Specific antibody fragments for the human carcinoembryonic antigen (cea)

Abstract

The invention relates to mono- and bivalent (diabody) single-chain Fv-type (scFv) antibody fragments which are obtained using recombinant DNA techniques from the carcinoembryonic anti-antigen (CEA) monoclonal antibody (McA) CB / ior-CEA.1. The aforementioned McA has a high affinity for the CEA and is used in the diagnosis and monitoring of colorectal tumours in humans. As with the original McA, diabody and monovalent scFv fragments exhibit high affinities for the human CEA and recognise an epitope that is dependent on carbohydrate conservation. The diabody and monovalent scFv fragments have affinity constants for the CEA of (5.0 + / - 0.4) x 10<9> L mol<-1> and (2.8 + / - 0.3) x 10<10> L mol<-1> respectively. The two aforementioned fragments do not display cross-reactivity with normal human tissues and cells, except for the normal colonic mucosa where the CEA is occasionally present. Said fragments can be produced through expression in recombinant micro-organisms from the cloning of nucleic acid sequences that code for variable regions obtained from the hybridoma that is produced by the CB / ior-CEA.1 McA. As with the original McA, the diabody and the monovalent scFv have a capacity for the in vivo identification in rats of human CEA-producing cells which grow forming tumours. The monovalent scFv and diabody do not posses Fc domains and the molecular sizes of said monovalent scFv and diabody are 5 and 2.5 times, respectively, less than the rat McA. As a result, the aforementioned monovalent scFv and diabody can better penetrate tissues in vivo and are less immunogenic in humans.

Description

technical field [0001] The present invention relates to the branch of immunology and, in particular, to monovalent and bivalent (dimeric) forms of single-chain Fv-type antibody fragments obtained by recombinant DNA techniques from mouse monoclonal antibodies with demonstrated clinical efficacy, and is specific to human carcinoembryonic antigen. Background of the invention [0002] Carcinoembryonic antigen (CEA) is a 180 kDa glycoprotein that is preferentially secreted by human gastrointestinal tumors and other cancer cells, however it can also be detected in some non-malignant tissues like colonic mucosa. Its physiological role has not been fully elucidated, so far it is considered to be related to the cell adhesion process in some aspects (Gold P, Freedman SO, Journal of Experimental Medicine, 122: 467, 1965; Zimmermann W et al., PNAS USA, 84: 2960 -2964, 1987; Paxton RJ et al., PNAS USA, 84:920-924, 1987; BeaucheminN et al., Molec Celluar Biol, 7:3...

AI Technical Summary

Problems solved by technology

scFv was produced in E. coli and demonstrated recognition of CEA in ELISA and cytochemical studies, but with a 200-fold lower affinity for the immobilized antigen than Fab obtained by natural methods (Pérez L et al., Applied Biochem Biotechnol, 24:79-82, 1996)

This same scFv fragment was cloned, expressed, and generated in Pichia pastoris (Freyre FM et al., J Biotechnol, 76(2-3):157-163, 2000), but without any improvement in affinity for human CEA , and studies using radiolabeled fragments in experimental animals indicated abnormal biodistribution (Pimentel GJ et al., Nucl MedCommun, 22: 1089-1094, 2001), so its further development was not pursued

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