Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-09-21
2001-07-17
Bansal, Geetha P. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S069600, C424S133100, C424S134100, C530S387300
Reexamination Certificate
active
06262238
ABSTRACT:
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:
impeded refolding
denaturation: (I) degradation and (II) immunogenicity even at 37° C. in vivo
impaired avidity
aggregation and loss of activity on storage
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
3
-10
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
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
k
protein REI described by Frisch is not a V
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
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
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 specifically stabilized, destabilized or can be restabilized after destabilizing measures such as for example the removal of disulfide bridges.
The object of the invention is a process for the production of a functional antibody, functional derivative or fragment thereof with an improved stability in a eukaryotic or prokaryotic organism by transformation with an expression vector which contains a recombinant gene which codes for the said immunoglobulin, derivative or fragment characterized in that
a) the gene of at least one of the variable domains of the immunoglobulin is compared with the consensus tables 1-6 and the table is selected which has the highest homology to this domain,
b) at least one codon of an amino acid is replaced in the gene of this variable domain and namely
aa) in the case that this amino acid is not mentioned at its position in this selected table 1 by a codon for one of the stated amino acids and/or
bb) in the case that this amino acid is mentioned at its position in table 1, by a codon for one of the stated amino acids with a higher frequency,
c) the prokaryotic or eukaryotic organism is transformed with the gene modified in this manner and the antibody, the fragment or derivative with the desired activity is expressed.
If necessary the antibody can be isolated from the organism and optionally purified according to methods familiar to a person skilled in the art.
In a preferred embodiment of the invention the process is carried out in such a manner that
a) at least one codon for an amino acid is replaced in the gene of the variable domain of the heavy chain of humans and namely
aa) in the case that this amino acid is not mentioned at its position in table 1, by a codon for one of the stated amino acids and/or
ab) in the case that this amino acid is mentioned at its position in table 1, by a codon for one of the stated amino acids with a higher frequency,
b) in the gene of the variable domain of the heavy chain of the mouse
ba) in the case that this amino acid is not mentioned at its position in table 2, by a codon for one of the stated amino acids and/or
bb) in the case that this amino acid is mentioned at its position in table 2, by a codon for one of the stated amino acids with a higher frequency,
c) in the gene of the variable domain of the light chain of the kappa type of humans
ca) in the case that this amino acid is not mentioned at its position in table 3, by a codon for one of the stated amino acids and/or
cb) in the case that this amino acid is mentioned at its position in table 3, by a codon for one of the stated amino acids with a higher frequency,
d) in the gene of the variable domain of the light chain of the kappa type of the mouse
da) in the case that this amino acid is not mentioned at its position in table 4, by a codon for one of the stated amino acids and/or
db) in the case that this amino acid is mentioned at its position in table 4, by a codon for one of the stated amino acids with a higher frequency,
e) in the gene of the variable domain of the light chain of the &lgr; type of humans
ea) in the case that this amino acid is not mentioned at its position in table 5, by a codon for one of the stated amino acids and/or
eb) in the case that this amino acid is mentioned at its position in table 5, by a codon for one of the stated amino acids with a higher frequency,
f) in the gene of the variable domain of the light chain of the &lgr; type of the mouse
fa) in the case that this amino acid is not mentioned at its position in table 6, by a codon for one of the stated amino acids and/or
fb) in the case that this amino acid is mentioned at its position in table 6, by a codon for one of the stated amino acids with a higher frequency,
g) and the prokaryotic or eukaryotic organism is transformed and the antibody, the fragment or derivative with the desired activity is expressed.
The process according to the invention is used in such a manner that the antibody which it is intended to stabilize is firstly sequenced an
Steinbacher Stefan
Steipe Boris
Arent Fox Plotkin Kintner Kahn
Bansal Geetha P.
Canella Karen A
Roche Diagnostic GmbH
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