The overall objective of this competing R01 renewal is the development of the Elastin thermal targeting technology for combination therapy of solid tumors. The objective will be achieved by conjugating Astatine-211 (211At) and Doxorubicin to a genetically engineered, thermally responsive elastin-like polypeptide (ELP). The proposed research builds upon results from the previous project period that a systemically administered ELP that was designed to undergo a hydrophilic-hydrophobic phase transition at 40 oC (ELPactive) formed micron size aggregates that adhered to the vasculature of tumors that were heated to 42 oC. In the first stage of delivery, we hypothesize that the formation of highly concentrated ELP microparticles only in the tumor vasculature will selectively ablate the tumor vasculature by 211At because it emits highly potent, short penetration a-particles that are concentrated to the vasculature. Upon cessation of hyperthermia and dissolution of the ELP aggregates, these particles dissolve and """"""""pump"""""""" soluble ELP out into the tumor mass, resulting in homogeneous penetration of the soluble ELP and significantly enhanced localization compared to a thermally insensitive ELP control. An important finding was that thermal cycling of the tumor between body temperature and 42 oC further amplified this effect. In the second stage of targeting the tumor cells with a chemotherapeutic, thermal cycling of tumors will be combined with affinity targeting of tumor cells to deliver doxorubicin to tumor cells. Affinity targeting will be carried out by incorporating luteinizing hormone releasing hormone (LHRH) at the gene level at the N-terminus of the ELP to allow receptor-mediated endocytosis, thereby enhancing the uptake of ELPs into tumor cells and bioavailability of Dox.
In Specific Aim 1, recombinant expression in E. coli will be used to synthesize a thermally responsive ELP with LHRH at the N-terminus (and control ELPs). Each ELP will be conjugated to 211At through a stable amide bond and to doxorubicin through an acid-labile hydrazone bond.
Specific Aim 2 will determine the optimized dose and hyperthermia treatment duration of 211At-ELPactive-Dox conjugate that exhibits the following features: (1) highest amount of vascular ELP aggregates during hyperthermia to maximize first-stage vascular ablation by 211At; (2) highest %ID/g in tumors; (3) highest tumor to normal tissue ratio; and (4) highest penetration of the ELP conjugate within the tumor mass post-hyperthermia to maximize second-stage tumor cell kill by Doxorubicin and 211At.
In Specific Aim 3, tumor growth delay studies will be carried out using the optimal dose and temperature-time profile determined from Specific Aim 2. The molecular mechanism underlying tumor regression will also be delineated by correlating therapeutic effects with microvessel function and tumor hypoxia. ? ? ?
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