Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1993-07-29
1999-01-12
Eisenschenk, Frank C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 7021, 435 711, 435 691, 435 91, 4352523, 435326, 5303873, 530867, 536 2353, C12N 510, C07K 1646, C07H 2104
Patent
active
058587250
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the preparation of chimaeric antibodies. The invention is typically applicable to the production of humanised antibodies.
Antibodies typically comprise two heavy chains linked together by disulphide bonds and two light chains. Each light chain is linked to a respective heavy chain by disulphide bonds. Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end. The light chain variable domain is aligned with the variable domain of the heavy chain. The light chain constant domain is aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises a framework of four regions, whose sequences are relatively conserved, connected by three complementarily determining regions (CDRs). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site. CDRs and framework regions of antibodies may be determined by reference to Kabat et al ("Sequences of proteins of immunological interest" U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1987).
The preparation of an altered antibody in which the CDRs are derived from a different species to the variable domain framework regions is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody may be derived from a human antibody. Such a humanised antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody. Humanised CAMPATH-1 antibody is disclosed in EP-A-0328404.
The technique of "overlap extension" involves the use of oligonucleotide primers complementary to a template nucleotide sequence and the polymerase chain reaction (PCR) to generate DNA fragments having overlapping ends. These fragments are combined in a "fusion" reaction in which the overlapping ends anneal allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. Ho et Al (Gene, 77, 51-59 (1989)) describe the use of this technique to introduce specific alterations in a nucleotide sequence by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, those variants of the mouse major histocompatibility complex class-I gene were generated cloned and analysed.
Horton et a (Gene, 77 61-68 (1989)) describe a technique of gene splicing by overlap extension (SOE). The technique allows the production of a hybrid length of DNA, AD, by splicing two pieces of DNA, AB and CD, which are produced by a PCR using primers A, B, C and D. At least part of the primers B and C are complementary to each other. The fragments AB and CD produced by PCR are mixed to allow the positive strand of AB to anneal to the negative strand of CD. The overlap between B and C allows the two strands to prime extension of each other. Primers A and D are used to prime a PCR reaction of the extended strands.
The above technique was used to splice a portion (CD) of the mouse H-2K.sup.b gene between upstream and downstream regions (AB and EF respectively) of the corresponding upstream and downstream parts of the H-2L.sup.d gene. All three fragments, AB, CD and EF were produced by PCR, using primers A to F. The three fragments were joined by two rounds of SOE, the first one producing a fragment AD (ie. AB-CD)
REFERENCES:
Colman, Research in Immunology, 145:33, 1994.
Horton, et al., Gene, 77 (1989), pp. 61-68.
Horton, et al., Biotechniques, 8(5) (May 1990), pp. 528-535.
Lewis, et al., Gene, 101:2 (1991), pp. 297-302.
LeBoeuf, et al., Gene, 82 (1989), pp. 371-377.
Crowe James Scott
Lewis Alan Peter
Eisenschenk Frank C.
Glaxo Wellcome Inc.
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