Introduction: Breast cancer is a heterogeneous disorder characterized and second lethal cancer in women. Approximately 15% to 30% of breast cancer tumors show over-expressed HER2 protein as a result of gene amplification. Cancer treatment strategies are divided into two groups, traditional and modern strategies. Traditional approaches such as chemotherapy, radiotherapy, and surgery are rather non-specific. On the other hand, modern strategies or targeted therapies focus on the specific biomarkers that target cellular molecular pathways to stop the cancer cells. One of the targeted-therapy approaches against HER2 is Antibody-drug conjugates (ADCs). ADCs allow selective targeting of cytotoxic drugs directly to cells presenting tumor-associated surface antigens, thereby minimizing systemic toxicity. First-generation ADCs use linking technologies that conjugate drugs non-selectively to either cysteine through sulfhydryl groups or lysine through amine group residues on the antibody, resulting in a heterogeneous mixture of ADCs. This approach leads to suboptimal safety and efficacy properties and makes optimization of the biological, physical and pharmacological properties of ADCs challenging. Due to this, second-generation ADCs are being made to be more specific through approaches such as cysteine engineering which are challenging as well.
Genetic code expansion (GCE) is a novel and promising technology. It is used to incorporate non-proteogenic, unnatural amino acids (UAA). GCE, as the name suggests, expands the genetic code by using modified tRNAs/aminoacyl tRNA synthetase to assign UAAs to new or existing codons present within a protein of interest. This technology has various applications depending on the UAA incorporated. By applying GCE to ADCs we aim to improve homogeneity and the precision of both conjugation sites and stoichiometry. We believe that more precise conjugation will remarkably reduce the required dosage of cytotoxic agents, therefore, helping to improve therapeutic windows.
Methods: We designed corresponding vectors to introduce two unnatural amino acids to the selected monoclonal antibody. The genetic code assignment is done by means of a false amber stop codon. The setup was first established in a synthetic model protein reporter construct. Subsequently, the genetic code of the monoclonal antibody was expanded in two positions and the efficiency of these two sites was analyzed using biorthogonal chemistries of unnatural amino acids. Purification and mass spectrometry are done to confirm genetic code expansion.
Results: The confocal microscopy images of cells that were transfected with model protein demonstrated transmembrane fluorescence signal only in the presence of unnatural amino acid in the medium which suggests the expansion was done successfully. For the monoclonal antibody, the IDDEA and SPAAC reaction between unnatural amino acid residues and specific fluorophores proved genetic code expansion and the presence of unnatural amino acid residues in the antibody.
Conclusion: Here, we demonstrate that our two developed constructs are productive for the corresponding unnatural amino acids in vitro and the Genetic Code Expansion of the selected monoclonal antibody is possible with these constructs. Besides, the incorporated unnatural amino acids are accessible for biorthogonal chemical reactions.