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Surface expression libraries of heteromeric receptors

a surface expression and receptor technology, applied in the field of recombinant expression of heteromeric receptors, can solve the problems of significant loss of sequences from the population, laborious and time-consuming monoclonal antibody generation, and unfavorable human developmen

Inactive Publication Date: 2005-01-06
APPLIED MOLECULAR EVOLUTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generation of monoclonal antibodies is laborious and time consuming.
A disadvantage to this method is that undesired combinations of vector portions are brought together when generating the coexpression vector.
Although these undesired combinations do not produce viable phage, they do however, result in a significant loss of sequences from the population and, therefore, a loss in diversity of the number of different combinations which can be obtained between heavy and light chains.
This makes the lambda system inherently more difficult to manipulate as compared to other available vector systems.

Method used

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  • Surface expression libraries of heteromeric receptors
  • Surface expression libraries of heteromeric receptors
  • Surface expression libraries of heteromeric receptors

Examples

Experimental program
Comparison scheme
Effect test

example i

Construction, Expression and Screening of Antibody Fragments on the Surface of M13

[0037] This example shows the synthesis of a diverse population of heavy (Hc) and light (Lc) chain antibody fragments and their expression on the surface of M13 as gene VIII-Fab fusion proteins. The expressed antibodies derive from the random mixing and coexpression of a Hc and LC pair. Also demonstrated is the isolation and characterization of the expressed Fab fragments which bind benzodiazapam (BDP) and their corresponding nucleotide sequence.

[0038] Isolation of mRNA and PCR Amplification of Antibody Fragments

[0039] The surface expression library is constructed from mRNA isolated from a mouse that had been immunized with KLH-coupled benzodiazapam (BDP). BDP was coupled to keyhole limpet hemocyanin (KLH) using the techniques described in Antibodies; A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor, New York (1988), which is incorporated herein by reference. Briefly, 10.0 milligrams (...

example ii

Cloning of Heavy and Light Chain Sequences Without Restriction Enzyme Digestion

[0083] This example shows the simultaneous incorporation of antibody heavy and light chain fragment encoding sequences into a M13IXHL-type vector with the use of restriction endonucleases.

[0084] For the simultaneous incorporation of heavy and light chain encoding sequences into a single coexpression vector, a M13IXHL vector was produced that contained heavy and light chain encoding sequences for a mouse monoclonal antibody (DAN-18H4; Biosite, San Diego, Calif.). The inserted. antibody fragment sequences are used as complementary sequences for the hybridization and incorporation of Hc and LC sequences by site-directed mutagenesis. The genes encoding the heavy and light chain polypeptides were inserted into M13IX30 (SEQ ID NO: 1) and M13IX11 (SEQ ID NO: 2), respectively, and combined into a single surface expression vector as described in Example I. The resultant M13IXHL-type vector is termed M13IX50.

[00...

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Abstract

A composition of matter comprising a plurality of procaryotic cells containing diverse combinations of first and second DNA sequences encoding first and second polypeptides which form a heteromeric receptor exhibiting binding activity toward a preselected molecule, said heteromeric receptors being expressed on the surface of filamentous bacteriophage.

Description

[0001] This application is a continuation-in-part of U.S. Ser. No. 07 / 590,219, filed on Sep. 28, 1990.BACKGROUND OF THE INVENTION [0002] This invention relates generally to recombinant expression of heteromeric receptors and, more particularly, to expression of such receptors on the surface of filamentous bacteriophage. [0003] Antibodies are heteromeric receptors generated by a vertebrates organism's immune system which bind to an antigen. The molecules are composed of two heavy and two light chains disulfide bonded together. Antibodies have the appearance of a “Y”—shaped structure and the antigen binding portion being located at the end of both short arms of the Y. The region on the heavy and light chain polypeptides which corresponds to the antigen binding portion is known as variable region. The differences between antibodies within this region are primarily responsible for the variation in binding specificities between antibody molecules. The binding specificities are a composit...

Claims

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Application Information

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IPC IPC(8): C07K14/01C07K14/705C07K16/00C07K16/44C12N15/10C12N15/13C12N15/64C12N15/65C12N15/70C40B40/02
CPCC07K14/005C07K14/705C07K16/00C07K16/44C07K2319/00C40B40/02C12N15/64C12N15/65C12N15/70C12N2795/10022C12N2795/14022C12N15/1037
Inventor HUSE, WILLIAM
Owner APPLIED MOLECULAR EVOLUTION
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