Engineering Antibodies to Avoid Escape Mutants

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For infectious diseases, both bacteria and viruses have the ability to escape the host immune system and specific antibody treatments via mutations that change their surface proteins or structure, creating so-called escape mutants that are no longer neutralized by the specific antibody. The best way to circumvent this is by using a combination of antibodies directed at different viral targets. The use of cocktails of two or more antibodies was shown to provide synergism or additive effects in neutralizing hepatitis B virus (HBV) and RSV infections. Combinations of antibodies have also been used in HIV, targeting GP41 and GP120 viral proteins, and rabies. And are part of the strategy in most anti-virtal drug development companies.

Therefore, part of the early preclinical development of such antibody combinations consists of serial passage of virally infected cells (>20 generations) to ensure continued efficacy and the absence of escape mutants. This is combined with testing against known patient isolates, when available. Cocktails of antibodies would also be an interesting approach to target groups of infectious agents often seen in parallel in (i.e. burn wounds)

Another strategy to increase efficacy, the ability of the treatment to reach the intended target, and avoid unwanted side effects resulting from the killing of non-target cells is antibody engineering. More precisely, genetic manipulation of the Fc domain (mainly in the CH2 domain) or changes to the glucosylation pattern of the N-linked oligosaccharide moieties attached at antibody N297 in the Fc part of the heavy chain.

For generating antibodies with enhanced effector functions, different mutations have been identified that have increased affinity to the FcγIIIa receptor and a significant enhanced cellular cytotoxicity (S239D/A330L/I332E) These antibodies either directly or indirectly enhance binding of Fc receptors and thus significantly enhance cellular cytotoxicity. Enhanced effector function can also be achieved by modulating the oligosaccharide moieties. Removal of fucose from the A297 linked oligosaccharide moietites, which creates so-called afucosylated Fc domains, has been shown to greatly increase the potency for inducing antibody-dependent cellular cytotoxicity. This is achieved by manufacturing the antibodies in cell lines lacking the enzyme fucosyl transferase, which renders them unable to add fucose to the oligosaccharide moieties.

Similarly, ways to reduce or ablate the ability of antibodies to trigger effector functions have been described and are being used broadly in cases where the aim is to block specific membrane-bound receptors/targets and where killing of the cell harboring the target is not desired. Again, mutations in the Fc part (L234A and L235A), also called the LALA mutation, greatly reduce but do not completely remove effector functions by removing amino acids important for the C1q factor of complement.

Modulation of the glycosylation pattern, in this case creating completely aglycosylated antibodies, has also been shown to remove the ability to properly bind Fc receptors on effector cells and trigger effector functions. One alternative approach used especially when developing immuno-modulatory agonistic antibodies is the use of antibodies of the IgG4 isotype, which does not trigger effector functions. Finally, mutations in the Fc part that increase the affinity to the FcRn receptor have also been used to create antibodies with an increased half-life. Introduction of three mutations in the Fc domain (M252Y, S254T, and T2556E, also called the YTE) has been shown to provide a half-life extension of 3- to 4-fold. From a convenience point of view, a long half-life is obviously attractive, but it can be a down-side in the case of severe adverse effects due to the long duration of action.