Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-01-14
1998-12-29
Feisee, Lila
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4241331, 4241341, 5303873, A61K 39395
Patent
active
058540270
DESCRIPTION:
BRIEF SUMMARY
This application is a 35 U.S.C. 371 national stage filing of PTC/EP95/02626, filed Jul. 6, 1995.
The invention concerns a process for modifying the stability of antibodies (AB) and their use especially in diagnostics and therapeutics.
Antibody biotechnology is a rapidly expanding field with focus on diagnostics (in vitro: e.g. antigen detection, in vivo: e.g. imaging) in therapy (in this case particularly humanized antibodies with increased serum half-life and reduced immunogenicity) and in toxicology (e.g. anti-digoxin antibodies as a specific antidote for a cardiac glycoside overdose). Further areas of application are under development for the induction of transplant tolerance (e.g. by anti-CD4 AB), for immunotherapy (e.g. CAMPATH in non-Hodgkin lymphoma) and for catalytic antibodies which in particular enable stereoselective and regioselective catalysis.
Natural antibody sequences are not optimized for stability, genetically engineered sequence hybrids (e.g. humanized antibodies or single-chain Fv fragments) are frequently considerably destabilized. The consequences can for example be: in vivo
In order to stabilize antibodies in solutions it is for example known that proteins from the DNAJ protein family (EP-A 0 556 726) or from the HSP90 protein family (EP-A 0 551 916) can be added. By contrast no process is known up to now by which antibodies can be stabilized by specific mutations of the amino acid sequence. It is indeed theoretically possible to introduce numerous point mutations in antibodies and to screen these mutants for stability. However, in the case of other proteins it has turned out that only one in 10.sup.3 -10.sup.4 mutants has an improved stability. Such screening methods are thus very tedious and in addition are limited to proteins which have identifiable functions such as enzymatic activity (Rollence, 1988; Chen, 1989; Turner, 1992; Risse, 1992; Arase, 1993).
The genes of the variable domains of immunoglobulins have undergone diverse changes due to multiple gene duplications and mutations during their development. They are optimized for the ability of antibodies to bind selectively and with high affinity (Tonegawa, 1983; Berek, 1988; French, 1989). In this process the sequences which code for the domains are randomly mutated and those B cells are selected and propagated which exhibit improved antigen binding (Berek, 1993). Although the optimization of the antigen binding ability plays a dominant role, the quality of an antibody depends on the sum total of numerous factors such as antigen affinity, domain stability, interaction between the heavy and light chain, variable and constant domains, protease sensitivity and the ability to export and secrete the antibodies from the cell. Accordingly natural antibodies are not necessarily optimized for stability.
It is known from Frisch (1994) that a human V.sub.k protein is destabilized after a substitution of cysteine 23 which prevents the formation of the cysteine 23/cysteine 88 disulfide bridge. This destabilization can be partially reversed again by a substitution of tryptophan 32 for histidine. However, this is only a chance result which moreover is not consistent with the teaching of the invention.
The reason for this is that the V.sub.k protein REI described by Frisch is not a V.sub.k domain fragment of a naturally occurring antibody but rather a protein which is overexpressed as such in a myeloma cell line. REI is a protein whose composition differs substantially from V.sub.k domains that are fragments of naturally occurring antibodies. REI has for example unusual amino acids at positions 50 (E) and 93 (Q). Due to the spatial arrangement of the amino acids it is presumably possible for a salt bridge to form between E 50 and H 32 and a hydrogen bridge to form between Q 92 and H 32. Such a hydrogen bridge bond which does not occur in natural antibodies then stabilize this V.sub.k protein.
The object of the invention is to provide a process which enables the stability of antibodies to be modified in such a way that these antibodies are spe
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Steinbacher Stefan
Steipe Boris
Boehringer Mannheim GmbH
Feisee Lila
Reeves Julie E.
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