Genetically Engineered Nanoparticles of Asymmetric Triblock Polypeptide with a Platinum(IV) Cargo Outperforms a Platinum(II) Analog and Free Drug in a Murine Cancer Model.

The development of platinum(Pt)-drugs for cancer therapy has stalled, as no new Pt-drugs have been approved in over a decade. Packaging small molecule drugs into nanoparticles is a way to enhance their therapeutic efficacy. To date, there has been no direct comparison of relative merits of the choice of Pt oxidation state in the same nanoparticle system that would allow its optimal design. To address this lacuna, we designed a recombinant asymmetric triblock polypeptide (ATBP) that self-assembles into rod-shaped micelles and chelates Pt(II) or enables covalent conjugation of Pt(IV) with similar morphology and stability. Both ATBP-Pt(II) and ATBP-Pt(IV) nanoparticles enhanced the half-life of Pt by ∼45-fold, but ATBP-Pt(IV) had superior tumor regression efficacy compared to ATBP-Pt(II) and cisplatin. These results suggest loading Pt(IV) into genetically engineered nanoparticles may yield a new generation of more effective platinum-drug nanoformulations.

Heuristics for the Optimal Presentation of Bioactive Peptides on Polypeptide Micelles.

Bioactive peptides describe a very large group of compounds with diverse functions and wide applications, and their multivalent display by nanoparticles can maximize their activities. However, the lack of a universal nanoparticle platform and design rules for their optimal presentation limits the development and application of peptide ligand-decorated nanoparticles. To address this need, we developed a multivalent nanoparticle platform to study the impact of nanoparticle surface hydrophilicity and charge on peptide targeting and internalization by tumor cells. This system consists of micelles of a recombinant elastin-like polypeptide diblock copolymer (ELPBC) that present genetically encoded peptides at the micelle surface without perturbing the size, shape, stability, or peptide valency of the micelle, regardless of the peptide type. We created the largest extant set of 98 combinations of 15 tumor-homing peptides that are presented on the corona of this ELPBC micelle via 8 different peptide linkers that vary in their length and charge and also created control micelles that present the linker only. Analysis of the structure-function relationship of tumor cell targeting by this set of peptide-decorated nanoparticles enabled us to derive heuristics to optimize the delivery of peptides based on their physicochemical properties and to identify a peptide that is likely to be a widely useful ligand for targeting across nanoparticle types. This study shows that ELPBC micelles are a robust and convenient system for the presentation of diverse peptides and provides useful insights into the appropriate presentation of structurally diverse peptide ligands on nanoparticles based on their physicochemical properties.

Author Correction: Injectable tissue integrating networks from recombinant polypeptides with tunable order.

In the version of this Article originally published, one of the authors' names was incorrectly given as Jeffery Schaal; it should have been Jeffrey L. Schaal. This has been corrected in all versions of the Article.

Injectable tissue integrating networks from recombinant polypeptides with tunable order.

Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.

Brachytherapy via a depot of biopolymer-bound 131I synergizes with nanoparticle paclitaxel in therapy-resistant pancreatic tumours.

Locally advanced pancreatic tumours are highly resistant to conventional radiochemotherapy. Here we show that such resistance can be surmounted by an injectable depot of thermally responsive elastin-like polypeptide (ELP) conjugated with iodine-131 radionuclides (131I-ELP) when combined with systemically delivered nanoparticle albumin-bound paclitaxel. This combination therapy induced complete tumour regressions in diverse subcutaneous and orthotopic mouse models of locoregional pancreatic tumours. 131I-ELP brachytherapy was effective independently of the paclitaxel formulation and dose, but external beam radiotherapy (EBRT) only achieved tumour-growth inhibition when co-administered with nanoparticle paclitaxel. Histological analyses revealed that 131I-ELP brachytherapy led to changes in the expression of intercellular collagen and junctional proteins within the tumour microenvironment. These changes, which differed from those of EBRT-treated tumours, correlated with the improved delivery and accumulation of paclitaxel nanoparticles within the tumour. Our findings support the further translational development of 131I-ELP depots for the synergistic treatment of localized pancreatic cancer.

Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers.

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

Nanoparticle formulation improves doxorubicin efficacy by enhancing host antitumor immunity.

Strategies that enhance the host antitumor immune response promise to revolutionize cancer therapy. Optimally mobilizing the immune system will likely require a multi-pronged approach to overcome the resistance developed by tumors to therapy. Recently, it has become recognized that doxorubicin can contribute to re-establishing host antitumor immunity through the generation of immunogenic cell death. However, the potential for delivery strategies to further enhance the immunological effects of doxorubicin has not been adequately examined. We report herein that Chimeric Polypeptide Doxorubicin (CP-Dox), a nanoparticle formulation of doxorubicin, enhances antitumor immunity. Compared to free doxorubicin, a single intravenous (IV) administration of CP-Dox at the maximum tolerated dose increases the infiltration of leukocytes into the tumor, slowing tumor growth and preventing metastasis in poorly immunogenic 4T1 mammary carcinoma. We demonstrate that the full efficacy of CP-Dox is dependent on CD8+ T cells and IFN-γ. CP-dox treatment also repolarized intratumoral myeloid cells towards an antitumor phenotype. These findings demonstrate that a nanoparticle drug is distinct from the free drug in its ability to productively stimulate antitumor immunity. Our study strongly argues for the use of antitumor immunotherapies combined with nanoparticle-packaged chemotherapy.

One-week glucose control via zero-order release kinetics from an injectable depot of glucagon-like peptide-1 fused to a thermosensitive biopolymer.

Stimulation of the glucagon-like peptide-1 (GLP1) receptor is a useful treatment strategy for type 2 diabetes because of pleiotropic effects, including the regulation of islet hormones and the induction of satiety. However, the native ligand for the GLP1 receptor has a short half-live owing to enzymatic inactivation and rapid clearance. Here, we show that a subcutaneous depot formed after a single injection of GLP1 recombinantly fused to a thermosensitive elastin-like polypeptide results in zero-order release kinetics and circulation times of up to 10 days in mice and 17 days in monkeys. The optimized pharmacokinetics leads to 10 days of glycemic control in three different mouse models of diabetes, as well as to the reduction of glycosylated hemoglobin levels and weight gain in ob/ob mice treated once weekly for 8 weeks. Our results suggest that the optimized GLP1 formulation could enhance therapeutic outcomes by eliminating peak-and-valley pharmacokinetics and improving overall safety and tolerability. The design principles that we established should be broadly applicable for improving the pharmacological performance of other peptide and protein therapeutics.

Injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy.

Intratumoral radiation therapy - 'brachytherapy' - is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide (131)I to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 min; second, the high energy ß-emissions of (131)I further stabilize the depot by introducing crosslinks within the ELP depot over 24h. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3 M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72h. The (131)I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p=0.001, ANOVA) and enhanced median survival to 27 days over controls.