Anti-idiotypic antibody Ab2/3H6 mimicking gp41: a potential HIV-1 vaccine?

Abstract

Anti-idiotypic antibodies (Abs) represent an alternative vaccination approach in human therapy. This approach is based on the idiotype (Id) network theory postulated by Jerne describing the Ab (Ab1) – anti-idiotypic Ab (Ab2) – anti-anti-idiotypic Ab (Ab3) cascade stimula-tion. Specific anti-Id Abs serve as an " internal image " of the target antigen and can be used to induce Abs able to bind to the cognate antigen [1]. The anti-Id Ab Ab2/ 3H6 [2], developed at our Institute was generated in mouse and is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates [3]. Ab2/3H6 which has been characterized previously [4,5] is able to mimic the anti-gen recognition site of 2F5 and therefore it is suggested as a putative candidate for an HIV-1 vaccine. We investigated the potential of Ab2/3H6 by immuni-zation of Fab fragments and fusion proteins with inter-leukin 15 (IL15) and tetanus toxin (TT) tags as immune modulators. After three prime/boost administrations rabbit sera were purified and analyzed for 2F5-like spe-cific Abs. Further, the 2F5-like Abs from the sera were enriched by affinity purification and characterized for their binding affinity to 2F5. In an additional approach we applied different huma-nization methods to reduce the immunogenicity of the originally mouse derived Ab2/3H6. The mouse variable regions of Ab2/3H6 were subjected to three different humanization methods, namely resurfacing, CDR-graft-ing and superhumanization. Four differently humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the original Ab2/3H6. Results To evaluate the humoral immune response of Ab2/3H6 we designed Ab2/3H6 Fab fusion proteins with IL15 and TT. Recombinant CHO cell lines were established and after protein purification New Zealand white rabbits were immunized with the Ab2/3H6 Fab variants. Ten days after the final boost sera were collected and ana-lyzed for total rabbit IgG levels. After proteinA affinity purification of the sera the isolated rabbit IgGs were tested for Ab2/3H6 Fab and recombinant gp140 (UG37) specificity (Figure 1A). Further an affinity enrichment step using a UG37/ELDKWA column was performed and the obtained Ab3 fraction was tested on UG37 (Fig-ure 1B) and additionally on the original 2F5 epitope ELDKWA (Figure 1C). Finally the Ab3 fraction was tested for binding affinity to the UG37 in a bio-layer interferometry assay which showed that the Ab3 fraction has a 6.6 fold reduced affinity towards UG37 compared to the mAb 2F5 (Table 1). For the humanization approach three different meth-ods were chosen. The " resurfaced " variant (RS3H6) was developed by a computer model and surface exposed amino acids in the murine framework (FR) were substi-tuted by residues usually found at equivalent positions in human Abs. The " superhumanized " form (SH3H6) was designed by structural homologies between the murine Ab2/3H6 CDRs and human germline CDRs.