CP-scFvKRAS, Lung Cancer

A Novel Cell-Penetrating Antibody, CP-scFvKRAS For The Efficient Delivery Into Cancer Cells

The RAS proteins (KRAS, HRAS and NRAS) are signaling proteins regulated by GTP binding and hydrolysis. RAS, located intracellularly anchored to the plasma membrane, is activated upon binding of ligand (i.e. epidermal growth factor) to a receptor tyrosine kinase (RTK). RAS gene mutations are found in as many as 30% of human tumors, and most frequently in tumors of the pancreas, colon and lung. Mutant RAS proteins are constitutively active (GTP-bound) and promote cell proliferation in a signal-independent manner. Thus, inhibiting active RAS function has been proposed as a human cancer therapy. Farnesyltransferase inhibitors (FTIs), shown to inhibit membrane localization of RAS proteins, have demonstrated significant anti-tumor activity. However, the FTIs do not solely target RAS but also affect farnesylation of other proteins as well.

Therefore, an ideal RAS-based anti-cancer therapy would involve strategies to directly target oncogenic RAS

Single-chain variable fragment (scFv) consists of the smallest functional antigen-binding domain of an antibody, in which variable heavy and variable light chains are joined together by a flexible peptide linker. scFv retains the binding specificity of the parent antibody and offer several advantages over monoclonal antibodies. scFv displays improved pharmacokinetic properties, such as better tissue penetration, rapid blood clearance, and low immunogenicity which makes better therapeutic agents.

While recent studies have shown a substantial anti-cancer effect of a single chain fragment variable region (scFv) which specifically binds to activated GTP-bound RAS in cancerous cells harboring a RAS mutation, targeting intracellular RAS continues to pose a major challenge. Therefore, TSDT can provide a solution to efficiently deliver scFv intracellularly to solely target RAS mutant to prevent cell division and proliferation.

Cell-Permeable (CP)-scFvKRAS proteins were expressed in CHO cells and screened to identify those with the best solubility and yield on purification. The engineered proteins maintained the ability to bind mutant KRAS proteins with low nM affinity and the antigen antibody complexes were stable for days at 37℃. All of the aMTD sequences enabled a high degree of cell-permeability, and much of the internalized protein colocalized with RAS on the plasma membrane (Figure 1). These results validate the use of TSTD to deliver potentially therapeutic single chain antibodies into cells. CP-scFvKRAS proteins would strongly inhibit cell phenotypes associated with malignant change, specifically cell cycle progression, proliferation and cell viability. In addition, while some of CP-scFvKRAS proteins suppressed constitutive KRAS signaling in NCI-H358 cells, as assessed by decreased steady state levels of phosphorylated Raf, ERK, and AKT, the inhibitory effects were variable and did not always correlate with reductions in cell viability.

Although scFv has higher tissue penetration compared to antibodies, it still requires delivery technology for higher efficacy. Delivering scFv to targeting area is one of the main obstacles in developing scFv as a therapeutic candidate.

To intracellularly localize scFv, many research organizations have tried variety delivery technologies such as nanoparticles, adeno-associated virus (AAV), and cell-penetrating peptides. Therefore, upon successful validation of the technology, further scientific/business collaboration opportunity may be considered with regard to other of candidate therapeutic proteins.

  • scFvKRAS
  • aMTD-scFvKRAS

Figure 1. aMTD-scFvKRAS internalizes into the cell & binds to active Ras mutant in living cells (red: scFv, green: KRAS).