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

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.

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

The identification of antibody variable regions in the heavy (V H ) and light (V L ) 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 V L genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma V K and a unique, full-length V L . After finding that the unique V L 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.

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci

Trans-chromosomic (Tc) mice carrying mini-chromosomes with human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies (Abs); however, we previously observed that mitotic instability of human mini-chromosomes in mice has limited the efficiency of hybridoma production. Here, we established a new generation of human Ab producing Tc mice (TC-mAb mice), which stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. Comprehensive, high-throughput DNA sequencing revealed that the human Ig repertoire, including variable gene use, was well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, immunized TC-mAb mice exhibited more subsets of antigen-specific plasmablast and plasma cells compared with wild-type mice, leading to high efficiency hybridoma production. Thus, TC-mAb mice offer a valuable platform to obtain fully human therapeutic Abs and to elucidate the regulation of human Ig repertoire formation.

Production and Characterization of a Monoclonal Antibody Neutralizing Poliovirus Type 2

Monoclonal Antibodies (mAbs) are used for biomedical research, diagnosis, treatment, production, and the quality control of biological products. mAbs are also very helpful in poliovirus research studies because it is still one of the major public health problems in developing countries. The main objective of this study was the production of mAbs against Poliovirus Type 2 (PV2) to be prepared and respond to the re-emergence of this virus. After fusion of immunized B cells prepared from mice with myeloma tumor cells and screening of about 250 hybridoma colonies, 22 with the highest antibody titer and without cross-reaction with others types were selected and cloned by limiting dilution. In the end, two colonies capable of secreting mAbs against epitopes of PV2 were used to produce mAbs. The mAbs were characterized by antibody assays, isotyping, and epitopes analysis using western blotting test, the crossreactivity with other types, as well as stability, sterility, and mycoplasma tests. The results indicated that the produced mAbs had high specificity, sensitivity and stability against PV2 without any cross-reactivity and were of IgG1 Kappa chain with similar bands at 26 kDa during electrophoresis associated with viral protein VP3 neutralization. These mAbs were specific in serum neutralization tests for PV2 vaccine strain, and therefore, they are potentially valuable for routine polio research, diagnosis, isolation, production, and control of poliovirus vaccines.

Comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing

ABSTRACTThe humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoire of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.