Phage-display of immunoglobulin heavy chain libraries for identification of inhibitors of intracellular constituents

• US 5,824,520 A
• Filed: 07/19/1997
• Issued: 10/20/1998
• Est. Priority Date: 05/09/1995
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Mulligan-Kehoe, Mary Jo
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/897,040
• Time in Patent Office: 458 Days
• Field of Search: 435/6, 435/172.3, 435/91.4, 435/91.41, 435/320.1, 435/375, 514/44, 935/22, 935/23
• US Class Current: 435/91.41
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

One aspect of the invention relates to a phage-display library that expresses single-chain recombinant binding proteins. Inserts in the library comprise immunoglobulin heavy chain framework regions flanking highly divergent, synthetically produced hypervariable regions. A second aspect of the invention relates to the use of single-chain recombinant binding proteins to inhibit the activity of an intracellular constituent. In the exemplary case presented, the activity of intracellular glucose-6-phosphate dehydrogenase was inhibited by intracellular expression of a cloned single-chain recombinant binding protein.

Claims

1. A method for preparing a eukaryotic expression construct that codes for an intracellular constituent-binding polypeptide, comprising the steps of:

(a) obtaining a phage-display library comprising a plurality of recombinant phage, each of said phage having incorporated therein a polynucleotide coding for a single-chain recombinant polypeptide encompassing a region spanning from upstream of an immunoglobulin heavy chain CDRI to a position downstream of CDRIII, wherein a DNA sequence encoding either CDRI or CDRIII is replaced by a randomly ordered DNA sequence, a fusion protein comprising the polypeptide encoded by said polynucleotide is expressed in the absence of an immunoglobulin light chain protein or portions thereof on an outer surface of recombinant phage of the library;

(b) screening said library to identify a phage clone which binds an intracellular constituent and thereby inhibit a measurable biological activity thereof;

(c) purifying phage DNA from said phage clone;

(d) isolating from said phage DNA a polynucleotide encoding said single-chain recombinant polypeptide, but lacking a secretion leader; and

(e) incorporating said polynucleotide into a eukaryotic expression vector to create said eukaryotic expression construct.

6. The method of claim 1, wherein the phage-display library obtained in step (a) is constructed in an M13-derived cloning vector.

7. The method of claim 1, wherein the intracellular constituent is immobilized to a solid support in step (c).

2. A method of inhibiting activity of an intracellular constituent within a cell in vitro, comprising the steps of:

(a) obtaining a phage-display library comprising a plurality of recombinant phage, each of said recombinant phage having incorporated therein a first polynucleotide coding for a single chain recombinant polypeptide encompassing a region spanning from upstream of an immunoglobulin heavy chain CDRI to a position downstream of CDRIII, wherein a DNA sequence encoding either CDRI or CDRIII is replaced by a randomly ordered DNA sequence, and wherein a fusion protein comprising the polypeptide encoded by said first polynucleotide is expressed in the absence of an immunoglobulin light chain protein or portions thereof on an outer surface of recombinant phage of the library;

(b) screening said library to identify a phage clone expressing a polypeptide which binds the intracellular constituent, said intracellular constituent having a measurable biological activity;

(c) purifying phage DNA from said phage clone;

(d) isolating from said phage DNA a second polynucleotide encoding a region of said polypeptide that includes framework and hypervariable regions but not a secretion leader;

(e) incorporating said second polynucleotide into a eukaryotic expression vector to create a eukaryotic expression construct encoding said polypeptide;

(f) inhibiting an activity of the intracellular constituent within a cell in vitro by introducing said eukaryotic expression construct into said cell and allowing expression of said polypeptide in the absence of an immunoglobulin light chain protein or portions thereof.

3. The method of claim 2, wherein the intracellular constituent has detectable enzyme activity.

4. The method of claim 2, after step (b) fiber comprising the step of measuring a change in biological activity of said intracellular constituent on binding of said polypeptide thereto.

5. The method of claim 3 wherein a reaction product produced in an enzymatic assay is detected optically.

8. The method of claim 2, wherein the DNA purified in step (c) is double-stranded.

9. The method of claim 2, wherein the DNA purified in step (c) is single-stranded.

10. The method of claim 2, wherein step (d) comprises amplification of said second polynucleotide by polymerase chain reaction.

11. The method of claim 2, wherein step (e) includes ligating said polynucleotide into said eukaryotic expression vector.

12. The method of claim 11 wherein the eukaryotic expression vector is selected from the group consisting of a plasmid vector and a viral vector.