Expression of functional antibody fragments

• US 7,018,809 B1
• Filed: 09/18/1992
• Issued: 03/28/2006
• Est. Priority Date: 09/19/1991
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Carter, Paul J.
• Primary Examiner(s): Ungar, Susan
• Assistant Examiner(s): DAVIS, MINH TAM B
• Application Number:
  US08/199,268
• Time in Patent Office: 4,939 Days
• Field of Search: 530/387.1, 530/387.3, 435/69.6, 435/70.21, 435/172.3, 435/69.1, 435/325, 435/69.7, 435/70.1, 435/972
• US Class Current: 435/69.1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional

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.

Claims

1. A method for the production of a Fab′

antibody fragment coupled to a heterologous molecule, comprising the steps of;

(a) expressing nucleic acid encoding a Fab′

antibody fragment in a prokaryotic host cell culture comprising the nucleic acid such that the Fab′

is secreted to a periplasmic space of a prokaryotic host cell;

(b) recovering the Fab′

from the host cell culture, wherein the recovered Fab′

comprises a CH1 fused to the amino acid sequence of CysXX, wherein X is Ala, Arg, Asp, or Pro, and wherein the cysteine of Cys XX is present as a free thiol, and the amount of the Fab′

recovered from the host cell culture exceeds the amount of F(ab′

)₂ recovered therefrom; and

(c) coupling the cysteine of CysXX that is present as a free thiol of the recovered Fab′

to a heterologous molecule.

2. The method of claim 1 wherein the Fab′

is recovered from host cell culture medium or a periplasmic preparation in step (b).

3. The method of claim 1 wherein a metal ion chelating agent is present during the recovery of the Fab′

.

5. The method of claim 1 wherein a protease inhibitor is present during the recovery of the Fab′

.

6. The method of claim 1 wherein the prokaryotic host cell is an E. coli cell.

7. The method of claim 1 wherein the recovered Fab′

is able to be covalently bonded to a second Fab′

to form a functional F(ab′

)₂.

8. The method of claim 1 wherein the Cys XX sequence has an amino acid sequence of CAA.

9. The method of any of claim 1, wherein the heterologous molecule is a different Fab′

.

10. The method of any of claim 1, wherein the heterologous molecule is a detectable label, cytotoxic drug, toxin, or solid support.

11. The method of any of claim 1, wherein the heterologous molecule is a radionuclide or a fluorescent probe.

4. The method of clam 3 wherein the metal ion chelating agent is EDTA.