Modular assembly of antibody genes, antibodies prepared thereby and use

• US 5,693,493 A
• Filed: 05/25/1995
• Issued: 12/02/1997
• Est. Priority Date: 11/01/1985
• Status: Expired due to Term

• Current Assignee: Xoma Technology Ltd. (Xoma Corp.)
• Original Assignee: Xoma Corp.
• Inventors: Horwitz, Arnold H., Robinson, Randy R., Lei, Shau-Ping, Liu, Alvin Y., Wall, Randolph, Better, Marc, Wilcox, Gary L.
• Primary Examiner(s): Ziska, Suzanne E.
• Application Number:
  US08/450,731
• Time in Patent Office: 922 Days
• Field of Search: 435/240.2, 435/172.3
• US Class Current: 435/69.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/43 Sweetening agents, e.g. thaumatin, monellin
• CPC Class Codes: C07K 16/3046 Stomach, Intestines
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• 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: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/88 Lyases (4.)

Abstract

The invention relates to cDNA genetic sequences, vehicles containing same as well as hosts transformed therewith, for the production of chimeric immunoglobulin molecules, functional fragments thereof and immunoglobulin derivatives exhibiting novel functional properties comprising human constant region modules and non-human variable region modules, or for class switching antibody molecules and/or chains. The invention also relates to DNA coding for pectate lyase signal peptide has been cloned on a plasmid to create a secretion vector which is capable of producing a chosen protein which is transported across the bacterial membrane. The secretion vector has been used to secrete extracellular thaumatin and extracellular chimeric antibody fragments. The proteins produced by this vector have biological activity. The thaumatin is properly folded and the antibody fragments are capable of binding antigens on target cancer cells. The invention also relates to the secretion of chimeric antibodies and fragments thereof from yeast in functional form.

Claims

1. A process of producing an immunoglobulin fragment, said process comprising:

(1) culturing an E. coli host that has been transformed with a polynucleotide molecule encoding said immunoglobulin fragment under conditions that express said immunoglobulin fragment, and

(2) producing and secreting said immunoglobulin fragment, wherein said polynucleotide molecule comprises;

(1) a first DNA encoding a first pectate lyase secretion signal sequence operably linked to a DNA sequence encoding an immunoglobulin Fd molecule or a fragment thereof comprising the variable region, and

(2) a second DNA sequence encoding a second pectate lyase secretion signal sequence operably linked to a DNA sequence encoding an immunoglobulin light chain or a fragment thereof comprising the variable region,

wherein said first and second DNA sequences are operably linked to a single prokaryotic promoter so as to form a dicistronic transcription unit, and said immunoglobulin fragment is capable of binding to an antigen.

2. The process of claim 1, wherein said light chain is full-length.

3. The process of claim 1, wherein said immunoglobulin fragment is Fab.

4. The process of claim 1, wherein said immunoglobulin Fd molecule and/or said immunoglobulin light chain are chimeric.

5. The process of claim 4, wherein said immunoglobulin Fd molecule is chimeric.

6. The process of claim 4, wherein said immunoglobulin light chain is chimeric.

7. The process of claim 4, wherein both said immunoglobulin Fd molecule and said immunoglobulin light chain are chimeric.

8. The process of any one of claims 1-7, wherein the produced immunoglobulin fragment is in the periplasmic space.

9. The process of any one of claims 1-7, wherein the produced immunoglobulin fragment is in the culture medium.