Mutant-specific antibodies: improvements and new targets

Sequence variations of single nucleotides in our genomes are responsible for the phenotypic variation of humans. If a single nucleotide change in a gene alters the function of the encoded protein, then the phenotypic consequences of such a mutation may reach from an increased susceptibility for to the manifestation of disease. The analysis of the molecular mechanisms of autosomal-dominant inherited diseases caused by single amino acid changes is difficult because of the lack of specific tools that allow to distinguish between the mutant variant and its wild-type counterpart, which are present in the same cell. Thus, we proposed in the previous project to generate monoclonal antibodies specific for laminopathies-associated mutant proteins. We chose these diseases because most laminopathies are caused by single point mutations, are autosomal-dominant diseases, show heterogenous phenotypes, and the knowledge of disease pathogenesis is still limited. Furthermore, we had the unique opportunity to pursue an in-house collaboration with Dr. Roland Foisner, a leading scientist in this field, which was highly beneficial for the project progress. With our antigen display we succeeded in eliciting lamin A mutant-specific immune responses but due to the instability of the hybridoma clones, a well-known but unresolved technical problem of the hybridoma fusion method, we only obtained two mutant-specific monoclonal antibodies. Because of this uncertainty of the method the generation of a monoclonal antibody is still a time-, labor- and thus cost- intensive process as reflected in the high costs of custom monoclonal services. To solve the "instability problem" I propose a novel approach for the generation of stable antibody-secreting hybridoma clones. We will apply this novel method to generate monoclonal antibodies against the previously proposed as well as new lamin A mutants associated with phenotypic different laminopathies. With the novel hybridoma fusion method it should be possible to significantly reduce the expenditure of time, labour and costs of monoclonal antibody production. Moreover, the lamin A mutant-specific monoclonal antibodies will help the researchers to analyse the disease pathogenesis on the molecular level and to answer the key question, how different point mutations in lamin A can cause so many different disease phenotypes. Hence, the outcome of the project is expected to be of great scientific and commercial value both for basic as well as potential clinical applications.

Monoclonal antibodies are among the most important analytical research tools in the life sciences. Even more important is their diagnostic and therapeutic use in medicine, which makes monoclonal antibodies a multi-billion dollar business. The standard method of generating monoclonal antibodies still follows a protocol that remained almost unchanged since its development in the 70ies despite methodic imponderabilia such as the genomic instability of the hybridoma cells that are dealt with by time-, material- and labor-intensive measures. Large numbers of hybridoma cells have to be generated and screened until cells can be isolated that produce monoclonal antibodies of the desired antigen specificity making monoclonal antibody production a cost-intensive process. Therefore, the prime goal of this project was to improve the (efficiency of) current protocol of monoclonal antibody generation. Our novel approach significantly reduces the time, labor and costs from hybridoma creation to monoclonalization. In the second project part we continued the work on the major goal of the precedent project, which dealt with the generation of monoclonal antibodies specific for laminopathies-causing mutant proteins. Laminopathies are autosomal-dominant inherited, phenotypically diverse diseases that are caused by single point mutations in the lamin A/C gene. We could show that antibodies with such exquisite specificity are excellent tools for the analysis of the disease pathogenesis on the molecular level. For two of the now five different lamin A/C mutant-specific monoclonal antibodies we already concluded licensing agreements with 4 international biotech companies making the project a commercial success.