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.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

“…DISCUSSION A key step in the development of antibody therapy is the identification of target antigen-specific antibodies. Hybridoma [25][26][27] technology is commonly used in screening and selecting these antibodies. In most situations, they are nonhuman antibodies, which need to be humanized for further development as therapeutics.…”

Comparison of two functional kappa light‐chain transcripts amplified from a hybridoma

“…The major problem in rapidly obtaining chain variable genes from a hybridoma is the occurrence of aberrant mRNAs that can be transcribed but that have no function. If the aberrant gene is used in the construction of the chimeric or humanized antibody, it may produce immunoglobulin that has low affinity for the antigen and is completely nonfunctional . We encountered such a situation in our experiments.…”

“…X58634) believed to be derived from the fusion partner P3×63‐Ag8.653. Aberrant VH genes are less often reported but are more complicated and not as widely recognized as that of the MOPC21κ allele .…”

Development and application of monoclonal antibodies to Chikungunya virus proteins

“…The first step in attempting to create a chimeric or humanized mAb is to clone the functional immunoglobulin variable genes via PCR amplification using degenerate primers and the cDNA of a hybridoma as template. However, the occurrence of transcribing non-functional genes from aberrant heavy and/or kappa light chain mRNA transcripts is relatively high (Carroll et al, 1988;Kutemeier et al, 1992;Ding et al, 2010). The myeloma cell line P3X63Ag8.653 used for the creation of the 5.2H7 hybridomas was derived from MOPC-21 cells, which are known to express endogenous immunoglobulin heavy and light chain transcripts, as well as harbor aberrant, non-functional genes (Strohal et al, 1987;Irani et al, 2008;Ding et al, 2010).…”

“…Primer sets are available for amplifying variable region genes from mouse hybridomas (Morrison, 2002), but these primers are not exhaustive and some hybridoma antibody genes can prove to be difficult to amplify (Carroll et al, 1988;Ding et al, 2010). In this study, we…”