KR20040048935A - Treatment of hepatitis B virus infection with human monoclonal antibodies - Google Patents

Abstract

The pharmaceutical composition according to the present invention is used for treating or preventing hepatitis B virus infection, characterized in that it comprises a 1: 3 mixture of two human anti-HBsAg monoclonal antibodies 19.79.5 and 17.1.4. . The present invention also provides a preferred mode of administration. The pharmaceutical composition may be provided as a single therapy or in combination with other anti-viral agents.

Description

Treatment of hepatitis B virus infection with human monoclonal antibodies}

Despite the introduction of global hepatitis B vaccination across more than 100 countries, hepatitis B virus (HBV) infection is still a serious problem worldwide, causing a death rate of one million people each year (Kane, Lancet 1996; 348). -696). It took several decades to reduce transmission and incidence through vaccination. Meanwhile, relevant HBV infected patients are demanding new anti-viral therapies that are better than currently available. In the United States, approximately 300,000 new acute HBV infections occur each year, 10% of which become carriers of HBV, and 50% of them develop chronic liver disease, increasing the risk of developing HCC. Serag and Mason, N Eng J Med 1999; 340 745-750).

Hepatitis B vaccine is effective in preventing primary infection but has not shown significant effect in already infected patients.

Two currently approved therapies for the treatment of chronic HBV infection are Interferon alpha 2b (IFNα) (Wong et al., Ann Intern Med 1993; 119, 312-323) and lamivudine (Dienstag et al., N Eng J Med 1999 341, 1256-1263). However, due to the relatively low response rate, severe side effects of IFNα and the development of lamivudine resistant strains, only a partial resolution of both treatments is possible (Liaw et al., Hepatology 1999; 30, 567-572).

In order to prevent liver re-infection after liver transplantation, passive immunotherapy using human highly immune immunoglobulin preparations in HBV-immune patients is commonly used. To prevent vertical infection of HBV from infected mothers, the preparation is administered to the newborn by intramuscular route. However, it is not used for the treatment of chronic patients.

The use of plasma-derived polyclonal antibodies is limited. This is because these preparations are diverse in activity, difficult to obtain, and carry the potential risk of transmission of infectious agents.

In contrast, monoclonal antibodies (mAb) can be produced consistently and there is no risk of infection associated with plasma-derived products.

Previous studies using a single human mAb to treat HBV infected patients with liver transplantation have generated escape mutants (McMahon et al., 1992 Hepatology 15 (5) 757-766). The same antibody was administered to patients with chronic hepatitis B who had been previously treated with lamivudine for a period of two weeks, forming a complex with HBsAg that lowered the level in the patient. Three months after treatment, HBsAg levels returned to pretreatment levels (Heijtink et al., 2001 J. Med. Virol. 64 427-434).

In another study, two human monoclonal antibodies have been developed for different epitopes of the hepatitis B virus surface antigen (HBsAg) (PCT / IL97 / 00184 and PCT / IL97 / 00183). A single administration of a mixture of these antibodies to HBV chronic carrier chimpanzees immediately reduced HBsAg levels and returned to initial levels within a few days (Eren et al., 2000 Hepatology 32, 588-596).

The present invention relates to pharmaceutical compositions for the treatment or prevention of hepatitis B virus infection comprising a mixture of two human monoclonal antibodies.

1A and 2B show HBsAg and HBV-DNA serum levels of two patients with a single dose of the HBV-Ab ^XTL mixture. HBV-Ab ^XTL mixture was administered at time zero. The time range was not scaled. FIG. 1A is patient number 303, dose is 0.26 mg, Ab: Ag molar ratio is 1:14, FIG. 1B is patient number 310, dose is 39 mg, and Ab: Ag molar ratio is 1: 2. In these graphs, the division of HBV-DNA and HBsAg was as follows.

2A-2D show the HBsAg and HBV-DNA serum levels of four patients with multiple infusions of HBV-Ab ^XTL mixture. HBV-Ab ^XTL mixtures were administered at time points 0, 8, 15 and 22 (day) and the time of administration was indicated by the arrow. Figure 2a is patient number 303, dosage is 4 x 10 mg, Figure 2b is patient number 308 and dosage is 4 x 20 mg, Figure 2c is patient number 105 and dose 4 x 40 mg, Figure 2d is patient number 301 Dose 4 x 80 mg. In these graphs, the division of HBV-DNA and HBsAg was as follows.

3A-3D show serum levels of HBsAg and anti-HBsAg antibody in four patients with multiple infusions of the HBV-Ab ^XTL mixture. HBV-Ab ^XTL mixtures were administered at time points 0, 8, 15 and 22 (day) and the time of administration was indicated by the arrow. FIG. 3A is patient number 303, dose is 4 × 10 mg, FIG. 3B is patient number 308, dose is 4 × 20 mg, FIG. 3C is patient number 105 and dose 4 x 40 mg, FIG. 3D is patient number 301 Dose 4 x 80 mg. In these graphs, the distinction between HBsAg and anti-HBsAg antibodies was as follows.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present invention.

Summary of the Invention

The pharmaceutical composition according to the present invention comprises a combination of two fully human high-affinity monoclonal antibodies against different epitopes of hepatitis B virus surface antigen (HBsAg). A pharmaceutical composition according to a preferred embodiment of the present invention comprises, as an active ingredient, human monoclonal antibody 19.79.5 or a fragment thereof which retains the antigen binding properties of the antibody and a human monoclonal antibody 17.1.41 or A mixture of the fragments retaining the antigen binding properties of the antibody is included with a pharmaceutically acceptable carrier. Antibody 19.79.5 is secreted by a hybridoma cell line deposited with European No. 96052168 to European Collection of Cell Cultures (ECACC), and antibody 17.1.41 is from a hybridoma cell line deposited with EC No. Are secreted and their sequences are disclosed in PCT / IL97 / 00184 and PCT / IL97 / 00183. Fragments retaining the antigen binding properties of the antibodies are, for example, Fab or F (ab) ₂ fragments obtained by digesting the entire antibody with various enzymes well known and disclosed in the art. Antigenic characteristics of an antibody are determined by testing the binding of the antibody to a specific epitope using standard assays such as RIA, ELISA or FACS analysis.

The invention also relates to various prophylactic and therapeutic uses of antibody mixtures. In this respect, the pharmaceutical composition comprising the antibody mixture comprises a therapeutically effective amount of the antibody or a mixture of fragments thereof capable of binding HBVsAg, alleviating the symptoms of HBV infection or viral particles in an individual. An amount effective to reduce the number of cycles can be used to treat chronic hepatitis B patients by administering to patients with chronic hepatitis B. Means for measuring the alleviation of symptoms of HBV infection include measuring the level of the enzyme alanine aminotransferase (ALT) or measuring serum conversion, so-called depletion of HBeAg, or examining liver biopsies and tissue fibers by methods well known in the art. Including but not limited to measurement of liver function made by determining the level of formation. The number of circulating viral particles can be measured by measuring HBV DNA levels using PCR or by detecting HBsAg levels in the blood.

The pharmaceutical composition according to one embodiment of the present invention is administered at a dosage ranging from 0.26 mg to 80 mg. Preferably 10 mg or 40 mg.

Pharmaceutical compositions according to one preferred embodiment of the invention comprise antibody 19.79.5 to 17.1.41 in a ratio of approximately 1: 3.

In addition to the antibody mixture, the pharmaceutical composition according to the present invention may optionally also comprise a carrier selected from carriers known in the art. One example of such a carrier is liposomes. The pharmaceutical compositions according to the invention may also comprise various adjuvants and diluents known per se.

Compositions according to the invention can be administered in a variety of modes of administration. For example, intravenous, intramuscular and subcutaneous administration can be performed.

The pharmaceutical compositions according to the invention can be administered in combination with other anti-viral agents. Such anti-viral agents include, but are not limited to, interferon, anti hepatitis B monoclonal antibodies, anti hepatitis B polyclonal antibodies, nucleoside analogs, DNA polymerase inhibitors and therapeutic vaccines. . In the case of such combination therapies, the antibodies may be administered simultaneously with the anti-viral agent or sequentially, such as before or after treatment of the anti-viral agent.

The pharmaceutical compositions according to the invention can be used, for example, in the prophylactic treatment of newborns born from HBV infected mothers or healthcare workers exposed to viruses or liver transplant recipients (to prevent recurrence of HBV infection in transplanted liver). .

Materials and methods

Virology and Immunological Assays

Serum HBsAg Levels: HBsAg levels were measured as standard by modified automated immunoassay (IMX system, Abbott GmbH Diagnostika) using purified HBsAg preparations (Bio-Hep-B, Biotechnology General, Ness-Ziona, Israel). .

Serum Anti-HBs Levels: Anti-HBs levels were measured by AUSAB RIA and compared to the WHO reference for anti-HBs. Reference serum of anti-HBs was obtained from Red Cross Blood Transfusion Service, CLB, The Netherlands.

Serum HBV-DNA Levels: HBV-DNA levels in patient serum were determined by manufacturer's instructions by HBV-DNA PCR using Amplicor HBV Monitor ^tm Test (Hoffman-La Roche Inc., Roche Diagnostics, Branchburg, NJ, USA). Analyzed accordingly.

Formulations of HBV-Ab ^XTL

Each batch of HBV-Ab ^XTL is a 250 ml normal saline solution of two antibodies of 19.79.5 and 17.1.41, with a ratio of approximately 1: 3 between each antibody (ie 1 mg of antibody 19.79.5 To about 3 mg of antibody 17.1.41).

Example 1

First, HBV-Ab ^XTL was tested in a gradual increase in dose (single-dose) Phase 1A study for patients with previously untreated chronic hepatitis B virus infection (Galun et al., 2000 Hepatology 32 (4). Pt. 2): p221A). The study was performed on a total of 15 patients, with each patient receiving a single dose of HBV-Ab ^XTL . Dosage ranged from 0.26 to 40 mg. Dosing levels were based on the molar ratio of antibody to antigen (Ab: Ag) (Table 1a and Table 1b). HBV-Ab ^XTL was administered intravenously over 2-8 hours. Tables 1A and 1B below show the pre-treatment clinical characteristics of patients in Phase 1A. The contents of Table 1b are linked to the contents of Table 1a.

A decrease in HBsAg and HBV-DNA levels was detected immediately after initiation of administration, but this was only observed in patients receiving the antibody at high Ab: Ag ratios. In the fifth group (Ab: Ag molar ratio 1: 2), HBsAg levels dropped to undetectable levels and then began to increase 24 hours after initiation of administration, reaching pretreatment levels only 8 days after administration (Figures 1a and 1b). HBV-DNA levels also decreased after initiation of HBV-Ab ^XTL administration and reached pretreatment levels after 1 day. The decrease in HBV-DNA levels was between one and three orders of magnitude. The most common adverse event reported was mild muscle pain observed in six patients (40%).

Example 2

Subsequently, for patients infected with chronic hepatitis B virus, in a multi-dose, dose escalation Phase 1B study, 12 patients were tested and 3 patients in each of the four sequential dose groups (Table 1). 2). Each patient received HBV-Ab ^XTL at a dose of 10-80 mg per four doses per week. Intravenous administration was over 2 or 4 hours. Table 2 below shows the pre-treatment clinical characteristics of phase 1B patients.

The first group of patients received four doses of 10 mg each week. Two of the three patients had their HBsAg levels dropped to undetectable levels immediately after dosing and then returned to nearly original levels prior to the next dosing. Similar patterns were observed after each dose, resulting in a gradual decrease in HBsAg levels during repeated doses. In one patient, HBsAg levels remained undetectable 24 hours after dosing, but in the other two patients began to increase. Similarly, HBV-DNA levels decreased by 3 logs upon administration, with a gradual decrease observed with each administration. This level remained undetectable for 24 hours after each administration (FIGS. 2A-2D).

Patients in the second group received HBV-AB ^XTL four times a week, 20 mg each (FIG. 2A). Similar patterns were observed in the three patients, where HBsAg levels decreased to undetectable limits. HBV-DNA levels dropped to 1-4 logs. The third group received 40 mg of HBV-AB ^XTL four times a week, and the fourth group received 80 mg of HBV-AB ^XTL four times a week. This administration showed a similar effect on HBsAg and HBV-DNA kinetics (Figures 2c and 2d). In all cases, HBV-DNA was markedly reduced and HBsAg levels decreased to undetectable levels immediately after administration.

The antibody was well tolerated: there were no serious side effects and myalgia was reported in only one patient (8%). The most common adverse events were hematuria and mild chest pain, reported in 3 of 12 patients (25%). There was no evidence of immune complex disease.

HBV-Ab ^XTL levels were followed after 4 weekly administrations to patients in Phase 1B. The kinetics of the increase and decrease of anti-HB (hepatitis B) antibody levels showed an opposite pattern when compared to HBsAg levels. In each patient, the anti-HB antibody level increased after each dose to reach a peak and then returned to the pre-treatment level before the next dose (FIGS. 3A-3D). In patients who received doses of 40 mg and 80 mg repeatedly, anti-HB antibody levels decreased slightly slower.

Example 3

In the following study, HBV-Ab ^XTL was administered in combination with lamivudine. Lamivudine was administered at a dose of 100 mg / day (dose of lamivudine recommended for the treatment of chronic hepatitis B virus infection). HBV-Ab ^XTL was administered intravenously at a 10 mg or 40 mg dose.

The formulation of this particular single dose is shown in Table 3 below. Table 3 below shows the amounts of HBV-Ab 17.1.41 and HBV-Ab 19.79.5 in the HBV-Ab ^XTL .

Patients were treated according to the following dosing schedule:

A. HBV-Ab ^XTL was administered 10 mg weekly for 4 weeks, followed by 10 mg every 4 weeks for 48 weeks, with lamivudine 100 mg once daily for 64 weeks.

B. HBV-Ab ^XTL was administered 40 mg weekly for 4 weeks, followed by 10 mg every 4 weeks for 48 weeks, with lamivudine 100 mg once daily for 64 weeks.

C. HBV-Ab ^XTL was administered 40 mg weekly for 4 weeks followed by 40 mg every 4 weeks for 48 weeks, with lamivudine 100 mg once daily for 64 weeks.

D. 40 mg HBV-Ab ^XTL three times a week for two weeks, then 40 mg once a week for two weeks, then 10 mg every four weeks for 48 weeks, with lamivudine for 64 weeks 100 mg once daily.

E. HBV-Ab ^XTL was administered 40 mg three times a week for two weeks, then 40 mg once a week for two weeks, followed by 40 mg every four weeks for 48 weeks, with lamivudine 1 for 64 weeks. 100 mg once daily.

Claims (23)

As the active ingredient, Human monoclonal antibody 19.79.5 or a fragment thereof that retains the antigen binding properties of the antibody; And Human monoclonal antibody 17.1.41 or a fragment thereof that retains the antigen binding properties of the antibody, A pharmaceutical composition comprising with a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 1, wherein the concentration of the antibodies is 0.26 to 80 mg. The pharmaceutical composition of claim 1, wherein the concentration of antibodies is 10 mg. The pharmaceutical composition of claim 1, wherein the concentration of antibodies is 40 mg. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration ratio (mg) of the human monoclonal antibody 19.79.5 and the human monoclonal antibody 17.1.41. Is about 1: 3. Composition. The pharmaceutical composition of claim 3 comprising 2.38 mg of human monoclonal antibody 19.79.5 and 7.6 mg of human monoclonal antibody 17.1.41. The pharmaceutical composition of claim 4 comprising 9.5 mg human monoclonal antibody 19.79.5 and 30.5 mg human monoclonal antibody 17.1.41. A pharmaceutical composition according to any one of claims 1 to 7 for the treatment of hepatitis B virus (HBV) infection. A pharmaceutical composition according to any one of claims 1 to 7 for the prevention of hepatitis B virus (HBV) infection. Use of the pharmaceutical composition according to any one of claims 1 to 7 in combination with an anti-viral agent for the treatment or prevention of HBV infection. The group of claim 10, wherein the anti-viral agent consists of interferon, anti hepatitis B monoclonal antibody, anti hepatitis B polyclonal antibody, nucleoside analogue, inhibitor of DNA polymerase and therapeutic vaccine And selected from. Use according to claim 10, wherein the anti-viral agent is lamivudine. A method of treating HBV infection comprising administering the pharmaceutical composition of claim 1 to a subject in need thereof. A method of preventing HBV infection comprising administering to a subject a pharmaceutical composition according to any one of claims 1 to 7 in order to prevent further infection of a patient treated with HBV. A method of treating HBV infection comprising administering a pharmaceutical composition according to any one of claims 1 to 7 in combination with an anti-viral agent to a subject in need thereof. The group of claim 15, wherein the anti-viral agent consists of interferon, anti hepatitis B monoclonal antibody, anti hepatitis B polyclonal antibody, nucleoside analogue, inhibitor of DNA polymerase and therapeutic vaccine HBV infection treatment method characterized in that it is selected from. The method of claim 16, wherein the anti-viral agent is lamivudine. 18. The method of any one of claims 15 to 17, wherein the pharmaceutical composition is administered once or three times for four weeks, followed by 48 weeks for four weeks, in combination with a therapeutically effective amount of an anti-viral agent. HBV infection treatment method characterized in that administered once a week. 19. The method of claim 18, wherein said anti-viral agent is lamivudine. 20. The method of claim 19, wherein lamivudine is administered in a 100 mg dose once daily. 21. The method of any one of claims 13-20, wherein the pharmaceutical composition is administered by subcutaneous injection. 21. The method of any one of claims 13-20, wherein the pharmaceutical composition is administered by intramuscular injection. 21. The method of any one of claims 13-20, wherein the pharmaceutical composition is administered by intravenous injection.