Construction of recombinant antibodies specific for Leishmania antigens

Abstract

The development of hybridoma technology generated an enormous expansion in the use of antibodies in diagnosis as well in therapeutic applications. Furthermore, developments in recombinant DNA technology, in addition to the use of suitable expression hosts, allowed the production of a new generation of designed antibodies. The single-chain Fvs (scFvs), comprising the antibody heavy (VH) and light (VL) chain variable domains connected by a flexible linker, are the smallest stabilized antibody fragments containing a complete antigen binding site. Due to their small size, scFvs have lower retention times in non-target tissues, a more rapid blood clearance and better tumor penetration than larger antibody fragments or complete immunoglobulins. In Portugal, visceral leishmaniasis, caused by Leishmania infantum, is a zoonosis in which dogs are the main reservoir. Leishmania/HIV co-infection is emerging with an increasing frequency as an extremely serious association. AIDS and VL are locked in a vicious circle of mutual reinforcement. Construction of scFvs against antigens from Leishmania infantum would be very useful for the serological diagnosis of leishmaniasis in HIV infected patients. In this work, an hybridoma secreting a mAb to a 97 kDa protein fraction from L. infantum was used to construct a scFv. After mRNA isolation from IgM producing hybridoma cell line and cDNA synthesis, antibody VH and VL domain coding regions were amplified by PCR, producing several amplification products. Subsequently to the digestion with restriction enzymes, each variable antibody fragment coding region was cloned in the prepared pOPE 101-215 (Yol) vector and used to electrotransform E. coli. Clones with open reading frames were identified by immunoblotting of total cell lysates after 3 h induction with IPTG and the correct insertion of each gene fragment confirmed by DNA sequencing. Nucleotide sequences were translated to amino acids, using the HUSAR sequence analysis program from the German Cancer Research Center, and used to identify the complementaritydetermining regions (CDRs) for the heavy and light chain variable domains. Search for homology of the deduced amino acid sequences of the heavy and light chain variable domains in Kabat database showed significant homology with the amino acid consensus sequences from mouse Ig heavy chain variable region and mouse Ig kappa light chain variable region. Both variable domain coding regions were assembled in the pOPE vector and, after transformation of E. coli with the desired antibody constructs, selected expressing clones were used to extract scFvs from E. coli periplasm. scFv antibody fragments were purified from the periplasmic extracts by immobilized metal affinity chromatography (IMAC) and anion exchange chromatography. Functional studies of the constructed antibody fragments were performed by ELISA, Immunoblotting and Indirect Immunofluorescence.

O desenvolvimento da tecnologia de hibridomas tem permitido uma enorme expansão no uso dos anticorpos tanto em diagnóstico, como em terapia de patologias humanas e animais. O desenvolvimento da tecnologia de anticorpos recombinantes, conjugado com o uso de vectores de expressão adequados, tem vindo a permitir a obtenção de uma nova geração de anticorpos. Os scFvs, constituídos pelos domínios variáveis das cadeias pesada (VH) e leve (VL) dos anticorpos, unidos por um “linker” flexível, são os fragmentos de anticorpo estáveis, de menor dimensão, que possuem completo local de ligação ao antigénio. Os scFvs, devido à sua dimensão, apresentam um reduzido tempo de retenção nos tecidos “não-alvo”, uma rápida eliminação do sangue e uma melhor penetração nos tumores comparativemente aos fragmentos de anticorpo de maiores dimensões ou às imunoglobulinas completas. Em Portugal, a leishmaniose visceral, causada pelo protozoário da espécie Leishmania infantum, é uma zoonose na qual o cão é o principal reservatório. Casos de co-infecção Leishmania/HIV têm sido detectados com uma frequência surpreendente. SIDA e leishmaniose visceral estão associadas num ciclo de reforço mútuo. A construção de scFvs específicos para fracções antigénicas de Leishmania infantum poderá ser bastante útil no diagnóstico serológico da doença em pacientes infectados com o vírus da SIDA. O presente trabalho descreve a construção de scFvs a partir de um hibridoma secretor de anticorpo monoclonal de classe IgM para a fracção antigénica de 97 kDa de L. infantum. Após isolamento do mRNA a partir da linha celular e síntese do cDNA, as regiões que codificam para os domínios variáveis das cadeias [...]