Human monoclonal antibodies to HIV-1 gp140 from mice bearing YAC-based human immunoglobulin transloci

Abstract

Mice carrying human immunoglobulin transloci were immunised with HIV-1 gp140 antigen to gain insight into the range and nature of human monoclonal antibodies (mAbs) that can be elicited from such humanised mice. Using five-feature mice that harbour YAC-based germline-configuration human IgM, Igκ and Igλ transloci in a mouse background disrupted for endogenous mouse IgH and Igκ expression, gp140-specific human IgM mAbs were readily elicited following serial immunisation. These mAbs were converted to human IgG1 format and were found to bind diverse epitopes within gp140, exhibiting high functional affinity for the antigen-typically in the nanomolar or sub-nanomolar range. The number of specific, stable hybridomas per mouse was, however, low (typically around five) with the hybridomas within individual mice often being clonally related. Nevertheless, different mice used B cell clones expressing varied V(D)J combinations, with affinity maturation through somatic hypermutation making a critical contribution. Thus, a wide range of distinct high-affinity mAbs can be obtained by immunising multiple animals. The results confirm the utility of the translocus-mouse approach and give insight into strategies for possible future improvement.

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

“…A comparison of immunization results, based on Ag-binding and isotype (see Tables III, IV), would make it near impossible to identify whether the results were obtained from normal wt rats or from OmniRat. This appears to be very different from the selected transgenic human Ab results made available and from the experience we had respective to the relative performance of the transloci and endogenous loci in mice carrying fully human IgH transloci (15,19,55).…”

“…The molecular diversity of the Abs produced was considerable, with contributions as anticipated (44) from nearly all the V, D, and J gene sequences on the transloci segments. This was in stark contrast to some mice carrying fully human transloci where selective clonal expansion of relatively few precursor B cells was found to yield only limited molecular diversity (19,55). Thus, for example, five-feature mice expressing fully human Ab repertoires showed a substantial reduction in the frequency of IgM + B cells in the bone marrow from the pre-B-cell stage onward: frequencies were 21% of those observed in wt mice (56).…”

“…However, a distinct attraction of using an IgH translocus in which the IgH C regions are of endogenous origin is that regions toward the 39 end of the IgH locus are known to play a major role in achieving efficient Ig class switch recombination as well, probably as optimal IgV gene somatic hypermutation (19,51,52). There are substantial differences between mouse/rat and human in the sequences in this enhancer region of the IgH locus, located some 30 kb downstream of Ca (53).…”

“…Binding kinetics were analyzed by surface plasmon resonance using a Biacore 2000 with the Ags directly immobilized as described (19).…”

“…Indeed, it has been suggested that suboptimal performance of these humanized mouse strains with regard to the efficacy with which they yield human mAbs might result from imperfect interaction between the C region of the human Ig expressed on the B cell membrane and the mouse cellular signaling machinery (19). Because the transgenic mice can essentially be viewed as a source of Ag-specific IgV genes (with the desired IgC region provided at a later stage during the creation of cell lines for bulk Ab production), we wondered whether the transgenic approach could be improved if the germline configuration human IgV H -D-J H segments were linked to endogenous (rather than human) IgC H regions.…”