Bivalent binding molecules of 7 transmembrane G...

Details Bivalent binding molecules of 7 transmembrane G... Bivalent binding molecules of 7 transmembrane G...

1998-07-17

2004-01-27

Wessendorf, T. D. (Department: 1639)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S025400

Reexamination Certificate

active

06682886

ABSTRACT:

FIELD OF THE INVENTION
Described herein are bivalent binding molecules that can activate or inhibit 7 transmembrane G protein-coupled receptors. Also described herein are methods for identifying and preparing bivalent binding molecules to 7 transmembrane G protein-coupled receptors. The methods disclosed herein are based on the SELEX method for generating high affinity nucleic acid ligands, also termed aptamers. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The bivalent binding molecules of this invention comprise two or more binding domains to two or more different epitopes of the same 7 transmembrane G protein-coupled receptor. In a preferred embodiment, at least one binding domain is an aptamer. These bivalent binding molecules are useful as therapeutic and diagnostic agents.
BACKGROUND OF THE INVENTION
The seven transmembrane receptors (also known as G protein-coupled receptors or 7TM G protein-coupled receptors) comprise a superfamily of structurally related integral proteins. 7TM G protein-coupled receptors exhibit detectable amino acid sequence similarity and all appear to share a number of structural features (See, FIG.
1
). These features include: an extracellular amino terminus (EAT); seven predominantly hydrophobic alpha-helical domains (of about 20-30 amino acids) which are believed to span the cell membranes and are referred to as transmembrane domains (TMD 1-7); six loops which connect the transmembrane domains (three extracellular loops (ELs) and three intracellular loops (ILs)); and a cytoplasmic carboxy terminus (CCT).
Each 7TM G protein-coupled receptor is predicted to associate with a particular heterotrimeric G protein (composed of &agr;, &bgr; and &ggr; subunits) at the intracellular surface of the plasma membrane. Upon binding of an agonist to the receptor, a conformational change occurs in the receptor, which enables interaction of the intracellular loops of the receptor with its associated intracellular, membrane-anchored heterotrimeric G protein. This causes the alpha-subunit of the G protein to exchange a bound GDP molecule for a GTP molecule and to dissociate from the &bgr; and &ggr; subunits. The GTP-bound form of the alpha-subunit in turn stimulates specific intracellular signal-transducing enzymes and channels.
It has been proposed that 7TM G protein-coupled receptors adopt two major conformations: an active, G protein-coupled and thus transducing conformation and an inactive (non-transducing) conformation (Schwartz, T. W. et al.,
Cur. Pharmaceut. Design
, 1:325-342 (1995)). The binding of an agonist or antagonist selectively stabilizes the active and the inactive receptor conformations, respectively, as predicted by the allosteric regulation of proteins as suggested by Monod, Wymann and Changeux (
J. Mol. Biol
., 2:7439-7442 (1965)). Agonists are thus extracellularly acting allosteric ligands that increase the signal transduction rate at intracellular sites upon binding. Antagonists are extracellularly acting ligands that inhibit signal transduction upon binding.
The 7TM G protein-coupled receptors are the largest family of cell-surface receptors comprising several hundred distinct receptors, and over 100 receptors have been cloned. The transmembrane segments of 7TM G protein-coupled receptor family members exhibit considerable homology, whereas the extracellular connecting loops are less conserved, showing high homology only between closely related receptor subtypes. The 7TM G protein-coupled receptors can be grouped based on their homology levels and/or the nature of the ligands they recognize. For example, the interleukin-8 receptor, the angiotensin II receptor, the thrombin receptor, the endothelin receptors, the N-formyl peptide receptor and the C5a receptor all bind peptide ligands and share 20-40% amino acid similarity.
The 7TM G protein-coupled receptors bind a wide variety of ligands of different molecular size ranging from small monoamines and other small molecules, to large neurotransmitters and peptide hormones. The family of 7TM G protein-coupled receptors also includes the receptors for light (rhodopsin), for odors (olfactory receptors) and for taste (gustatory receptors). Additionally, the conserved structure among 7TM G protein-coupled receptors has allowed for the cloning of many novel genes encoding 7TM G protein-coupled receptors whose natural ligand and function are yet to be elucidated. These receptors are referred to as“orphan” receptors. Table 1 lists a number of 7TM G protein-coupled receptors which have been cloned and expressed.
Because of the involvement of 7TM G-protein-coupled receptors in the regulation of many critically important biological functions and disease conditions, many of these functions and conditions may be influenced or determined by the state of activation or inhibition (e.g., blockade) of a 7TM G protein-coupled receptor. However, these receptors are difficult to purify. The proteins can be removed from the membrane only by the action of detergents, which denatures some proteins. In addition, most membrane proteins are not soluble in water. To date, few novel agonists or antagonists to these receptors have been identified. Common methods have involved generating antibodies to 7TM G protein-coupled receptors expressed in cells which have been administered to a host. Lerner et al. (PCT Application No. WO 98/03632) have described peptide dimer agonists for 7TM G protein-coupled receptors. These dimers were comprised of two known peptide agonists or antagonists (e.g., natural ligands) to different 7TM G protein-coupled receptors.
It would be useful to be able to develop agonists and antagonists to the specific binding portions of 7TM G protein-coupled receptors. Attempts to achieve expression of only the ligand binding portion of a 7TM G protein-coupled receptor have been unreproducible or have resulted in inefficient and/or unpredictable levels of expression (Xie, U. B., et al,
J. Biol. Chem
. 265:21441-21420 (1990); Tsai-Morris, C. H., et al.
J. Biol. Chem
. 265:19385-19388 (1990)).
As suggested in Lemer et al., bivalent binding molecules can have utility as therapeutics. More specifically, bivalent and bispecific antibodies have many practical applications, including in immunodiagnosis and therapy. Bivalency can allow antibodies to bind to multimeric antigens with great avidity; multivalency theoretically can increase apparent binding affinity by several orders of magnitude (Crothers, D. M. et al.,
Immunochemistry
9: 341-351 (1972)). Bispecificity can allow the cross-linking of two antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell. Specific examples of bivalent molecules capable of binding to adjacent epitopes include small bivalent antibodies composed of either antibody fragments (F
ab
) or single chain antibodies (F
v
) (Pack, P. et al.,
Biochemistry
31, 1579-1584 (1992); Holliger, P. et al., Proc. Natl. Acad. Sci. USA 9, 6444-6448 (1993); Mallender, W. D. et al.,
J. Biol. Chem
., 269: 199-206 (1994)). Neri, D. et al. (
J. Mol. Biol
., 246:367-373 (1995)) developed a bispecific antibody fragment, binding two antibodies with a polypeptide chain, that recognizes adjacent and non-overlapping epitopes of lysozyme and is able to bind both epitopes simultaneously.
Bivalent peptides, such as receptor-adhesive modular proteins (“RAMPs”), have been used in an alternative approach to cell targeting. M. Engel et al., (
Biochemistry
30: 3161-3169 (1991)) and C. A. Slate et al., (
Int. J. Peptide Protein Res
. 45: 290-298 (1995)) have designed large synthetic peptides, which contain two ligand sites separated by a spacer region and a dimerization domain.
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential Enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment

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