Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1995-06-06
2001-03-20
Burke, Julie (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S069600, C435S069700, C435S328000, C435S091100, C530S387300, C424S133100, C536S023530
Reexamination Certificate
active
06204023
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to recombinant DNA methods of preparing immunoglobulins, genetic sequences coding therefor, as well as methods of obtaining such sequences.
BACKGROUND ART
The application of cell-to-cell fusion for the production of monoclonal antibodies by Kohler and Milstein (
Nature (London
), 256: 495, 1975) has spawned a revolution in biology equal in impact to the invention of recombinant DNA cloning. Hybridoma-produced monoclonal antibodies are already widely used in clinical diagnoses and basic scientific studies. Applications of human B cell hybridoma-produced monoclonal antibodies hold great promise for the clinical treatment of cancer, viral and microbial infections, B cell immunodeficiencies with diminished antibody production, and other diseases and disorders of the immune system.
Unfortunately, yields of monoclonal antibodies from human hydridoma cell lines are relatively low 1 ug/ml in human x human compared to 100 ug/ml in mouse hydridomas), and production costs are high for antibodies made in large scale human tissue culture. Mouse x mouse hybridomas, on the other hand, are useful because they produce abundant amounts of protein, and these cell lines are more stable than the human lines. However, repeated injections of “foreign” antibodies, such as a mouse antibody, in humans, can lead to harmful hypersensitivity reactions.
There has therefore been recent exploration of the possibility of producing antibodies having the advantages of monoclonals from mouse-mouse hybridomas, yet the species specific properties of human monoclonal antibodies.
Another problem faced by immunologists is that most human monoclonal antibodies (i.e., antibodies having human recognition properties) obtained in cell culture are of the IgM type. When it is desirable to obtain human monoclonals of the IgG type, however, it has been necessary to use such techniques as cell sorting, to separate the few cells which have switched to producing antibodies of the IgG or other type from the majority producing antibodies of the IgM type. A need therefore exists for a more ready method of switching antibody classes, for any given antibody of a predetermined or desired antigenic specificity.
The present invention bridges both the hybridoma and monoclonal antibody technologies and provides a quick and efficient method, as well as products derived therefrom, for the improved production of chimeric human
on-human antibodies, or of “class switched” antibodies.
INFORMATION DISCLOSURE STATEMENT*
Approaches to the problem of producing chimeric antibodies have ben published by various authors.
Morrison, S. L. et al.,
Proc. Natl. Acad. Sci., USA,
81: 6851-6855 (November 1984), describe the production of a mouse-human antibody molecule of defined antigen binding specificity, produced by joining the variable region genes of a mouse antibody-producing myeloma cell line with known antigen binding specificity to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed, wherein the heavy chain variable region exon from the myeloma cell line S107 well joined to human IgG1 or IgG2 heavy chain constant region exons, and the light chain variable region exon from the same myeloma to the human kappa light chain exon. These genes wee transfected into mouse myeloma cell lines and transformed cells producing chimeric mouse-human antiphosphocholine antibodies were thus developed.
Morrison, S. L. et al., European Patent Publication No. 173494 (published Mar. 5, 1986), disclose chimeric “receptors” (e.g. antibodies) having variable regions derived from one species and constant regions derived from another. Mention is made of utilizing cDNA cloning to construct the genes, although no details of cDNA cloning or priming are shown. (see pp 5, 7 and 8).
Boulianne, G. L. et al.,
Nature,
312: 643 (Dec. 13, 1984), also produced antibodies consisting of mouse variable regions joined to human constant regions. They constructed immuoglobulin genes in which the DNA segments encoding mouse variable regions specific for the hapten trinitrophenyl (TNP) were joined to segments encoding human mu and kappa constant regions. These chimeric genes were expressed as functional TNP binding chimeric IgM.
For a commentary on the work of Boulianne et al. and Morrison et al., see Munro,
Nature,
312: 597 (Dec. 13, 1984), Dickson,
Genetic Engineering News,
5, No. 3 (March 1985), or Marx,
Science,
229: 455 (August 1985).
Neuberger, M. S. et al.,
Nature,
314: 268 (Mar. 25, 1986), also constructed a chimeric heavy chain immunoglobulin gene in which a DNA segment encoding a mouse variable region specific for the hapten 4-hydroxy-3-nitrophenacetyl (NP) was joined to a segment encoding the human epsilon region. When this chimeric gene was transfected into the J558L cell line, an antibody was produced which bound to the NP hapten and had human IgE properties.
Neuberger, M. S. et al., have also published work showing the preparation of cell lines that secrete hapten-specific antibodies in which the Fc portion has been replaced either with an active enzyme moiety (Williams, G. and Neuberger, M. S.
Gene
43:319, 1986) or with a polypeptide displaying c-myc antigenic determinants (
Nature,
312:604, 1984).
Neuberger, M. et al., PCT Publication WO 86/01533, (published Mar. 13, 1986) also disclose production of chimeric antibodies (see p. 5) and suggests, among the technique's many uses the concept of “class switching” (see p. 6).
Taniguchi, M., in European Patent Publication No. 171 496 (published Feb. 19, 1985) discloses the production of chimeric antibodies having variable regions with tumor specificty derived from experimental animals, and constant regions derived from human. The corresponding heavy and light chain genes are produced in the genomic form, and expressed in mammalian cells.
Takeda, S. et al.,
Nature,
314: 452 (Apr. 4, 1985) have described a potential method for the construction of chimeric immunoglobulin genes which have intron sequences removed b the use of a retrovirus vector. However, an unexpected splice donor site caused the deletion of the V region leader sequence. Thus, this approach did not yield complete chimeric antibody molecules.
Cabilly, S. et al.,
Proc. Natl. Acad. Sci., USA,
81: 3273-3277 (June 1984), describe plasmids that direct the synthesis in
E. coli
of heavy chains and/or light chains of anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd′) fragment in
E. coli
. Functional CEA-binding activity was obtained by in vitro reconstitution, in
E. coli
extracts, of a portion of the heavy chain with light chain.
Cabilly, S., et al., European Patent Publication 125023 (published Nov. 14, 1984) describes chimeric immunoglobulin genes and their presumptive products as well as other modified forms. On pages 21, 28 and 33 it discusses cDNA cloning and priming.
Boss, M. A., European Patent Application 120694 (published Oct. 3, 1984) shows expression in
E. coli
of non-chimeric immunoglobulin chains with 4-nitrophenyl specificity. There is a broad description of chimeric antibodies but no details (see p. 9).
Wood, C. R. et al.,
Nature,
314: 446 (April, 1985) describe plasmids that direct the synthesis of mouse anti-NP antibody proteins in yeast. Heavy chain mu antibody proteins appeared to be glycosylated in the yeast cells. When both heavy and light chains were synthesized in the same cell, some of the protein was assembled into functional antibody molecules, as detected by anti-NP binding activity in soluble protein prepared from yeast cells.
Alexander, A. et al.,
Proc. Nat. Acad. Sci. USA,
79: 3260-3264 (1982), describe the preparation of a cDNA sequence coding for an abnormally short human Ig gamma heavy chain (OMM gamma
3
HCD serum protein) containing a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion removes the remainder of the V and the entire C
H
1 domain. This is cDNA coding for an i
Better Marc
Horwitz Arnold H.
Lei Shau-Ping
Liu Alvin Y.
Robinson Randy R.
Burke Julie
Sterne Kessler Goldstein & Fox P.L.L.C.
XOMA Ltd.
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