Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

Abstract

Murine monoclonal antibodies (mAbs) are widely used but have limitations if administered in humans. The use of chimeric or humanized mAbs can reduce immunogenicity. The first step in producing such mAbs is to clone murine variable genes from a hybridoma, but it is possible to amplify both functional and aberrant variable genes, as they coexist in the hybridoma. During the development of a murine-human chimeric antibody, we have cloned from a hybridoma the functional heavy chain variable region (V(H)) and light chain variable region (V(L)) genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen. In this study, we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1, the development of a method to distinguish between the functional and abundant aberrant V(L) transcripts, and the origins of these aberrant genes. The aberrant V(L) gene is derived from OUR-1 cells, while the aberrant V(H) gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells. The aberrant V(H) and V(L) genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells.

Figure 1

Agarose gel electrophoresis of PCR products of variable genes. Murine V_H and V_L genes were amplified from FR1 to FR4 by reverse transcription PCR from hybridoma cells. GAPDH was an internal control. Lane M: DNA marker; lane 1: GAPDH; lane 2: V_H; lane 3: V_L. FR, framework region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Agarose gel electrophoresis of the V_L PCR product before and after BciVI digestion. Purified PCR product of the V_L gene was digested by BciVI to isolate the functional V_L gene. After incubation overnight at 37 °C, most of the product was cut to form two bands of approximately 180 and 190 bp. Non-cut products were rare.

Figure 3

Agarose gel electrophoresis of eight TA plasmids containing V_L digested again by BciVI. Eight TA plasmids containing V_L were digested by BciVI. Three (nos. 2, 5 and 7) were cut into two fragments, indicating no cut in V_L (functional V_L gene). Five plasmids (nos. 1, 3, 4, 6 and 8) cut into three fragments carried the same aberrant gene.

Figure 4

Agarose gel electrophoresis of PCR products of V_H genes using different sense primers. Murine V_H genes were amplified from signal sequence to FR4 by reverse transcription PCR using different sense primers and the same antisense primer. Lane M: DNA marker; lane 1: mixture of three MHALT primers; lane 2: MHALT1; lane 3: MHALT2; lane 4: MHALT3. FR, framework region.

Figure 5

Agarose gel electrophoresis of PCR products of V_L genes using different sense primers. Murine V_L genes were amplified from signal sequence to FR4 by reverse transcript PCR using different sense primers and the same antisense primer. Lane M: DNA marker; lane 1: mixture of five MLALT primers; lane 2: MLALT1; lane 3: MLALT2; lane 4: MLALT3; lane 5: MLALT4; lane 6: MLALT5. FR, framework region.

Figure 6

Alignment of the DNA sequences of abVH-LF8 (HM046413) with other V_H genes. The DNA sequence of aberrant abVH-LF8 was aligned with an aberrant V_H gene (EU121635) and two functional V_H genes (U60820 and AY646832). Signal sequences are underlined and complementarity-determining regions of functional genes are shadowed. Gaps are represented by dashes, identical bases by *.

Figure 7

Agarose gel electrophoresis of functional and aberrant V_H and V_L genes using different sense primers and corresponding antisense primer. Lane M: DNA marker; lane F: functional variable genes (sense primer MHALT1 for V_H and sense primer MLALT2 for V_L); lane A: aberrant variable genes (sense primer MHALT3 for V_H and sense primer MLALT1 for V_L).