Isolating cells expressing secreted proteins

Abstract

A method for identifying and isolating cells which produce secreted proteins. This method is based upon a specific characteristic or the expression level of the secreted protein by transiently capturing the secreted protein on the surface of an individual cell, allowing selection of rare cell clones from a heterogeneous population. Also provided is the use of this method to generate cells which produce a desired level of secreted protein or secreted protein of a particular characteristic(s), and organisms which possess such cells. In particular, the method allows rapid isolation of high expression recombinant antibody-producing cell lines, or may be applied directly to rapid isolation of specific hybridomas, or to the isolation of antibody-producing transgenic animals. This method is applicable for any cell which secretes protein.

Description

[0001] This application claims priority to U.S. provisional application No. 60 / 261,999, filed Jan. 16, 2001. Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.FIELD OF THE INVENTION [0002] The field of this invention is a method for identifying and isolating cells which produce secreted proteins. This method is based upon a specific characteristic or the expression level of the secreted protein by transiently capturing the secreted protein on the surface of an individual cell, allowing selection of rare cell clones from a heterogeneous population. The field also encompasses the use of this method to generate cells which produce a desired level of secreted protein or secreted protein of a particular characteristic(s), and organisms which possess such cells. In particular, the method allows rapid isolation of high expression recombinant antibody-producing...

AI Technical Summary

Benefits of technology

The present invention describes a method for quickly identifying and isolating cells that produce a specific protein by directly screening for the protein on the surface of cells. This method can be used to detect and isolate cells that produce any secreted protein, including antibodies. It allows for the convenient monitoring of cells that secrete the protein of interest (POI) during the manufacturing process. The invention also allows for the isolation of rare cells that produce the desired protein. The method can be applied to transgenic animals and can be used to detect and isolate cells that produce any secreted protein. Overall, this invention provides a faster and more efficient way to identify and isolate cells that produce specific proteins.

Problems solved by technology

These procedures are laborious, inefficient, expensive, and the number of clones that can be analyzed is usually limited to a few hundred.

Moreover, the collection of clone pools, or hand-picked colonies, risks losing high expression cells, which often grow more slowly, to faster growing low expression cells.

Incorporation of flow cytometry into methods used for the isolation of stable expression cell lines has improved the capability of screening large numbers of individual clones, however, currently available methods remain inadequate for diverse reasons.

Diffusion of the POI between cells of different characteristics was also a problem.

Because this method depends on the coupling of GOI expression to the reporter gene by use of an IRES in a recombinant construction, it is not applicable to the isolation of hybridomas.

Moreover, use of flow cytometry significantly reduces the direct handling of cells.

Unfortunately, the level of GFP production is not a direct measure of the production level of the POI.

Moreover, some cells may be sensitive to the encapsulation process.

However, diffusion of POI between neighboring cells is problematic, and this method also requires a high viscosity medium to reduce diffusion of POI away from expressing cells.

The problems associated with identification and isolation of high expression recombinant cell lines especially applies to the isolation of hybridomas that express an antibody of interest.

However, the identification of useful hybridomas includes several additional problems; they must be screened first for antigen-binding activity, then for immunoglobulin isotype.

Moreover, GFP-based methods are not applicable to the identification and isolation of hybridomas because construction of hybridomas does not include a recombinant construct such that expression of the antibody genes can be linked to a transcriptional reporter such as GFP.

Hybridoma screening is a slow, laborious endeavor where the number of clones screened is limited by existing technologies.

A similar problem involves the selection of rare cells producing an antibody, an ScFv, a fragment thereof, or anything fused to an antibody constant region, with a desired specificity, isotype, and avidity for a particular antigen, from a heterogeneous population of cells expressing different antibodies, ScFvs, fragments thereof, or anything fused to antibody constant regions.

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