No 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.

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.