Methods and compositions for inducible extracellular membrane capture of monoclonal immunoglobulins secreted by hybridomas
AssigneeLankenau Institute for Medical Research
InventorsScott K. Dessain, Rama Devudu Puligedda
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Abstract
Compositions and methods are provided for making and using a cell having expressed on its outer plasma membrane surface an Anchor designed to form a first complex with a selected Linker. The Linker is designed to form a second complex with a target protein that is not recognized by the Anchor. These cells can be primary cells or immortalized cells, and can function as fusion partners to create hybridoma cells.
Claims18
A method of identifying a hybridoma cell that secretes a monoclonal antibody that has a pre-selected characteristic, said cell residing among a population of cells that do not secrete a monoclonal antibody with said characteristic, comprising:
- (a) contacting
- (i) a population of hybridoma cells, prepared by fusing B lineage cells with immortalized cells having expressed on their outer plasma membrane surface an Anchor designed to form a first complex with a selected Linker;
- with
- (ii) the Linker designed to form the first complex with the Anchor and a second complex with a single monoclonal antibody secreted by a single hybridoma cell, wherein the monoclonal antibody is not recognized by the Anchor;
- the contacting permitting the Linker to adhere to the outer plasma membrane of the hybridoma cells by binding in the first complex with the Anchor;
- (b) maintaining the cells of (a) in the presence of the Linker under conditions in which the Linker forms a second complex with the monoclonal antibody secreted by a hybridoma cell and the monoclonal antibody binds to the hybridoma cell that secretes it via the adhered first and second complexes;
- and
- (c) identifying the existence, antigen specificity, antigen binding affinity, titer, amount, or biological activity of the monoclonal antibody secreted by the hybridoma cells and bound thereto by the cell bound multiple immune complex formed by Anchor-Linker-monoclonal antibody.
A nucleic acid molecule encoding an Anchor in operative association with regulatory sequences that direct expression of the Anchor on the surface of a cell containing the nucleic acid molecule, the Anchor designed to form a first complex with a selected Linker;
- the Linker designed to form a second complex with a target protein that is not recognized by the Anchor.
A cell having expressed on its outer plasma membrane surface an Anchor designed to form a first complex with a selected Linker;
- the Linker designed to form a second complex with a target protein that is not recognized by the Anchor, said cell characterized by at least one of:
- (a) wherein the Anchor comprises
- i. an extracellular region polypeptide that binds the Linker;
- ii. an optional polypeptide tag;
- and
- iii. a transmembrane amino acid sequence derived from an integral membrane protein;
- or
- (b) wherein on the cell's outer plasma membrane surface is the first immune complex comprising the Anchor and the Linker;
- or
- (c) wherein the Linker is an antibody or antibody fragment;
- or
- (d) wherein the antibody fragment is Fv fragment, Fab fragment, Fv or a single chain Fv, or a single domain antibody, and recombinant versions thereof;
- or
- (e) wherein on the cell's outer plasma membrane surface is a multiple immune complex comprising the Anchor bound to the Linker, the Linker also bound to the target protein;
- or
- (f) wherein the Linker can bind to both the Anchor and the Target Protein, under conditions wherein the Target Protein and the Anchor do not bind to each other in the absence of the Linker;
- or
- (g) wherein the target protein is an antibody.
Dependencies are inferred from the claim text.