Streptavidin mutants having secondary functional domains

Details Streptavidin mutants having secondary functional domains Streptavidin mutants having secondary functional domains

1999-08-25

2002-07-02

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000

Reexamination Certificate

active

06413934

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is directed to streptavidin molecules having secondary functional domains such as a cell adhesion domain.
Streptavidin, a protein produced by
Streptomyces avidinii
, forms a very strong and specific non-covalent complex with the water soluble vitamin biotin. Streptavidin is a tetrameric protein that binds biotin with an affinity that is among the highest displayed for non-covalent interactions between a ligand and protein, with an association constant (Ka) estimated to be in the range of 10
13
M
−1
to 10
15
M
−1
. This binding affinity is strong enough to be essentially irreversible under normal physiological solution conditions, and provides the basis for streptavidin and biotin's usefulness in a wide variety of clinical and industrial applications. See, Green,
Adv. Prot. Chem
. 29:85-143 (1975).
Both streptavidin and the homologous protein avidin, which shares its high affinity for biotin, have been studied as paradigms of strong ligand-protein interactions. The X-ray crystal structures of streptavidin and avidin, both in their apo and holo forms, have been described. The sequences of both have also been reported, as have the construction of several streptavidin fusion proteins (Sano and Cantor,
Biochem. Biophys. Res. Commun
. 176:571-577 (1991); U.S. Pat. No. 4,839,293).
In addition to the extremely high binding affinity, the usefulness of streptavidin also arises from the unique architectural properties of the protein. Streptavidin is a tetramer of four identical subunits, with each subunit contributing a binding site for biotin. Because the tetramer has approximate two-fold symmetry, the binding sites are positioned in pairs on opposite sides of the molecule, making the protein an efficient molecular adaptor. This structural feature, along with the high affinity of streptavidin for biotin, has made the protein an important component in many technologies.
Streptavidin is a key components in four technological areas of great significance: 1) bioseparations/cell sorting; 2) imaging; 3) drug delivery; and 4) diagnostics (Wilchek and Bayer, in
Meths. Enzymol
. 184:5-45 (1990)). In the separations area, this protein has been used extensively in important cell sorting applications, where, for example, it is used to remove contaminating cells from hematopoietic stem cells prior to marrow transplantation. Berenson et al.,
Prog. Clin. Bol. Res
. 377:449-459 (1992). Streptavidin has found similar wide use in cancer diagnostics, where it is used in both research and clinical settings to test for the presence of various tumor specific biomarkers.
The imaging and drug delivery applications of streptavidin and biotin arise from the capability for simultaneous targeting and delivery of imaging agents or therapeutics to tumor cells. There is particularly significant emerging interest in the use of streptavidin for targeted delivery of imaging agents and therapeutics in vivo. Streptavidin has been used to deliver drugs, toxins and imaging agents to targeted cells both in vitro and in vivo. See, for example, Meyer et al.,
Exp. Hematol
. 19:710-713 (1991). In these systems, streptavidin plays the crucial role of molecular adaptor between an antibody that serves as the targeting component, and a biotinylated therapeutic or imaging agent. With some strategies, cells are pre-targeted with the antibody-streptavidin conjugate, with subsequent delivery of the biotinylated agent. In other applications, a biotinylated antibody is first used to pre-target cells, with subsequent delivery of the streptavidin-biotinylated agent conjugate. A three-step delivery is also possible, using biotinylated antibody followed by streptavidin and then the biotinylated agent.
It would be advantageous to have a streptavidin molecule that contains a secondary functional domain so that biotin binding is retained but the streptavidin molecule also has another function. For example, it would be advantageous to have a streptavidin molecule that binds to a cell so that, for example, molecules can be selectively delivered to that cell. It would be advantageous to have streptavidin that selectively binds to a particular cell type or to a particular protein so that selective purification can be performed.
BRIEF SUMMARY OF THE INVENTION
Streptavidin molecules are disclosed that contain a secondary functional domain. In preferred embodiments, the secondary domain is a cell adhesion peptide incorporated in the streptavidin amino acid sequence at a site not interfering with biotin binding. In a preferred embodiment, the cell adhesion peptide is arginine-glycine-aspartate (Arg-Gly-Asp) (RGD). The peptide is preferably placed on an exposed loop of the streptavidin molecule, such as within the loop defined by residues 63 to 69. The mutant streptavidin molecule can have other characteristics such as reduced biotin binding due to a modification of an amino acid at the biotin binding site. Preferred uses for the disclosed streptavidin molecules are as adaptors to bring, via a streptavidin/biotin interaction, the secondary functional domain into proximity with a cell or molecule to be affected and as a coating for substrates such as vascular devices or prostheses.


REFERENCES:
patent: 4458066 (1984-07-01), Caruthers et al.
patent: 4839293 (1989-06-01), Cantor et al.
patent: 5328985 (1994-07-01), Sano et al.
patent: 5443955 (1995-08-01), Cornell et al.
patent: 5492890 (1996-02-01), Ginsberg et al.
patent: WO 96/24606 (1996-08-01), None
McDevitt, et al., “Constrained cell recognition peptides engineered into streptavidin,”Chem. Abs.131:57810 (1999).
Katz & Cass, “In crystals of complexes of Streptavidin with peptide ligands containing the HPQ sequence the pKaof the peptide histidine is less than 3.0,”J Biol Chem272(20):13220-28 (1997).
Rezania & Healy, “Biomimetic peptide surfaces that regulate adhesion, spreading, cytoskeletal organization, mineralization of the matrix deposited by osteoblast-like cells,”Biotechnol Prog15(1):19-32 (1999).
Yamada, et al., “Structure of a conformationally contrained Arg-Gly-Asp sequence inserted into human lysozyme,”J Biol Chem270(11):5687-90 (1995).
Alon, et al., “Cell-adhesive properties of streptavidin are mediated by the exposure of an RGD-like RYD site,”Eur J Cell Biol.58(2):271-9 (1992).
Alon, et al., “Streptavidin blocks immune reactions mediated by fibronectin-VLA-5 recognition through an Arg-Gly-Asp mimicking site,”Eur J Immunol.23(4):893-8 (1993).
Alon, et al., “Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin,”Biochem Biophys Res Commun.170(3):1236-41 (1990).
Argarana et al., “Molecule Cloning and Nucleotide Sequence of the Streptavidin Gene,”Nucl. Acids. Res.14:1871-1882 (1986).
Barbas, et al., “High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site, ”Proc Natl Acad Sci U S A.90(21):10003-7 (1993).
Beaucage, et al., “Deoxynucleoside phosphoramidites-a new class of key intermediates for deoxypolynucleotide synthesis,”Tetra. Lett.22:1859-1862 (1981).
Berenson, et al., “Transplantation of stem cells enriched by immunoadsorption,”Prog Clin Biol Res.377:449-59 (1992).
Brown, et al., “Chemical synthesis and cloning of a tyrosine tRNA gene,”Meth. Enzymol.68:109-151 (1979).
Chilkoti, et al., “Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120,”.Proc Natl Acad Sci USA92:1754-1758 (1995).
D'Souza, et al., “Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif,”Trends Biochem Sci.16(7):246-50 (1991).
Green, “Avidin,”Adv Protein Chem.29:85-133 (1975).
Gross & Meienhofer, The Peptides; Analysis, Synthesis, Biology vol. 2. Special Methods in Peptide Synthesis, Part A, Academic Press:London, 1980.
Hashino, et al., “Engineering of artificial cell adhesion proteins by grafting the Arg-Gly-Asp cell adhesive signal to a calpastatin segment,”J Biochem(Tokyo). 112(4):547-51 (1992).
Maeda, et al., “A novel cell adhesive protein engineered by insertion

Affiliated with

Also associated with

Rating

Streptavidin mutants having secondary functional domains does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Streptavidin mutants having secondary functional domains, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Streptavidin mutants having secondary functional domains will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2895229