Immunochemical and structural analysis of llama anti-idiotypic VHHs bearing DNA internal image. (Poster)

LAURA M. ZAREBSKI; SEBASTIÁN KLINKE; MARIELA URRUTIA; FERNANDO A.GOLDBAUM

Angra dos Reis, estado de Río de Janeiro, Brasil

Congreso; 1st Latin American Protein Society Meeting (LAPSM); 2004

Protein Society

In camelids, a subset of the immunoglobulins consists of heavy chain homodimers devoid of light chains, and are thus called heavy chain IgGs (hcIgGs). Their variable region (VHH) is the smallest antigen-binding fragment possible, and being just one polypeptide chain it is especially suitable for engineering. From llamas immunized with an anti-DNA mouse IgG, we sought to obtain anti-idiotypic VHHs (a-Id VHHs) able to back-elicit anti-DNA antibodies by molecular mimicry. Upon immunization of llamas with ED84, a monoclonal anti-DNA antibody with high affinity and sequence specificity to a ds18-mer (Site35), high titers towards ED84 Fab fragment were obtained. Total IgGs were purified and hcIgGs and conventional IgGs were isolated. By ELISA, both fractions showed significant binding to ED84. Subsequently, VHHs cDNA was prepared from total RNA of mononudear cells and was used to construct a phage display library of 108 individual clones. After two rounds of solid phase panning with specific elution (i.e., with Site35), a 58% of a-Id VHHs was obtained. By contrast, non-specific elution (with glycine 0.1 M pH 2.2) resulted in a significantly smaller proportion of a-Id VHH. The sequence from the a-Id dones eluted with Site35 revealed a strong selection (90%) of clones bearing a consensus sequence. Confirming this specific selection, their binding to ED84 was inhibited by Site35, indicating they are Ab2α. In contrast, non-specific elution yielded a 30% of anti-Id clones with this consensus sequence. From the inhibition and affinity measurements, clone VHH79 was chosen to continue structural and functional studies. Clones VHH79 (Ab2α, "private a-Id") and VHH28 (Ab2β, "public a-Id") were injected in mice. Preliminary results show that it is possible lo obtain anti-DNA antibodies from the response to private anti-idiotypic antibodies, confirming that a VHH is capable of bearing an "internal image" of DNA. X-ray crystallographic data from VHH79-ED84Fab complex was collected at 2.90Å at the Laboratório Nacional de Luz Síncrotron in Campinas, Brazil. The structural analysis of this Id-anti-Id complex would allow to define at the atomic level the molecular basis of mimicry by a single domain antibody.