Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting
Limitations To Current Antibody Therapeutics
Two problems revolve around standard antibodies, each unique to outer cell membranes targeting antibodies and CPP tagged antibodies.
Monoclonal antibodies (mAbs) are outer cell antibodies that mimic the activities of the body's immune system and attack cancer cells. Naked mAbs bind to antigens located on the surface of irregular cells. These act as a marker for the body’s immune system to trigger an immune response and bind to cell growth receptors located on the outer surface of the cell.
While the immune system is the natural protection against bacteria and disease in the human body, it has been proven efficient, especially in immunosuppressed patients. The antibody destroys cancerous cells after it has been produced and accumulated to then try and bind to the numerous growth receptors on each cell. The lack of target specificity of monoclonal antibodies also causes immune reactions and adverse side effects such as infections and cardiotoxicity.
mAbs are not effective in stopping the uncontrolled cell proliferation, while the success of the attempts to destroy and block cell receptors may work in strict conditions limited to a person with a strong internal immune system and the magnitude of cancer cell accumulation is of small scale.
To overcome the limitations of monoclonal antibodies, CPP tagged scFv antibodies were engineered in order to create a system that will directly interfere with intracellular protein interactions. However, the disulfide bridge linking the VH and VL of the antibody dissolves in the reducing environment that normally contributes about 4-5 kcal/mol of antibody domain stability.
Consequently, immunoglobulin antibody folding is not properly executed, restricting the antibodies with promising target specificity and affinity to properly function within the cell. The disulfide bond antibodies are thus insoluble and result in cytotoxicity when entered a normal cell. High thermodynamic stability is required for cytoplasmic expression; however, the effect of the reducing environment disables such work.
Significance of scFv(F8)
The hyperstable scFv(F8) antibody is one of the few antibodies that do not rely on disulfide bonds for the regular process of protein folding. The scFv(F8) antibody is tagged by CPP derived from Porcine circovirus type 2(PCV2).
The nuclear localization signals (NLS) produced by the PCV2 capsid protein (Cap) create positively charged residues and act as CPP. Multiple endocytic processes of the CPP tagged scFv allows rapid internalization of the antibody across the cell interior. While the antibody engages in target-specific binding, it avoids binding to any other cellular components that could possibly lead to side effects.
Ras and scFv(Ras)
scFv(Ras) is an engineered form of scFv(F8) made to target the Ras protein by grafting antigen-binding sites of anti-Ras antibody to scFv(F8). The grafting is based on the anti-Ras antibody and Ras protein complex structure (PDB ID: 2vh5). The oncogenic Ras protein is involved in a major signaling pathway mediated through the guanosine triphosphate (GTP) bound active and guanosine diphosphate (GDP) inactive states. When Ras or the downstream effectors such as PI3K and RALGDS is mutated, the Ras protein is left in a constant active state leading to abnormal cell division.
Ras mutation has been found in 30 percent of humans and is of the highest frequencies in colon, lung, pancreas, and adenocarcinoma cancer. When the CPP tagged hyperstable scFv(Ras) penetrates the cell, the antibody can bind to the Ras protein limiting the constitutive signal transduction and cell’s ability to propagate. Furthermore, scFv(Ras) can limit the side effects found in other cancer therapeutics posed to patients by preserving normal cells through target specificity while effectively shrinking the population of cancerous cells.
The eligibility of engineered antibody is being tested through different assays (including binding assay, cell penetration assay, and signal inhibition assay), and protein-protein docking (scFv(Ras) – Ras) will be performed to analyze the binding modes to further improve the effect of antibod