Dimeric scFv antibody construct of ior-CEA1 as potential agent for therapeutic application

The classic radioimmunotherapy is based on transporting to the tumour, the necessary activity of a therapeutic isotope using a monoclonal antibody (MAb) against tumour-associated antigen, which constitutes an ideal vehicle given its inherent specificity. In this sense, the whole immunoglobulins (Ig) have some severe practical limitations due to their unhappy pharmacokinetics, their relatively high molecular weight (150 kDa) and slowed clearance. Likewise the Ig molecule presents a poor diffusion through the tumour mass and a relative high immunogenicity. The solution to these problems has been encountered by genetic engineering, methods where the multivalent recombinant fragments have become the paradigm of design of constructed molecules. They are able to retain the the parental antibodies specificity and affinity, with reduced immunogenicity and improved pharmacokinetics. The monoclonal antibody ior-CEA1 has been employed for the diagnosis of primary tumours, recurrences and metastasis of colorectal tumours and has also been used extensively in the daily clinical practice as well. Inserted in this rebirth of the antibodies, this report explores the use of a new smaller molecular weight multivalent analogue of the parent ior- CEA1 antibody to be used for the diagnosis and therapy of CEA-expressing cancer tumours such as colorectal, lung, ovary among others. The dimeric scFv-ior-CEA1 construct was produced in the Centre for Genetic Engineering and Biotechnology and displayed to bind CEA epitope with a similar binding affinity to that of the murine IgG. Labelling method was achieved using Chloramine T, with a molar ration of Chloramine T:tyrosine of 2,5. The methodology proposed for the radioiodation attained incorporation over 90% of radioiodine to the protein. Although it is a relative high labelling yield, for the immunoreactivity studies and biodistribution the fragment was purified by means of FPLC. For the comparison of the immunoreactivity of labelled and unlabelled diabody, was plotted the specific union vs the antibody concentration for both molecules and adjusted to a straight line. The results showed that the 131I-(scFv)2 retain 85% of its immunoreactivity after labelling. This decreasing in the immunoreactive capacity could be attributable to the iodine/tyrosine binding in the region of recognition of the molecule. That is, from the 16 tyrosine present in each scFv, four of them are in the complementarity determinant regions (1 in VL CDR1 and 3 en VL CDR3), and due to the voluminous size of the iodine atom, it could produce steric impediments that hinder the formation of the antigen-antibody complex. The non-specific union in the study was of 6%. The Scatchard analysis was used to calculate the apparent affinity constant. The binding affinity was of 3,5x107 M-1, similar to other biomolecules of this construct. The immunoreactivity was also assayed using size-exclusion FPLC to evaluate their antigenbinding capacities after radiolabelling. Seventy-five nanograms of radiolabelled 131I-(scFv)2 were incubated with 15-fold excess of CEA for 1 h at room temperature and the sample was analysed by size-exclusion FPLC. The radiochromatograms of Diabody and immunoconjugate CEA/(scFv)2, where a radioactive bulk is eluted with a lower retention time suggesting the immunocomplex formation is shown. The immunoreactive fraction of the radioiodide fragment can be calculated from radiochromatogram by means of the integration of the area under the curve for the corresponding fractions to the complex antigen-antibody and the 131I-diabody. The obtained result was 87%, similar to 85% obtained by means of the classic technique. In order to study the 'in vivo' stability and immunoreactivity of the diabody, as well as to check the possible cross-reaction with the antigens NCA-95 expressed in granulocytes, whose previous variant of the scFv exhibited. It was carried out a study of biodistribution of the 131I ior-CEA1-(scFv)2 in MNRI healthy and nude mice bearing LS174T human CEA-positive tumours. The results showed a typical behaviour for this kind of biomolecules, without significant localisation in any critical organ. Being this, an advantage for its possible use as radioimmunotherapeutic agent, since the exposition levels in healthy tissues will be low. Both biodistribution exhibited a low uptake in bone marrow suggested the absence of crossed reactions with the NCA-95 antigen. It can also be observed a high uptake in tumour. The excellent tumour:blood ratios reflect the specific retention in the target tumour and avidity of the biomolecule for the CEA antigen