Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-11-12
1999-01-26
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352551, 5303873, 536 2353, C12P 2100, C12N 1513, C07K 1646, C07H 2104
Patent
active
058637656
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the production of antibody fragments and analogous entities. In recent years there has been considerable interest in the production of antibody fragments. One fragment of particular interest is the so-called Fv fragment which consists of the variable domain of the light chain of an antibody and the associated variable domain of the antibody's heavy chain. The inter-molecular forces which bring about the association of these domains in whole antibodies also hold them together in an Fv fragment.
The individual domains of an Fv fragment can each be expressed by a genetically transformed organism. Once they are present together in solution they will spontaneously associate to give the desired Fv fragment.
The expression of single antibody domains by a transformed microorganism is discussed at length in European Patent 368684 (Medical Research Council).
If the required variable domains from the light and heavy chains are present together in solution they will in general spontaneously associate together into the required Fv fragments; association is particularly favoured by a high concentration of the domains. It is not however straightforward to get both of the variable domains produced and in solution together in equal amounts and in appropriate concentrations which favour association. One possibility is to express them both by means of separate host organisms and then bring them together. This, however, requires the transformation of two host organisms and the amounts of the variable domains which are expressed may not match each other. Moreover, it is generally thought that the expression of the heavy chain without the light chain is often harmful to the cells which express it, making it difficult to obtain concentrations suitable for industrial production.
Another possibility is to transform a host organism so that a single transformed organism contains genetic information coding for both variable domains. This can be achieved by assembling the genetic information coding for both domains on a single vector as disclosed by Reichmann et al, J. express the two variable domains in equal amounts, thereby wasting cellular metabolism in unproductive synthesis, and again risking harm to the cells from the surplus of one chain.
A way around the difficulties is proposed in European Patent 281604B which discloses the production of a single polypeptide containing the binding portion of each variable domain along with a linking peptide sequence which joins them together. This linking peptide sequence is designed so that after the single polypeptide has been expressed, the binding portions of the two variable domains can associate together to form a molecule analogous to an Fv fragment which is the so-called single chain Fv fragment. European Patent 281604B brings two key advantages. First, the two domains are produced in equal quantities. Secondly, the two domains are produced at high "local" concentration--since they are linked--and therefore association is strongly favoured.
It is explained in this prior document that the design of this single polypeptide molecule necessitates some compromise. It is taught that the linking region should extend from the C-terminal region of the light chain to the N-terminal region of the heavy chain. However it should not join the extremities of these terminal regions because they are relatively far apart in a natural antibody, (and likewise in a complete Fv fragment).
Instead the peptide link should extend from a point spaced somewhat inwardly from the C-terminal of the light chain to a point spaced somewhat inwardly of the N-terminal of the heavy chain, these being points which are somewhat closer together in the natural antibody. The consequence of this is that a portion of the light chain adjacent its C-terminus and a portion of the heavy chain adjacent its N-terminus is not expressed and instead is replaced by the linking peptide. Even so the peptide link must be designed with some care so that it is of sufficient length to permit the two variable domains
Berry Mark John
Davis Paul James
Van Der Logt Cornelis Paul
Whitelam Garry Clark
Ketter James
Quest International BV
Sandals William
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