNA
Patient Cohort
(mg) (U/L) (pg/ml) (copies/ml)
303 I 4 x 10 14 0.02 2.0 x 10'
101 I 4x10 123 3.2 4.6x10'
304 I 4x10 69 4.4 4.0x10'
102 II 4 x 20 56 0.2 2.2 x 10'
302 II 4 x 20 49 2.7 4.0 x 10
308 II 4 x 20 94 9.4 7.0 x 10
202 III 4 x 40 19 41.4 4.0 x 108'
105 III 4 x 40 47 1.7 6.0 x 10'
203 III 4 x 40 38 1.5 5.0 x 10"
301 IV 4 x 80 137 4.6 3.0 x 10
311 IV 4 x 80 120 5.2 3.0 x 10'
106 IV 4 x 80 87 0.93 2.0 x 10'
Patients from the first cohort had received 4 weekly infusions of 10 mg each.
In two
out of the three patients, HBsAg levels decreased to undetectable levels
immediately
after administration and returned back almost to the original levels prior to
the next
infusion. A similar pattern was observed following each administration
resulting in a
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trend of progressive decrease in HBsAg levels during repeated administration.
At
24 hours following injection, HBsAg levels were still undetectable in one
patient but
started to increase in the other 2 patients. Similarly, upon infusion HBV-DNA
levels
decreased by 3 logs and a progressive decline was observed with every
administration. These levels remained undetectable for 24 hours after every
infusion
(Figure 2).
The second cohort of patients received four weekly infusions of 20 mg of HBV-
ABxTL each (Figure 2B). A similar pattern of reduction of HBsAg levels to
undetectable limit was also observed in these three patients. HBV-DNA levels
have
also dropped by one to four logs. The third cohort received four weekly
infusions of
40 mg of HBV-ABxTL and the forth cohort received four weekly infusions of 80
mg
of HBV-ABX~~L, each. These administrations showed similar effects on HBsAg and
HBV-DNA dynamics (Figure 2 C, D). In all cases HBV-DNA decreased
significantly, and HBsAg levels were reduced to undetectable levels
immediately
following infusion.
The antibody was well tolerated: there were no serious adverse events and
myalgia
was reported in only one patient (8%). The most common adverse events were
hematuria and mild chest pain, each reported in 3 out of 12 patients (25%).
There was
no evidence for immune complex disease.
We have followed the levels of HBV-AbxTL after four weekly infusions in
patients
from phase 1B. The kinetics of increase and decrease of anti-HB (hepatitis B)
antibody levels have opposite patterns as compared to that HBsAg levels. In
all
patients, after each infusion anti-HB antibody levels increased and reached a
peak,
then returned to pretreatment levels prior to the next administration (Fig.
3). In
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patients who received repeated doses of 40 mg and of 80 mg the decrease in
anti-HB
antibody levels was slightly slower.
Example 3
In the following study HBV-AbXTL is given in combination with lamivudine.
Lamivudine is given in a dose of 100 mg/day (The recommended dose of
lamivudine
for treatment of chronic hepatitis B virus infection) HBV-AbXTL is given
intravenously either as a 10 mg or 40 mg dose.
The preparation of these specific doses is shown in Table 3.
Table 3: Amount of HBV-Ab 17.1.41 and HBV-Ab 19.79.5 in HBV-AbXTL
Total HBV-Ab HBV-Ab
mAb 17 19 .79.5
.1.41
(2 mg/mL) (1.25
mg/mL)
(mg) (IU) mL mg mL mg
IU IU
9,310 3.8 7.6 4,560 1.9 2.38 4,750
40 37,240 15.2 30.4 18,2407.6 9.50 19,000
Patients are treated according to the following dosing regimen:
A. HBV-AbXT~ 10 mg weekly for 4 weeks followed by 10 mg every four weeks
for 48 weeks plus lamivudine 100 mg once daily for 64 weeks.
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B. HBV-AbXT~ 40 mg weekly for 4 weeks followed by 10 mg every four weeks
for 48 weeks plus lamivudine 100 mg once daily for 64 weeks.
C. HBV-AbXTL 40 mg weekly for 4 weeks followed by 40 mg every four weeks
for 48 weeks plus lamivudine 100 mg once daily for 64 weeks.
D. HBV-AbXTL 40 mg three times weekly for 2 weeks, followed by 40 mg once a
week for two weeks followed by 10 mg every four weeks for 48 weeks plus
lamivudine 100 mg once daily for 64 weeks.
E. HBV-AbXTL 40 mg three times weekly for 2 weeks, followed by 40 mg once a
week for two weeks followed by 40 mg every four weeks for 48 weeks plus
lamivudine 100 mg once daily for 64 weeks.
