. Cancer is the 2nd leading cause of death in the USA. Unfortunately, most small chemotherapeutics have profound side effects and hence the major thrust in the field is to develop targeted chemotherapeutics (e.g. Antibody drug conjugates, ADC). A major bottleneck in the development of such ADCs is the need to perform synthetic chemistry on drug candidates to enable linker attachment and covalent antibody bioconjugation while preserving drug activity and maximizing on target delivery. The Isaacs group is a leader in the chemistry of cucurbit[n]uril (CB[n]) molecular containers which are distinguished from other synthetic molecular containers by their outstanding molecular recognition properties (e.g. Ka routinely in the 106 ? 109 M- 1 range) toward hydrophobic and cationic species in water. Our central hypothesis is that the bioconjugation of CB[n] molecular containers to antibodies (e.g. trastuzumab, rituximab, anti GRP78) will result in Antibody- CB[n] conjugates that are able to non-covalently bind unfunctionalized anticancer agents to give Antibody- CB[n]drug complexes that enable targeted delivery of the drug. Secondarily, we hypothesize that Antibody- CB[n] conjugates will enable a powerful pre-targeting approach of (radio)therapeutics based on the ultratight (Ka > 1012 M-1) and highly selective binding between CB[7] and adamantane ammonium ion tagged species.
In Specific Aim 1, we synthesize monofunctionalized derivatives of CB[n] (n = 7, 8, 10) and CB[n] rotaxanes that are endowed with clickable cyclooctyne and cyclooctene functional groups. The clickable CB[n] species will be covalently attached to site specifically labeled trastuzumab and rituximab to deliver Antibody-CB[n] conjugates whose binding properties toward unfunctionalized anticancer agents and other guests will be determined by spectroscopic or calorimetric methods.
In Aim 1 we also prepare derivatives of adamantane amine tagged with fluorophores which bind tightly to CB[7] and thereby enable monitoring the uptake and localization of the Antibody-CB[n]Ad-fluorophore conjugates.
In Specific Aim 2, we will test the conjugates created in Specific Aim 1 using ex vivo and in vivo approaches. First, we will use specific cancer cell lines that either highly express the targeted protein (positive control) or do not/low level expression of the targeted protein (negative control). Next, we will use Antibody-CB[n]Ad-fluorophore conjugates to label the cancer cell lines and quantify the cell surface labeling via flow cytometry and follow the internalization of the conjugates via immunofluorescence microscopy. Antibody-CB[n] conjugates which demonstrate specific binding will be selected for in vivo tumor targeting studies using small animal in vivo imaging approaches. The innovation of the proposed work lies in the strategic merger of the targeting ability of antibodies with the recognition properties of CB[n] whereas the significance rests on the ability to accelerate the development of ADCs by allowing the targeted delivery of unfunctionalized anticancer agents and thereby have a major impact on the treatment of cancer.
Targeting of drugs to cancer cells increases efficacy and decreases the side effects of chemotherapeutic agents used to treat tumors in cancer patients. Here, we propose Antibody-Molecular container bioconjugates that non-covalently complex chemotherapeutic and/or imaging agents as a means to achieve targeted delivery of unmodified cancer drugs.
