Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Treatment of induced murine SLE with a peptide based on the CDR3 of an anti-DNA antibody reverses the pattern of pathogenic cytokines.

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA monoclonal antibody that bears the 16/6 idiotype (Id) was shown previously to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated responses. When injected, concomitant with active immunization with the pathogenic human anti-DNA, 16/6 Id+ mAb, pCDR3 inhibited the proliferation of LN-derived T cells stimulated in vitro with the 16/6 Id mAb. The inhibition of the specific proliferative responses could be reversed by the addition of exogenous IL-2 to the cultures. Analysis of secreted cytokine profile in supernatants of these cultures demonstrated that pCDR3 treatment reduced significantly the levels of both IL-2 and IFN-gamma that were elevated further in cells of the 16/6 Id-immunized mice. The CDR3-based peptide was shown here to immunomodulate in vivo experimental SLE, induced by the human anti-DNA 16/6 Id+ antibody. The beneficial effects of pCDR3 on the clinical manifestations of SLE were associated with downregulation of the Th1-type (IL-2, IFN-gamma) and proinflammatory (TNF-alpha) cytokines, whereas the immunosuppressive cytokine TGF-beta was up regulated.