Expression of functional antibody fragments
First Claim
1. A method for the production and purification of a Fab'"'"' having a hinge region comprising the steps of:
- (a) expressing nucleic acid encoding the Fab'"'"' in a microbial host cell comprising said nucleic acid such that the Fab'"'"' is secreted to the periplasmic space of the host cell; and
(b) recovering the Fab'"'"' from said host cell, wherein the amount of Fab'"'"' recovered from the host cell is higher than the amount of F(ab'"'"')2 recovered from the host cell and the recovered Fab'"'"' is able to be covalently bonded to a second Fab'"'"' to form a functional F(ab'"'"')2.
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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.
514 Citations
47 Claims
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1. A method for the production and purification of a Fab'"'"' having a hinge region comprising the steps of:
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(a) expressing nucleic acid encoding the Fab'"'"' in a microbial host cell comprising said nucleic acid such that the Fab'"'"' is secreted to the periplasmic space of the host cell; and (b) recovering the Fab'"'"' from said host cell, wherein the amount of Fab'"'"' recovered from the host cell is higher than the amount of F(ab'"'"')2 recovered from the host cell and the recovered Fab'"'"' is able to be covalently bonded to a second Fab'"'"' to form a functional F(ab'"'"')2. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 38, 39, 40, 44, 45, 46, 47)
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11. A method for the preparation of a polypeptide comprising F(ab'"'"')2 comprising the steps of:
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(a) expressing nucleic acid encoding a first Fab'"'"' in a microbial host cell comprising the nucleic acid such that the first Fab'"'"' is secreted to the periplasmic space of the host cell; (b) expressing nucleic acid encoding a second Fab'"'"' in a microbial host cell comprising the nucleic acid such that the second Fab'"'"' is secreted to the periplasmic space of the host cell; (c) recovering said first and second Fab'"'"' from said host cells, wherein the amounts of first and second Fab'"'"' recovered from said host cells are significantly higher than the amounts of F(ab'"'"')2 recovered therefrom; and (d) forming a covalent bound between cysteine residues which are present in the hinge regions of said first and second Fab'"'"' to form functional bivalent F(ab'"'"')2. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 41, 42, 43)
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- 19. A method for high yield production of an antibody fragment comprising culturing a microbial host cell transformed with nucleic acid encoding the antibody fragment under the transcriptional control of an inducible promoter/operator system, wherein the nucleic acid encoding the antibody fragment is preceded by nucleic acid encoding a signal sequence which directs secretion of the antibody fragment to the periplasmic space of the host cell and whereby expression of the antibody fragment is repressed prior to induction sufficient to permit post-induction antibody fragment levels in the cell culture of about 1 gram to in excess of about 2 grams of antibody fragment per liter of cell culture.
Specification