A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain

Abstract

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

Fig 1

(a) Schematic of PCR amplification of V_H and V_L cDNAs by signal peptide (SP) primers. Right panel shows light chain PCR, which typically results in amplification of myeloma cDNA by some primers (primarily SP2) and antigen specific cDNA by other SP primers. (b) DNA gels. For the heavy chain (left panel), SP4 gave the major product, which was sequenced and revealed a full-length V_H cDNA. For the light chain (right panel), SP2 amplified the non-productive Sp2/0 myeloma kappa chain, whereas SP7 primer amplified a full-length V_L cDNA.

Fig 2

(a) Flow cytometry histogram in far red channel showing staining of Cho-hCD63-eGFP cells, but no Cho WT cells, with 25 nM AF647-modified H5C6 hybridoma-derived mAb. (b) Binding curve derived from flow cytometry data (n = 3).

Fig 3. Characterization of recombinant mAbs.

(a) Schematic of recombinant mAb constructs. VL sequences were cloned in frame with human kappa CL, while VH was cloned in frame with human CH1-CH3. The sortag sequence, LPETGG, was appended at the C-terminus, immediately after the CH3 domain. (b) SDS-PAGE of recombinant mAbs, 1 = non-boiled, non-reduced, 2 = boiled, reduced. (c) Size exclusion HPLC of SP2/SP4 and SP7/SP4 mAbs, showing a single peak on the A280 detector at the expected size for immunoglobulin (7.9 min). (d) Flow cytometry histogram showing binding of 20 nM SP2/SP4 recombinant mAb, but not 20nM SP7/SP4 mAb, to Cho-hCD63-eGFP cells. (e) Flow cytometry-based binding curve of Cho-hCD63-eGFP cells (neither mAb showed any binding to Cho WT cells). (f) The affinity of SP2/SP4 mAb was similar to that of H5C6 hybridoma-derived monoclonal antibody (n = 3).

Fig 4. Identification of frameshift-corrected analog of Sp2/0 V_L cDNA.

(a) Results of Edman sequencing of H5C6 hybridoma-derived mAb—summary, standards, and first three residues are shown. (b) Comparison of amino acid sequences of full-length SP2 V_L cDNA and aberrant Sp2/0 myeloma sequence. Both the frameshift mutation and the conserved cysteine (C23Y) mutation are corrected in the full-length gene product.