Solving Drug Discovery Challenges:

Overview

The two most common general methods for discovering monoclonal antibodies are immunization of animals and molecular evolution, such as phage or yeast display. While there are limitations to both methodologies, the use of display technologies has many important advantages to the discovery of therapeutic drugs.

First, antibody libraries can be created “synthetically” thereby allowing for diversity that is not limited by in vivo biological systems. Also, display technologies are less dependent on target homology as there is no immune system to protect against self-reativity. Next, binding can be enhanced or removed for specific regions with biochemical positive and negative selection. Finally, yeast display technology allows for the selection of characteristics that are important for drug development such as increased expression, Tm, and stability. Yeast display thus increases the probability that an antibody will have good biophysical and manufacturing properties necessary for an antibody drug.

Herein, we provide examples of antibody optimizations using yeast display that provide advantages beyond binding potency to the human target.

Method Highlights

Molecular Evolution with Yeast Display:Antibody optimization libraries were created by either error-prone PCR techniques for the light and heavy chains (p95 and 1 TM) or synthetic design of CDRs and humanization framework toggle-points (soluble target).

Protein Generation:Protein production utilized expi293 cells with protein A purification.

Binding Assays:SPR/BLI-based binding was done by affixing antibodies to the surface with an anti-human antibody and flowing over the targets, using either an Octet or Biocore

Results

• A truncated form of HER2, the P95 variant, is specifically overexpressed on cancer cells and represents an interesting target for cancer therapies, particularly T cell engaging bispecifics
• Current anti-P95 antibody is poorly expressed and has low potency
• We used error-prone PCR on the heavy chain to improve expression and affinity

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Yeast display improved binding potency of p95 antibody

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Anti-p95 expression levels were increased by 50-fold in the higher potency variants and SEC shows high monomericity

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Conclusion:

Using yeast surface display, a poorly expressed p95-binding mAb was optimized for nearly an order of magnitude improvement in potency and nearly two orders of magnitude increased expression.

These characteristics provide advantages for antibody drug devlopment beyond binding potency to the human target.