Antibody optimization is the goal pursued by EvoGenix (Mountain View, CA and Melbourne, Australia). The company has focused on the goal of superhumanization, as explained by David Wilson, Ph.D., vp of antibody products.
Over the years, antibody engineers have grappled with the problem of the antigenicity of therapeutic antibodies, with varying degrees of success. In the early days of clinical trials with mouse monoclonals, patient reactions were so severe that the antibodies were ineffective after a first round of treatment.
The situation improved with the introduction of chimeric antibodies, in which murine constant regions were replaced with human sequences. Later humanized antibodies provided even fewer immunogenic sequences, in which the mouse framework regions of the variable portions of the antibody molecule were replaced by human amino acid sequences.
This technology enabled even more successful responses in patients. Finally, mice engineered with human immune systems offer the possibility of fully human recombinant antibodies containing no murine sequences whatsoever. Fully human monoclonal antibodies are produced by transgenic mice, developed by Medarex and Abgenix.
While fully human antibodies have provided reagents of low antigenicity, they carry with them certain disadvantages, including the tightly locked intellectual property. On the other hand, humanized antibodies retain their original epitope specificity, and according to Dr. Wilson, rarely provoke a significant immunogenic response in patient trials. In fact, 7 of 16 therapeutic antibodies currently on the market are humanized.
Humanization improved the response of patients to the injection of a foreign protein, but it frequently resulted in a depression of the antibody's affinity, as the molecule was stretched into an inappropriate configuration.
A more sophisticated approach is to introduce human framework regions that occur naturally in association with complementarity determining regions (CDRs) that are structurally similar to those of the mouse antibody. This sort of grafting technique results in a modest drop (approximately 14-fold) in affinity.
As Dr. Wilson explained, this loss of affinity could be compensated for by the EvoGene affinity improvement technology. This approach combines RNA-based mutagenesis and ribosome display.
In searching for an ideal mutagenesis platform, Wilson's colleagues were attracted to the RNA viruses, well known for their genetic instability. This feature of the RNA viruses has confounded the search for successful vaccines for diseases caused by them. An RNA virus that infects bacteria, called Qb, copies its genome with an error-prone replicase that has ideal properties to be exploited for directed molecular evolution.
When a gene for an antibody fragment or any other protein is cloned between the recognition sequences of the Qb replicase, it can be rapidly copied, yielding 12 mutations per gene. The striking feature of this replicase is that there are virtually no biases in the positions or types of mutations that arise, which is far from the case using DNA-based or chemical mutagenesis methods.
This allows the best range of point mutations to be obtained, with minimal alteration of the overall sequence. By isolating improved antibodies and repeating the whole process, better and better antibodies can be isolated.
As Dr. Wilson puts it, "the important point is to follow a minimum mutation pathway' to identify optimized variants of the starting molecule while minimizing the overall number of mutations.
"The best way to ensure this is to be thorough in the search of sequence variants in the immediate neighborhood of the starting human or humanized protein, which is made possible by exploiting the unique properties of the Qb RNA replicase."
EvoGenix has employed the Q optimization technology for a number of projects, including improving the enzymatic performance of -lactamase, selective reduction of binding of a growth factor to one of two interacting proteins, and increasing the affinity of antibody fragments.
This approach allowed a 23-fold improvement in affinity of a single domain antibody directed against the malaria antigen AMA-1.
The Evogenix approach offers an innovative means to achieve potent molecular improvement through a minimalist approach that preserves as much as possible the native sequence of the human or humanized antibodies and other proteins, thus minimizing the likelihood of immunogenicity in patients. Although none of these evolved proteins has yet entered the clinic, the biochemical validation of the approach reveals great promise.
Therapeutic antibody technology has now matured to a point at which earlier successes are being advanced into the next generation of improved products.