Lectin Type Fold as a Scaffold for Massive Sequence Variation
Simplified scaffold affords the creation of a population of binding proteins with highly diverse binding specificities and affinities through the modification of a single polypeptide chain. Optimized profile of variable residues within the CLec-fold scaffold, which are manipulable without influence on tertiary structure. Variable residues provide ~1020 possible combinations of amino-acid side chains in the binding site, allowing the generation of high affinity binding reagents for a variety of molecules, including nucleic acids, polysaccharides, lipids, small molecules, or a combination thereof. Can be readily displayed using phage display or other common methodologies to screen populations of scaffolds for structures that interact with a particular ligand. These scaffolds are soluble in E. coli and can be produced quickly and at far less expense than immunoglobulins. The structure of these scaffolds is tolerant to a reducing environment (relative to immunoglobulins, CTL4-like sandwich domains, V-like domains, and tendamistatin-based structures). The binding site is distant from both the N- and C-termini, allowing for the structure of these scaffolds to remain intact when attached to an immobilization surface (such as a bead) or otherwise modified at one end (such as pegylation). Selected scaffolds can be readily modified with a moiety for detection including a detectable label, a toxin, or an activatable pro-drug.
This invention could be used to target a variety of ligands; including, but not limited to, a viral antigen, a bacterial antigen, a fungal antigen, an enzyme, an enzyme inhibitor, a cell surface molecule of any composition, a reporter molecule, a serum protein, or a receptor. Library of scaffolds or binding proteins can be expressed as a phage display, ribosome display, polysome display, cell surface display, array, or microarray format, and scaffolds with your specific affinity of interest can be produced: A labeled scaffold can be used to identify its high affinity binding partner in samples from, cells, serum, tissue, body fluid, etc; using microscopy, flow, or a variety of other techniques available in the existing art. A labeled scaffold can be generated for the detection of a diagnostic molecule in serum, tissue, body fluid, or another assayable material (such as the detection of hCG in urine samples as an indicator of pregnancy). A labeled scaffold could be used to detect pathogenic microorganisms, or a diseased or infected cell. A scaffold selected to interact with a marker for a diseased or infected cell could be used for the targeted delivery of therapeutics. Selected scaffold molecules could be attached to a toxin or an apoptosis signal to target a cell surface marker such as a marker of a cancer cell. Scaffolds can be selected which bind antibodies from a particular species of animal for use as a secondary reagent in assays using antibodies as the primary detection agent. The composition and methods of this invention could be used to build a kit, consisting of a particular scaffold molecule or molecules, or for the expression and production of a library of these scaffolds.
This invention provides a new class of high affinity binding proteins, based on a modified C-type lectin (CLec)-fold like structure, with a broad range of binding specificities (~1020 unique variations). Variation at select amino acid positions within the variable binding site of the CLec-fold results in a scaffold with a protein binding interface highly amenable to directed manipulation. This technology is based upon the natural ability of the Bordetella bacteriophage to produce extensive amino acid sequence variation in its receptor-binding protein, the major tropism determinant. As these scaffolds are native to bacteriophage, they can be effectively produced in common bacterial strains. The stability of these scaffolds is superior to that of immunoglobulins and other existing antibody alternatives. The variable positions in this invention may be readily modified using standard oligonucleotide based cloning methodologies to produce a library of binding proteins with unique binding specificities and affinities. The library may be screened to identify one or more scaffolds which bind a ligand of interest.
Patent Number: US7749694B2
Application Number: US200427323A
Inventor: Ghosh, Partho | McMahon, Stephen | Miller, Jason | Lawton, Jeffrey
Priority Date: 31 Dec 2004
Priority Number: US7749694B2
Application Date: 31 Dec 2004
Publication Date: 6 Jul 2010
IPC Current: C12Q000100 | A61K003816 | C12Q000104
US Class: 435004 | 435034 | 530350 | 530395
Assignee Applicant: The Regents of the University of California
Title: C-type lectin fold as a scaffold for massive sequence variation
Usefulness: C-type lectin fold as a scaffold for massive sequence variation
Summary: For producing several proteins with different binding specificies; selecting a protein with binding specificity for a molecule of interest (e.g. a cell surface molecule); decreasing the viability of a cancer cell, including mammalian and human cells; for detecting a bacterial cell; and targeting a cell expressing a cell surface molecule (claimed) e.g. for diagnosis of several diseases including cancers, infections and other diseases; and in the in vivo and ex vivo treatment or prevention of diseases.
Novelty: New non-naturally occurring protein useful for producing a protein library containing proteins with different binding specificities comprises variable binding sites and binding specificities, useful for treating cancer
Disease Diagnostic/Treatment
Cancer/Tumor
7749694
State Of Development This technology is offered exclusively or nonexclusively for U.S. and/or certain foreign countries. A commercial sponsor for potential future research is sought. See patent application 20070275367. Related Materials The inventor is Partho Ghosh, UCSD Professor, Chemistry and Biochemistry. Technology-relevant papers are listed below. Tech ID/UC Case 19502/2005-108-0 Related Cases 2005-108-0
USA
