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
2000-01-12
2002-12-17
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069500, C435S069520, C424S085100, C424S085200, C536S023400, C536S023500, C536S023510, C530S351000
Reexamination Certificate
active
06495346
ABSTRACT:
BACKGROUND OF THE INVENTION
Complex forming proteins are widespread in nature.
Fundamentally, these proteins can be assigned to the following groups:
antibodies which, with their antigen-binding sites, can bind specifically to the corresponding epitope of an antigen (which can also be another antibody)
protein complexes, such as occur, for example, in the activation of the clofting system or of the complement system
ligand receptor interactions of very different types, for example
of antigens or their epitopes with the Tcell or B-cell receptor
of growth factors, cytokines, chemokines, peptide hormones, steroid hormones and mediators with their respective receptors
of enzymes such as, for example, uPA or tPA with its receptor
of virus proteins with their respective cell receptor
adhesions between adhesion molecules
protein complexes in signal transmission, control of the cell cycle and control of the transcription of genes
Numerous examples of this are summarized in the literature, i.e. by Hardie et al., The Protein Kinase Facts Book I and II, Academic Press 1995, Callard et al., The Cytokine Facts Book, Academic Press 1994, Pigott et al., The Adhesion Molecule Facts Book, Academic Press 1994, Barclay et al., The Leukocyte Antigen Facts Book, Academic Press 1994, Watson et al., G-Protein Linked Receptor Facts Book, Academic Press 1994, Hesketh, The Oncogene Facts Book, Academic Press 1995, Leid et al., Cell 68, 377 (1992), Murre et al., Cell 58, 537 (1989), Bbeneza et al., Cell 61, 49 (1990), Brugge, Curr. Top. Microbiol. Immunolbiol. 123, 1 (1981), Callebaut et al., Proc. Natl. Acad. Sci. USA 89, 6270 (1992), Nadeau et al., J. Biol. Chem. 268, 1479 (1993), Burbach et al., Proc. Natl. Acad. Sci. USA 89, 8185 (1992), Hoffman et al., Science 252, 954 (1991), Stress-induced Proteins, M. L. Pardue, J. R. Feramisco and S. Lindquist Ed, A. R. Liss, New York (1989), Eukaryotic Transcription Factor, D. S. Lachman, Academic Press (1991), Transcriptional Regulations, Eds. S. McKnight and K. R. Yamamoto, CSHL Press, Cold Spring Harbor, New York (1992).
In some cases, the largely specific naturally occurring complexes formed, between at least two proteins, are utilized for the analysis or detection of proteins in the diagnosis of diseases (for this see EP 0 491 362 B1) and the binding partners of such proteins are used for the prophylaxis or therapy of disorders. If one partner of the particular protein complexes is available, with the aid of this partner the amount of the second or further component, be it, for example, complement or clotting factors, antigens, receptors or signal proteins, can be determined. Moreover, specific protein complexes are utilized in the search for small-molecule activators or inhibitors. In addition, purified antibodies or ligands for receptors, such as, for example, cytokines and peptide hormones, are administered to patients or animals for the prophylaxis or therapy of disorders.
In the case of these different application possibilities for proteins which form protein complexes, the specificity with which the respective proteins enter into complex formation and in addition the distribution of the respective proteins in the organism is of crucial importance. The lower the specificity, the more frequently nonspecific binding of a partner to foreign proteins will occur.
For example, nonspecific, i.e. undesired, formation of complexes with foreign protein partners is, as is known, the main problem of analysis and diagnosis using antibodies. Undesired formation of complexes with foreign protein partners can also be the cause of side effects in vivo, for example after injection of antibodies. In addition, the specific exclusive binding between two proteins is an essential prerequisite for the production and specific function of complex transcription factors, in particular of synthetic transcription factor complexes, such as have been described in the Patent Application EP-A 0 805 209.
There is thus a considerable need for novel, highly specific complex-forming proteins which do not react with foreign partners.
SUMMARY OF THE INVENTION
The invention relates to a complex formed from specifically complex-forming proteins which are not naturally occuring, wherein the following components are contained in the complex:
a) at least one ligand specific for a target structure,
b) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived, i.e, obtained, by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically, i.e., bind specifically, with component c) and the component b) being bonded covalently to the component a),
c) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived, i.e., obtained, by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically, i.e., bind specifically, with component b) and the component c) being bonded covalently to the component d), and
d) at least one effector.
According to the invention, a characteristic of the components b) and c) is that in naturally occurring peptides or proteins of identical or different type amino acids are replaced or inserted such that the peptides or proteins mutated in this way can complex virtually only exclusively with one another. A complexation of peptides or proteins mutated in this way with the corresponding nonmutated wild-type proteins or peptides does not occur, however. In this way, homodimers or heterodimers of mutated monomers (mutated peptides or proteins) can be formed.
The mutations in the binding domains of the identical or different proteins therefor have the purpose of preventing the ability for complex formation between an unmutated monomer and a mutated monomer of a pair of identical or different proteins or peptides which would complex in the unmutated state. At the same time, the mutations impart to such a pair of mutated proteins the ability to bind to one another with high specificity. The mutations thus occur in pairs, where one mutation is present in component b), the other in component c), i.e. such that a molecular interaction is possible between the respective amino acids of such a pair.
Amino acids according to the invention inserted into naturally occurring peptides or proteins can be, for example (the listing in pairs is intended to indicate the molecular interaction in the context of a dimeric protein complex):
Component b) or c)
Component c) or b)
3-18 cysteines
3-18 cysteines
3-24 basic amino acids such as
3-24 acidic amino acids such as
histidine,
asparagine,
arginine,
asparic acid,
lysine
glutamine,
glutamic acid
3-24 hydrophobic amino acids such as
3-24 aromatic amino acids
methionine,
phenylalanine,
isoleucine,
tyrosine,
leucine,
tryptophan
valine
3-24 aromatic amino acids
3-24 aromatic amino acids
The starting proteins for components b) and c) can be identical or different here.
In a preferred embodiment, the binding constant of a complex of two proteins according to the invention is at least K
M
=10
−7
mol l
−1
, preferably at least K
M
=10
−8
mol l
−1
.
Within the meaning of this invention, the following identical or unidentical partners (for the production of component b) or c) and with the aim of binding heterodimers), for example, are preferably mutated in their respective binding domain and these or the entire protein are used:
Component b) or c)
Component c) or b)
Fos
Jun or Jun B or Jun D
FRAU-1
Jun or Jun B or Jun D
FRAU-2
Jun or Jun B or Jun D
FOS-B
Jun or Jun B or Jun D
OCT-1
Jun or Jun B or Jun D
NFKB (p65)
IKB
Ras
RAF
CD4
p56LcK
bcl-2
bad or bax
cyclin A
cdk1
E2F
DP
CD40
CD40L
Myc
Max
Myc N
Max
Myc L
Max
p105 (Rb1)
AFF-2, E1A
p107 (Rb2)
E7, E2F or Myc
p130
E7, E2F or Myc
CBL
Gag
TRP
TRP
Met
Met
Myb
p67 or p160
VAV
p67 or p160
APC
&agr;- or &bgr;-Catenin
APC
APC
VD receptor
h-(Retinoid x-receptor)RXR &agr; or &bgr;
T3 receptor
HRXR &agr; or &bgr;
MyoD
E12
Compo
Jerome Valerie
Müller Rolf
Sedlacek Hans-Harald
Aventis Pharma Deutschland GmbH
Chakrabarti Arun
Heller Ehrman White and McAuliffe
Jones W. Gary
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