Production of Antigen-Specific Human Monoclonal Antibodies: Comparison of Mice Carrying IgH/κ or IgH/κ/λ Transloci

Abstract

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/Ig loci and harbor human IgM + Ig (BAB) or human IgM + Ig + Ig transloci (BAB,). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BAB comprise human chains that are associated with mouse chains. In contrast, BAB, mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Ig translocus (in addition to the human IgH + Ig transloci) not only diminishes problems of endogenous mouse Ig expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.

Selection Strategies III: Transgenic Mice

“…4.4 Flow cytometry analysis of fi ve-feature mice (carrying three human transloci, IgH, Igκ, and Igλ and two disabled endogenous loci mouse IgH and Igκ). The analysis shows a 1/3 to 2/3 B-cell recovery rate with, for example, the number of Ig + splenic lymphocytes being ∼20% in the human Ig mice compared with ∼30% found in normal mice kept under the same conditions Brüggemann 2004). showed that human antibody titers are reduced compared with those of normal nonmanipulated animals Wagner et al 1994a,b;Jakobovitz et al 1995;Magadán et al 2002). Despite low-serum titers, human Ig loci mice are capable of mounting antibody responses to a wide range of antigens and, similar to normal mice, increased levels of specifi c antibodies are visible 2-3 weeks after primary immunization and can be further increased by secondary immunization.…”

“…A plausible reason is that maintenance of the transferred HAC as a separate human chromosome in the mouse cells may interfere with recognition by the cellular machinery affecting chromatin structure and locus accessibility (Jenuwein and Allis, 2001). In all the transgenic Ig strains a large proportion of the "human" antibodies contain mouse λ L chain and thus are in chimeric confi guration (Fishwild et al 1996;Mendez et al 1997;Nicholson et al 1999;Magadán et al 2002). Although the mouse Igλ locus has been silenced by gene targeting , human Ig mice with a background in which all three endogenous mouse Ig loci (IgH, Igκ and Igλ) have been rendered non-functional, and are thus unable to express any endogenous mouse Ig, have not yet been established by cross-breeding.…”

“…This is linked to a more effi cient B cell recovery and much reduced expression of endogenous λ L chain. Although human Igκ antibodies can be easily obtained from fi ve-feature mice, it may be an advantage to use individual four-feature strains, which either express human IgH,λ or human IgH,κ antibodies, to select the type of response Mendez et al 1997;Nicholson et al 1999;Magadán et al 2002;Protopapadakis et al 2005).…”

“…The formation of mixed molecules or polypeptides led to disappointment particularly when some immunizations produced a substantial number of chimeric human antibodies with mouse λ L chain (Russell et al 2000;Magadán et al 2002). Nevertheless antigen-specifi c fully human antibodies were produced and the use of hybridoma technology (for background and methods see King 1998) established a large number of human monoclonal antibodies (mAbs) of good affi nity (Table 4.2).…”

“…The V genes were diversifi ed by somatic mutation but most strikingly eight V H genes coded for an aspartate residue in CDR1 at position 33. Analysis of the fi ve-feature mice described by Nicholson et al (1999) Ishida et al 2002 revealed that the emergence of human IgH,κ or human IgH,λ Ig appears to be antigen-driven and that these mice do not produce signifi cant levels of chimeric human antibodies with mouse λ L chain (Magadán et al 2002;Brüggemann 2004). Immunization of transchromosomal mice resulted in human antibody responses including all Ig subclasses (Yoshida et al 1999;Ishida et al 2002).…”