Expression of functional antibody fragments
AssigneeGenentech, Inc.
InventorPaul J. CARTER
Life of this patent
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Abstract
Methods for the high yield production of antibody Fv-containing polypeptides, especially Fab′ and F(ab′)2 antibody fragments are provided. Expression of heavy and light chain Fv in a microbial secretory system is followed by recovery of Fv from the periplasm under conditions that maintain a cysteine residue as a free thiol. The free thiol is reacted with free thiol of an antibody fragment of the same or differing specificity, or with agents such as diagnostic labels or therapeutic moieties. The products offer advantages of homogeneity and purity not available through the use of known methods for preparing such derivatives.
Claims24
A method for the preparation of an immunoglobulin Fv fragment, comprising
- a. culturing recombinant microbial cells having nucleic acid encoding the Fv fragment comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, said light or heavy chain also comprising an unpaired cysteinyl residue C-terminal to the heavy chain variable region or the light chain variable region, whereby said culturing results in expression of said nucleic acid and secretion of the fragment into the periplasm of the microbial cells, and
- b. recovering the Fv fragment, wherein the recovered fragment has the cysteinyl residue present as a free thiol.
A method for the preparation of a Fab antibody polypeptide having at least one cysteine in a hinge region of the antibody present as a free thiol (Fab′-SH), comprising the steps of:
- a. expressing nucleic acid encoding an immunoglobulin amino acid sequence comprising Fab in microbial host cells transformed with a vector comprising said nucleic acid operably linked to control sequences which direct the secretion of Fab′ to the periplasmic space of the host cell;
- b. permitting the formation of Fab′-SH within the host cell;
- and
- c. recovering the Fab′-SH from said host cell.
A method for the preparation of F(ab′)2 comprising the steps of:
- a. expressing nucleic acid encoding a first Fab′, said first Fab′ being capable of binding a first epitope, in a microbial host cell, having a periplasmic space, transformed with a vector comprising the nucleic acid operably linked to control sequences which direct the secretion of said first Fab′ to the periplasmic space of the host cell;
- b. expressing nucleic acid encoding a second Fab′, said second Fab′ being capable of binding a second epitope, in a microbial host cell, having a periplasmic space, transformed with a vector comprising the nucleic acid encoding the second Fab′ operably linked to control sequences which direct the secretion of said second Fab′ to the periplasmic space of the host cell;
- c. permitting the formation of first and second Fab′-SH within the periplasmic spaces of said host cells;
- d. recovering said first and second Fab′-SH from the periplasmic spaces of said host cells;
- and
- e. forming a covalent bond between the cysteinyl free thiols of said first and second Fab′-SH to form bivalent F(ab′)2.
A method for high yield production of an immunoglobulin polypeptide comprising culturing a host cell transformed with nucleic acid encoding an immunoglobulin polypeptide under the transcriptional control of an inducible promoter/operator system wherein the promoter/operator system is subsequently induced, thereby resulting in polypeptide levels in the cell culture of greater than about 1 gram of polypeptide per liter of cell culture.
Dependencies are inferred from the claim text.