Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-03-03
2001-11-13
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C514S014800, C514S015800, C530S300000
Reexamination Certificate
active
06316409
ABSTRACT:
INTRODUCTION
The present invention relates to targeting, detection, immobilization, and purification of molecules using binding pairs. In particular, it relates to the use of calcium dependent binding proteins and ligands thereof. The present invention provides ligands that possess improved affinity for calcium-dependent binding proteins. The binding pairs of the present invention provide an attractive alternative to currently available binding pair systems utilised in biological procedures.
BACKGROUND TO THE INVENTION
The term “binding pair” refers to two molecules that bind each other with high affinity. Many binding pair systems presently exist for the purification and targeting of tagged compounds. The most widely-used system at present is the biotin-streptavidin binding pair that has also been applied to in vivo tumour targeting methods (Paganelli et al., 1991). In addition, recombinant fusion proteins expressing the myc or Flag tags have been isolated and purified using antibodies directed against these tags (Munro and Pelham, 1986; Hopp et al., 1988). Poly-his peptide tails have been genetically fused to recombinant proteins and purified on nickel-coated agarose or using affinity columns (Skerra et al., 1991). The streptavidin-binding strep tag (Schmidt and Skerra, 1993) has been used in similar applications.
However, the application of each of the above examples is restricted. Techniques such as affinity purification require a specific yet low affinity interaction so as not to impair the function of the recombinant protein with a harsh elution protocol. Conversely, targeting and specific immobilization both require high affinity binding of the tag with a long half-life of interaction of the binding pair. The systems described in the prior art that employ binding pairs do not possess this flexibility, and thus are not universally applicable.
Calmodulin is a calcium dependent binding protein that regulates a wide range of enzymes, and plays a key role in intracellular signal transduction. It is also one of the few examples of a small protein capable of binding ligands with a high affinity.
Due to their small size, calmodulin-ligand compelexes have been used as convenient models for the study of protein association and dissociation. Since the elucidation of three-dimensional structures of calmodulin in complex with high affinity ligand substrates, this system has been amenable to the study of the mechanisms by which proteins recognise one another in high affinity interactions.
Pending patent application WO95/12672 discloses the use of binding pairs comprising calmodulin and calmodulin ligands for the detection, immobilization, targeting and purification of recombinant polypeptides. This system is attractive since the binding interaction can be modified through changes in calcium concentration that can easily be controlled through the use of calcium chelators. This greatly broadens the applicability of binding pairs as experimental and therapeutic tools.
Stoko-Hahn et al., (1992) have made a fusion of a calmodulin binding ligand tag derived from the C terminus of rabbit skeletal muscle myosin light chain kinase (sMLCK), and a recombinant protein. They have used this in purification strategies on an affinity support. In the presence of high levels of calcium, this system displays a specific high affinity interaction, the dissociation constant for the binding pair being of the order of 3 nM. The addition of EGTA (a calcium chelator) lowers the affinity of the interaction, allowing a very mild elution protocol, and meaning that denaturation or disruption of the native protein structure is unlikely. However, although the affinity of calmodulin towards calmodulin ligands is high, it is not sufficiently high for many targeting, immobilisation and particularly therapeutic applications.
For many therapeutic applications, sub nanomolar or picomolar dissociation constants, high association rate constants and extremely low dissociation rate constants are necessary to ensure efficient targeting of the complex and a sufficiently long half-life of the binding interaction. The biotin-streptavidin binding pair is widely used both experimentally and therapeutically for the targeting, purification and immobilization of proteins. The main attraction of this system lies in the high affinity of interaction (K
d
=10
−15
M) of the binding pair.
However, a significant disadvantage of using this binding pair is the immunogenicity of streptavidin in the human body. This greatly limits the utility of this system for therapeutic applications. There is thus a great need for an experimentally malleable binding pair system that possesses both a high affinity interaction and low immunogenicity.
DESCRIPTION OF THE INVENTION
According to the present invention, there is provided a ligand capable of binding a calcium dependent binding protein comprising an amino acid sequence corresponding to that of a wild type ligand for the calcium dependent binding protein, with a modification which results in enhanced affinity of the ligand for the calcium dependent binding protein.
By wild type ligand is meant a naturally-occurring ligand capable of binding a calcium dependent binding protein. All ligands as defined by the present invention are peptides that comprise a modification of the binding domain of naturally-occurring peptide ligands that interact with calcium dependent binding proteins.
Examples of wild type ligands from which suitable ligands may be derived are skeletal myosin light chain kinase, smooth muscle myosin light chain kinase, mastoparan, melittin, AC-28 and NO-30, all of which exhibit high affinity binding to calmodulin. Preferably, the ligand of the present invention comprises a modified skeletal myosin light chain kinase ligand.
The said modification may consist of a substitution, insertion, or deletion of one or more amino acids in the wild type ligand that results in improved affinity of the ligand for the calcium dependent binding protein. The ligand of the present invention may comprise up to 20, preferably up to 10, more preferably up to 5 modifications to the amino acid sequence of the wild type ligand.
By insertion and deletion is meant the introduction or omission respectively of one or more amino acids, to lengthen or shorten the amino acid sequence of the wild type ligand, and thus modify its affinity for the calcium dependent binding protein. Preferably the modification comprises a substitution of one or more amino acids in the wild type ligand.
A substitution comprises the replacement of a naturally-occurring amino acid in the wild type peptide sequence for a surrogate residue. The preferred surrogate residue is alanine or valine. Both these amino acids are hydrophobic with relatively innocuous side chains, and both are thus ideal for substitution into a helical peptide with minimal perturbation of the peptide structure. Fully worked examples describing the production, identification and manipulation of such molecules are disclosed herein. Preferably the surrogate residue is alanine.
Individual alanine residues have previously been inserted in the sequence of proteins. Clackson and Wells, (1995) recently reported the systematic replacement of contact residues in the sequence of the human growth hormone receptor (hGHbp). This “alanine scanning” technique identified the residues involved in the binding interaction through quantification of the decrease in binding affinities exhibited by these mutant proteins.
Barstar, a physiological inhibitor of barnase (the extracellular RNase in
Bacillus amylolique
-
faciens
), has also been subjected to such experimental manipulation. The very high association rate constant possessed by the barnase-barstar complex (3.8×10
9
s
−1
M
−1
) is due to four acidic sidechain amino acid residues. When these were all individually mutated to alanine, this caused a decrease in the association rate constant. Concomitantly, a slight increase in the stability of barstar was seen, and it has therefore been hypothesized that protei
Neri Dario
Winter Gregory Paul
Darby & Darby
Low Christopher S. F.
Medical Research Council
Tu Stephen
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