Intrabody Drug Development

Currently, antibody drug therapy has achieved great success in treatment of cancer and autoimmune diseases. In addition, there is progress made in its use for treatment of metabolic and infectious diseases. The current antibody therapy is amenable to target only extracellular antigens that are accessible for antibody binding, which is the major limitation for further expanding therapeutic antibody development.

The antibody target space lies in their inability to efficiently penetrate cells and reach the cytosol. Under normal physiological conditions, antibodies, due to their large size and hydrophilicity, cannot readily cross intact cellular or subcellular membranes in living cells in order to reach the disease associated protein–protein interactions, occurring mainly in the cytosol of cells. In contrast, small molecules have been used to target those intracellular antigens with a functionality that is suitable for drug screening, but in comparison to antibodies, small molecules tend not to be as selective for their targets. They can exhibit unpredictable off-target activities, which consequently can lead to adverse side effects and may require a more individualized clinical development pipeline.

The antibodies that target intracellular antigens can open the door to a whole new realm of therapeutic targets, with potentially immense clinical benefits, which have broad clinical potential.

Overall, it is very important to develop a novel strategy and method for the generation of therapeutic antibody to recognize intracellular targeted proteins. Therefore, there is an increase in demand for efficient methods for antibody delivery into the cytosol of living cells applicable for a diverse array of functions. Many attempts have been made to directly deliver antibodies into intracellular compartments, and these can lead to the opportunities and challenges detailed below.

Currently, there are three broad approaches that antibodies have been used to target intracellular antigens.

(1) It is possible for antibodies (or their derivatives) to target antigens that are normally intracellular but become externalized (for example, during disease).

(2) It is also possible to engineer antibodies or antibody fragments that penetrate into cells, or those that are directly expressed within cells using a gene therapy style approach.

(3) Antibodies can also be generated to bind with cell surface major histocompatibility complex class I (MHC-I)-presented peptides that are derived from intracellular proteins.

(1) The first is a type of “gene therapy” approach using vectors that enable expression of the intracellular antibodies within the target cell—these can be either viral vectors or plasmids. 

(2) The second is direct administration of the antibody-based therapeutic—either alone, using electroporation or with dendrimers, liposomes, nanoparticles, or by fusing the antibody to protein-transduction domains that enable it to penetrate the cell .

      A) Microinjection, electroporation, liposome, nanoparticles and protein transfection (profection). 

      B) Using targeted receptor-mediated endocytosis and genetic or chemical conjugation with protein      

             transduction domains that directly penetrate intoliving cells. 

     C) The conjugation with protein transduction domains, which are represented by cell-penetrating        

             peptides (CPP) such as the HIV-1 TAT peptide has been extensively attempted in order to facilitated

             intracellular delivery of antibodies formatted as single chain variable fragments (scFvs), antigen

             binding fragments (Fabs), and full-length IgGs. 

A) These methods are successful for delivering antibodies into the cytoplasm of cultured living cells, but many issues, including cytotoxicity, loss of antibody stability, and difficulty of systemic administration, remain unresolved.

B) The large molecules, including antibodies that enter epithelial cells via receptor-mediated endocytosis, are usually retained in endosomes and are then recycled out of the cells or are degraded in lysosomes without being released into the cytosol.

C) Most of the CPP-conjugated antibodies inherited the intrinsic intracellular trafficking of the parent CPPs, which were either entrapped inside endocytic vesicles, translocated into the nucleus, or eventually degraded in lysosomes without efficient endosomal release into the cytosol.