(I-334) Brachytherapy by Elastin-Like Polypeptide Micelles Synergizes with Nanoparticle Immuno- and Chemotherapy for Treatment of Metastatic Cancer

Introduction::
As cancer develops from a primary tumor into metastatic disease, the stage at which the disease is diagnosed is a critical factor influencing survival. For patients diagnosed with early-stage, localized disease, locoregional interventions such as surgery or targeted radiotherapy are powerful tools that can eliminate the tumor while exerting minimal damage to surrounding tissue. Once tumors have metastasized, however, more comprehensive treatment strategies are needed to control systemic growth of metastases. We have developed a novel, multi-pronged approach using elastin-like polypeptides (ELPs) to inject radioactive micelles into the core of a tumor that, upon heating to body temperature, coacervate into a stable depot that induces necroptotic cell death in the tumor. We supplement this with either immunotherapy—using an ELP that is electrostatically complexed to a potent dendritic cell activating oligonucleotide, CpG—or nanoparticle chemotherapy—using a diblock chimeric polypeptide (CP) ELP chemically conjugated to paclitaxel (CP-PTX). When ELP-CpG micelles are injected at the core of a tumor, they coacervate into a depot that retains bound CpG within the tumor for over 20-fold longer than soluble CpG, driving the recruitment and activation of dendritic cells that present tumor neoantigens to naive T cells. ELP-paclitaxel nanoparticles, on the other hand, are injected intravenously and accumulate in tumor tissue, inducing tumor cell death. We have demonstrated that a combination of ELP brachytherapy with immunotherapy or chemotherapy synergistically inhibits primary tumor growth, reduces metastatic burden, extends survival, and even abolished tumors altogether in numerous mouse models.




Materials and Methods::
ELPs are protein biopolymers consisting of repeat units of the pentapeptide motif “Val-Pro-Gly-Xaa-Gly” derived from tropoelastin, where Xaa can be any amino acid except Pro. Our ELP brachytherapy comprises a ¹³¹I radionuclide conjugated to several Tyr residues in the C-terminal core of an ELP micelle. The radioactive ELP micelles undergo a phase transition at body temperature upon intratumoral injection and subsequently undergo radiation-induced crosslinking to create a crosslinked radioactive hydrogel in the tumor, such that the ¹³¹I is retained and irradiates the tumor from the inside-out. Our adjuvant therapies—ELP-CpG and CP-PTX—are based on similarly designed recombinant polypeptides. ELP-CpG comprises an ELP with a C-terminal 12-mer oligo-Lys tail that forms an electrostatic complex with CpG ODN-1826 (ELP-CpG), which binds to the negatively charged phosphate backbone of single-stranded CpG DNA. Upon coacervation within a tumor at body temperature, the resulting CpG depot facilitates controlled release of CpG from the depot with zero-order kinetics. Our nanoparticle-forming CP-PTX conjugate consists of a hydrophilic ELP block fused to eight C-terminal Cys residues for covalent drug conjugation. PTX is modified with levulinic acid (LEV) and further reacted with n-ɛ-maleimidocaproic acid hydrazide (EMCH) for covalent attachment to the Cys residues via an acid-labile linker. The ELP drug delivery platform is modular, allowing for separate dose and delivery optimization and administration of each component—brachytherapy, CpG, and PTX—for combination therapy.




Results, Conclusions, and Discussions::
In a combination of ¹³¹I-ELP brachytherapy with ELP-CpG, we observed significant efficacy against the aggressive, metastatic 4T1 model of murine breast cancer. Mice that received the combination therapy (¹³¹I-ELP + ELP-CpG) showed significant tumor growth regression compared to untreated mice or mice treated with either ¹³¹I-ELP brachytherapy or soluble/ELP-CpG monotherapy. Importantly, the combination therapy greatly enhanced survival, increasing median survival by 11 days over untreated mice, and significantly outperformed all other treatment groups. We also quantified lung metastases in mice treated with either monotherapy or combination therapy. We found that the addition of ELP-CpG significantly reduced lung metastatic burden over soluble CpG, the ¹³¹I-ELP monotherapy, and a sham ELP control. CP-PTX, similarly, has shown great efficacy when administered in combination with ¹³¹I-ELP brachytherapy in multiple metastatic models of murine pancreatic cancer. In the BxPc3-luc2 model of pancreatic cancer, we found that ¹³¹I-ELP administered with CP-PTX inhibits tumor growth and substantially extends survival over combinations of current standard of care treatments, including external-beam radiotherapy and Abraxane (an FDA approved nanoparticle of PTX bound to human serum albumin). In summary, our results demonstrate that ELP-based brachytherapy in combination with local delivery of immunostimulatory adjuvants or systemic delivery of chemotherapy shows a powerful and synergistic effect on primary tumors and metastases.




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