Preparation And Binding Characterization Of Chimeric Anti-CD154 Fab Expressed In Escherichia Coli

CD 154 (CD40L, gp39) is a 39kD glycoprotein expressed as a type II intergrel membrane protein on the surface of activated CD4+ T cells. CD 154, encoded by a gene located at Xq26.3-Xq27.1, belongs to tumor necrosis factor (TNF) superfamily.The contribution of CD40/CD154 interactions to the process of T cell priming, differentiation, and effector functions has been extensively reviewed. CD40/CD154 interactions play a critical role in T cell priming and have been suggested to prohibit tolerance induction. Therefore, interference with CD 154 mAbs has been extensively investigated to prolong the survival of experimentally transplanted organs. The treatment with anti-CD 154 monoclonal antibody prolonged survival of experimentally transplanted organs in some murine models including allografts of heart, skin, aorta, and pancreatic islets. It is important that in rhesus monkey model of kidney transplantation, exciting therapeutic effects of anti-CD 154 mAb treatment have been observed. Although mAbs have held great promise for the treatment of human disease such as cancer, viral infection and autoimmune disorders, the most important impediments in murine mAb have been the immune response against murine immunoglobulins and insufficient activation of human effector function. These problems could be overcome using genetic engineering techniques to produce humanized antibodies.In our study, an expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli.The single-strand cDNA was prepared by AMV reverse transcription from poly A+ RNA extracted from hybridoma cells (4F1) secreting mouse anti-human CD 154 mAb. Amplification of murine variable regions (mVH & mVL) was achieved by PCR using a pair of specific consensus primers. At the same time, constant regions of humanimmunoglobulin IgG1 (huFc, huCk and huCH1) were obtained from human spleen cells by RT-PCR using corresponding primers. By TP-PCR, the variable regions are fused directly to corresponding domains of the constant regions without any mutation or endonuclese sites to constitute a mouse and human chimeric H and L chains. For antibody expression, a novel co-expression vector was constructed containing a pelB signal sequence and at the C terminus of CHI there is an 11 amino acid c-myc peptide tag which could be recognized by a polyclonal anti-c-myc antibody and aids detection of the expressed Fab during purification. The pelB signal sequence directs chimeric heavy or light chains into the bacterial periplasm, where protein folding as well as heterodimer association occurred correctly to form functional Fab. Thus, the assembly pathway for the Fab fragment of CD154 mAb is similar to that of a whole antibody in the eukaryotic cell. The Fab fragment of CD 154 mAb was purified to homogeneity with affinity chromatography in a single step. The antigen-binding ability of the recombinant Fab fragment was demonstrated by competitive inhibitory assay showing that the affinity constant of the Fab fragment is a little lower than that of the parental murine antibody CD154mAb(4Fl).In conclusion, our studies indicated that mAbs from murine hybridomas may be simply and rapidly converted to chimeric Fab which can be expressed in bacteria and purified in reasonable quantities. This expression system should facilitate future protein engineering experiments on murine monoclonal antibodies. Besides, cloned antibody fragments also have the additional advantages in that they may be manipulated to alter affinity or to make fusion proteins with enzymes or toxins.