Pushing the Envelope of Synthetic Binding Proteins with Monobodies

Shohei Koide, PhD, Professor of Biochemistry and Molecular Pharmacology, New York University School of Medicine

Dr. Shohei Koide, PhD, opened the presentation on how to best target intracellular proteins. Biologics have made all surface molecules and extracellular proteins druggable, however, there are undruggable proteins within the cell. He explained the many specificity challenges associated with using a smaller molecule due to many more proteins in the cell with similar homologs.These findings are significant because T-cell receptors, (TCR) — like antibodies — combine the recognition of intracellular proteins with the versatility of monoclonal antibodies (MAb) to help expand the repertoire of therapeutic antibodies that are effective against chronic diseases like cancer. This presentation provides insight on overcoming the associated challenges and use-like-approaches for other undruggable proteins.

Targeting Undruggable Intracellular Proteins

Because it is challenging to target undruggable proteins, intracellular biologics can be used to help overcome the hurdles. This method allows you to define a best-case scenario and potential outcome to help acquire specificity when you biologically interfere with those processes. Dr. Koide introduced three main avenues for targeting intracellular proteins:

Exploiting a mechanistic link to a cell-surface target

Direct targeting

Target major histocompatibility complex (MHC) peptides

RAS is Among the Most Important and Most Challenging Drug Targets

The activated RAS protein pathway is a highly challenging direct drug target due to the inherent difficulties in disrupting the protein. Understanding the RAS pathway and mechanism of action is essential to cancer research primarily because one-third of all cancers derive from oncogenic RAS mutants. The mechanism of action is highly complex, and many protein interactions undermine its activation and function. There are additional hurdles as well.

Ras proteins are small GTPases responsible for cell growth, proliferation, and differentiation. The different Ras isoforms: HRas, NRas, and KRas, generate unique signal outputs, despite sharing common activators and effectors. Ras is activated by guanine nucleotide exchange factors (GEFs) that release GDP and allow GTP binding.Ras proteins also show extremely high affinity picomolar to GTP/GDP and difficulty discriminating single-point mutants from the wild-type. The hypothetical questions used to drive the research focus areas were:

Is it possible to develop highly selective, non-covalent inhibitors to major oncogenic RAS mutants?

Are such inhibitors efficacious?

Rapid Drug Prototyping with Intracellular Monobodies Methodology Explained

The mechanism of action for rapid drug prototyping involves choosing a target to grow a RAS-specific domain before beginning to develop monobodies. Following these procedural elements, the biochemical aspects are addressed:

Affinity/specificity

Epitope structure

At this point, the scientists genetically encode monobodies (vector transfection, viral transduction) and begin the process of protein expression in cells and animals. The cells are then implanted into tumor models for observation allowing scientists to further inform the drug discovery process. Dr. Koide noted that the development of monobodies requires exquisite specificity and requires precise methodology.

Monobody 12VC1 selectively and non-covalently binds to both KRAS(G12C) and KRAS(G12V)

Through experimentation, the clinical findings allude to the fact that monobodies structures hold enormous potential for targeting RAS-type undruggable proteins. Notable clinical findings include:

The monobody 12VC1 demonstrates the ability to bind to KRasG12B with high affinity.

The monobody recognizes the G12C mutation, upon which a shallow pocket forms, which may assist the recognition opportunity. Achieving this shallow pocket process is how to achieve very selective recognition of G12.

If KRasG12V is placed in the cell, a non-covalent inhibition of KRAS G12V is observed, which blocks signaling and inhibits growth in the mouse xenograft. The DOX promoter assists or decreases the desired biological interaction.

Future Direction

Dr. Koid shared that future research will include exploring more RAS-type undruggable proteins that may be able to be targeted using the same approach. The presented scientific findings support the cellular delivery of monobody inhibitors and degraders using protein delivery technologies can assist in developing new drug-specific targets to treat KRas cancer mutations.Additionally, knowing more about monobody structures has the potential for informing novel drug design. Although, it has taken roughly 30 years of historical research efforts on targeting RAS with intracellular biologics to achieve an FDA-approved product (Sotorasib), it paved the way for more clinically validated product offerings in this area.