(6b) Homogeneous Megamolecules for Targeting Heterogeneity in Breast Cancer | AIChE

(6b) Homogeneous Megamolecules for Targeting Heterogeneity in Breast Cancer


Metcalf, K. - Presenter, University of California, San Francisco
Kimmel, B., Northwestern University
Sykora, D., Northwestern University
Modica, J., Northwestern University
Parker, K., Northwestern University
Berens, E., University of California, San Francisco
Dai, R., University of California, San Francisco
Dravid, V. P., Northwestern University
Werb, Z., Northwestern University
Mrksich, M., Northwestern University
Tumors are composed of a collection of genetically and phenotypically distinct cancer cells. For example, expression of cancer cell surface markers, such as HER2, are heterogeneously expressed, making targeted therapies ineffective at completely eradicating all cancer cells. To improve therapeutic activity in heterogeneous tumors, we have developed nanobody-enzyme megamolecules to deliver chemotherapeutics to the tumor microenvironment. Megamolecules are proteins with site-specific crosslinking that enable homogeneous protein-protein conjugates with precise covalent bonding. We synthesized nanobody-enzyme megamolecules and showed specific binding of the anti-HER2 nanobody domain to HER2+, but not HER2-, cancer cells. Nanobody binding delivered the enzyme yeast cytosine deaminase, which elicited a cytotoxic effect by activating the non-toxic prodrug 5-fluorocytosine to the cytotoxic 5-fluorouracil. We varied the number of domains of nanobody and enzyme in the conjugate independently and measured their effect on cancer cell viability in both adherent and spheroid culture. Increasing the number of enzyme domains from one to two increased drug activation by allowing for intramolecular dimerization of the enzyme. The increased enzyme activity resulted in a 4- and 40- fold decrease in cytotoxicity in adherent and spheroid culture, respectively. Increasing the number of nanobody domains from one to two increased cancer cell affinity by 4-fold, but did not increase cytotoxicity significantly due to lowered enzyme activity. The most potent nanobody-enzyme megamolecule was cytotoxic to HER2- cells when greater than 30% HER2+ cells where present in an adherent co-culture, eliciting a bystander killing effect. This study determines design rules for constructing nanobody-enzyme conjugates for cancer therapy.