RECOMBINANT HUMAN ANTIBODY FRAGMENTS PRODUCED BY PHAGE DISPLAY TECHNOLOGY AGAINST THYMOCYTES RECOGNIZE PHERIPHERAL T CELL SURFAC

AC30

Thaís Barboza Bertolini (1) thabertolini@usp.com.br; Manuela Berto Pucca (1); Jaqueline Carlos (1); Eduardo Crosara Roncolatto (1); Carolina Mendes Fossa (1); Alessandra Souza de Paula (1); André Oliveira Mota Júnior  (1); José Elpidio Barbosa (2).

1- Pós-graduanda (o) Programa de Imunologia Básica e Aplicada. Faculdade de Medicina – USP2- Docente Departamento de Bioquímica e Imunologia. Faculdade de Medicina – USP

INTRODUCTION: The main treatment for the acute rejection of grafts consists of immunosupressant drugs which inhibit or lyse T lymphocytes. However, such treatment is followed by the immune susceptibility to infections and proneness to other diseases. Polyclonal anti-thymocytes antibodies (ATG) are also used; however, this procedure results in an increase of the susceptibility to linfoproliferative and infectious diseases and could lead to the development of the Serum Sickness Disease, due to heterologous antibodies. In an attempt to avoid the inconvenience of using heterologous antibodies, humanized chimerical antibodies have been developed. These antibodies, nonetheless, stimulate the activation of the complement system and the opsonization of cells by fagocytosis. OBJECTIVE: The aim of this work is to produce scFv portions of human anti-thymocytes immunoglobulins, capable to recognize pheripheral T cells, for a subsequent test in human lymphocytes proliferation in vitro assay and use as suppression therapy after allotransplants. METHODOLOGY: To produce human monoclonal antibodies, we used the phage display technology and the Griffin.1 library. This consists of the scFv phagemid library constructed by recloning the heavy and the light chain variable regions into the phagemid vector pHEN2. The thymocytes used in the selection were obtained from thymus of children submitted to cardiovascular surgery at the University Hospital of Faculty of Medicine, USP at Ribeirão Preto (HCFMRP-USP). To test whether the phage-antibodies produced against thymocytes were capable of recognizing peripheral T lymphocytes, the ELISA technique was used. The peripheral T lymphocytes were separated by using Ficoll and later isolated in columns of CD4 and CD8. A flow cytometry was performed to evaluate the separation. RESULTS: After 3 rounds of selection, the polyclonal ELISA was performed, and at rounds 2 and 3 thymocytes and peripheral T lymphocytes were recognized. Next, a monoclonal ELISA for each round was performed and a total of 162 positive clones to peripheral T lymphocytes were identified. CONCLUSION: The phage-antibodies produced recognize the peripheral T lymphocytes. New experiments are in progress to assess whether the fragments scFv produced by these phages are able to inhibit the proliferation of T lymphocytes becoming in the future a suppression therapy for allotransplants.