Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-27
2003-05-13
Wortman, Donna C (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023530, C435S320100, C435S173300, C514S04400A
Reexamination Certificate
active
06562599
ABSTRACT:
TECHNICAL FIELD
The present invention relates to single-chain antibodies against hepatitis B virus core protein, genes thereof, and therapeutic agents for hepatitis B using the same. In particular, the present invention relates to single-chain antibodies characterized in that they inhibit DNA synthesis of hepatitis B virus by binding to the core protein of said virus, DNAs encoding said single-chain antibodies, vectors comprising said DNAs, transformants transformed with said vectors, a process for producing said single-chain antibodies, and therapeutic agents for hepatitis B using such single-chain antibodies or genes thereof.
BACKGROUND ART
In Japan, hepatitis B virus (hereinafter sometimes abbreviated as HBV) accounts for a large part of the etiology of chronic hepatitis, as well as hepatitis C virus. Although the proportion of the virus carriers tends to decrease as a result of the recent preventive treatment by vaccination, there still exists a substantial number of patients with chronic hepatitis B, and medical treatments for this disease is quite important in terms of prophylaxis of primary hepatic cancer.
As medical treatments for patients with chronic hepatitis B, those treatments that aim to exclude the virus by eliciting the host immunity, such as the interferon therapy, the steroid withdrawal therapy, and the propagermanium therapy, or the antiviral agent therapies (adenine arabinoside monophosphate, hereinafter sometimes abbreviated as ara-AMP), Lamivudine) are now being used. In the interferon therapy, however, a long-term clinical improvement cannot be observed, although a transient decrease in amount of serological markers of viral activities occurs. Likewise, although ara-AMP has been used with some effect, it is hard to say that the results are satisfactory at present. A treatment in which interferon and ara-AMP are combined and alternately administered exhibits neurotoxicity, and therefore has problems in its use. Furthermore, fulminant hepatitis B, which is one of the forms of acute hepatitis B, is a disease causing a high lethality, and there exist no effective therapeutic agents for this disease presently. Thus, although there is a strong need for developing an anti-hepatitis B virus therapy based on a different mechanism from those of conventional methods, no useful therapies have not yet been developed.
As one of antiviral therapies, there is a method using antibodies specific for viral antigens. Although a method that intracellularly expresses such an antibody may possibly inhibit only the target antigen without damaging the host cells by virtue of the high specificity of the antibody, when IgGs are administered from outside of the cells, they can not inhibit the functions of the viral antigens existing within the cells due to the low efficiency of uptake into the cells. Furthermore, when genes for the H and L strands of an anti-viral antigen-IgG are introduced into cells that are not natural antibody forming cells, the H and L strands expressed from the transgenes will not necessarily form disulfide bonds as efficient as in antibody producing cells to produce IgG in its active form.
On the other hand, a protein produced by expressing a contiguous sequence of cDNAs, in which one cDNA corresponding to the variable region derived from the L strand of an antibody (hereinafter sometimes abbreviated as V
L
) and another cDNA corresponding to the variable region derived from the H strand of the antibody (hereinafter sometimes abbreviated as V
H
) are connected together through an appropriate oligonucleotide linker encoding a highly flexible peptide sequence, can specifically bind to the antigen while taking a structure containing no disulfide bonds. Such proteins are called “single-chain antibodies”, and characterized in that they do not require to be expressed in antibody producing cells in order to form their proper three-dimensional structures as in the above-mentioned IgG, and even when expressed in cells other than antibody producing cells, they have activity of specifically binding to the antigen (McCafferty J. et al.,
Nature
(London), 348, 552-554 (1990); and Marks J. D. et al.,
J. Mol. Biol
. 222, 581-597 (1991)).
As examples of researches on single-chain antibodies aiming at their application to diseases, for example, cancer, those studies may be mentioned in which (a) cDNAs for a single-chain antibody and for a physiologically active substance such as a toxin are ligated together and expressed with the expectation that they will attack and thereby reduce the target cells, or in which (b) the specific binding of the single-chain antibody to the antigen is expected in itself to produce a desired effect.
As specific examples of the above (a), studies have been reported on conjugates in which TGF-&agr; or a toxin such as the diphtheria toxin is coupled to an anti-EGFR single-chain antibody (Schmidt M. and Wels W.,
Br. J. Cancer
, 74, 853-862 (1996)), anti-erbB-2 single-chain antibody (Wels W. et al.,
Cancer Res
., 52, 6310-6317 (1992); and Schmidt M. and Wels W.,
Br. J. Cancer
, 74,853-862 (1996)), and anti-CD 40 single-chain antibody (Francesco J A. et al.,
Cancer Res
., 55, 3099-3104 (1995)), or on conjugates in which a neurotoxin derived from eosinophil is coupled to an anti-transferrin receptor single-chain antibody (Newton D L. et al.,
J. Biol. Chem
., 269, 26739-26745 (1994)). In these studies, conjugates between a toxin and a single-chain antibody were prepared and the single-chain antibodies were allowed to specifically bind, for example, to target cells with the expectation that only the target cells bound by the single-chain antibodies will be necrotized by the toxin.
As specific examples of the above (b), studies have been reported on single-chain antibodies against erbB-2 (Grim J. et al.,
Am. J. Respir. Cell. Mol. Biol
., 15, 348-354 (1996); Jannot C. B. et al.,
Oncogene
, 13, 275-282 (1996), EGFR (Jannot C B. et al.,
Oncogene
, 13, 275-282 (1996)), and Ras (Werge T M. et al.,
FEBS Lett
., 351, 393-396 (1994)). Furthermore, MMLV engineered so that it expresses an anti-MHC single-chain antibody on its surface has been used in an application for directly delivering it to MHC.
Examples of reported studies on single-chain antibodies against viruses aiming at their application to diseases are those studies against HIV (Wu Y. et al.,
J. Virol
., 70, 3290-3297 (1996); Levy-Mintz P.,
J. Virol
., 70, 8821-8832 (1996); Mhashilkar A M. et al.,
EMBO J
., 14, 1542-1551 (1995)), Foot-and-mouth disease virus (Mason P. et al.,
Virology
, 224, 548-554 (1996)), and Tick-borne fravivirus (Jiang W.,
J. Virol
., 69, 1044-1049 (1995)). In these studies, the targets of single-chain antibodies against HIV are nonstructural proteins such as Rev (Wu Y. et al.,
J. Virol
., 70, 3290-3297 (1996)) and Tat (Mhashilkar A M. et al.,
EMBO J
., 14, 1542-1551 (1995)). Since Rev protein binds to HIV-1 mRNA containing RRE expressed in HIV-1 infected cells and promotes expression of gag, pol, and env gene, binding of a single-chain antibody to this protein can inactivate Rev, and thereby suppress propagation of the virus by reduced expression of gag, pol, and env gene.
Tat protein has a transactivator activity, and specific binding of a single-chain antibody to this protein inhibits this activity and thereby suppresses propagation of HIV. In the case of the single-chain antibody against Tick-borne fravivirus, of which target is a viral surface antigen, it has a neutralizing activity and its binding to the structural protein on the viral surface decreases the viral infection and the synthesis of viral proteins.
However, all the antigens to which these single-chain antibodies are targeted are nonstructural proteins or structural proteins on the viral surface. A drawback of viral surface antigens is that the target antigens are prone to undergo mutation due to strong immuno-pressure exerted thereon, and the specific binding of single-chain antibody is thereby disturbed. For example, there exists a region prone to undergo mutation, called hypervariable region, an
Hayashi Norio
Tohdoh Naoki
Yamamoto Hiroko
Yamamoto Masato
Birch & Stewart Kolasch & Birch, LLP
Sumitomo Pharmaceuticals Company Limited
Wortman Donna C
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