Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

Intratumoral Treatment with 5-Androstene-3ß, 17α-Diol Reduces Tumor Size and Lung Metastasis in a Triple-Negative Experimental Model of Breast Cancer.

Breast cancer treatment failure is related to low response rates, high costs, and long-term toxicities. Thus, it is necessary to find less toxic, cheaper, and more effective treatments. In situ administration ensures drug delivery to tumor cells and decreases systemic toxic effects. The androstene-3ß, 17α-diol (α-AED) reduces breast tumor cell proliferation and is an ideal candidate to treat mammary tumors. This study aims to identify the in vitro and in vivo effects of α-AED on a triple-negative mammary tumor model. An in vitro biphasic steroid effect was observed in mouse and human mammary tumor cells treated with α-AED. In this sense, cells treated with higher doses (100 and 200 µM) showed an antiproliferative effect. The α-AED administrated intratumorally reduced average tumor weight and increased the percentage of natural killer cells (NK), plasmatic, and plasmablast cells in mice tumors. Of note, VEGF levels in all α-AED-treated tumors was lower than in the control and vehicle groups. The tumor in situ increased response was reflected systemically by higher anti-4T1 IgG concentration in serum from α-AED-treated mice, but no other associated systemic changes were detected. The reduction in tumor size for the local injection of α-AED is associated with the anti-proliferative effect of this steroid, and the lower local levels of VEGF may be related to the imperceptible macroscopic metastasis in α-AED-treated mice. The above suggests that α-AED may be used in clinical studies to prove its efficacy as an alternative breast tumor treatment or in conjunction with already established therapies.

Verteporfin-Loaded Polymeric Microparticles for Intratumoral Treatment of Brain Cancer.

Glioblastoma (GBMs) is the most common and aggressive type of primary brain tumor in adults with dismal prognosis despite radical surgical resection coupled with chemo- and radiotherapy. Recent studies have proposed the use of small-molecule inhibitors, including verteporfin (VP), to target oncogenic networks in cancers. Here we report efficient encapsulation of water-insoluble VP in poly(lactic- co-glycolic acid) microparticles (PLGA MP) of ∼1.5 µm in diameter that allows tunable, sustained release. Treatment with naked VP and released VP from PLGA MP decreased cell viability of patient-derived primary GBM cells in vitro by ∼70%. Moreover, naked VP treatment significantly increased radiosensitivity of GBM cells, thereby enhancing overall tumor cell killing ability by nearly 85%. Our in vivo study demonstrated that two intratumoral administrations of sustained slow-releasing VP-loaded PLGA MPs separated by two weeks significantly attenuated tumor growth by ∼67% in tumor volume in a subcutaneous patient-derived GBM xenograft model over 26 d. Additionally, our in vitro data indicate broader utility of VP for treatment for other solid cancers, including chordoma, malignant meningioma, and various noncentral nervous system-derived carcinomas. Collectively, our work suggests that the use of VP-loaded PLGA MP may be an effective local therapeutic strategy for a variety of solid cancers, including unresectable and orphan tumors, which may decrease tumor burden and ultimately improve patient prognosis.

Case Report: Radioactive Holmium-166 Microspheres for the Intratumoral Treatment of a Canine Pituitary Tumor.

Introduction: In this case study, a client-owned dog with a large pituitary tumor was experimentally treated by intratumoral injection of radioactive holmium-166 microspheres (166HoMS), named 166Ho microbrachytherapy. To our knowledge, this is the first intracranial intratumoral treatment through needle injection of radioactive microspheres. Materials and Methods: A 10-year-old Jack Russell Terrier was referred to the Clinic for Companion Animal Health (Faculty of Veterinary Medicine, Utrecht University, The Netherlands) with behavioral changes, restlessness, stiff gait, and compulsive circling. MRI and CT showed a pituitary tumor with basisphenoid bone invasion and marked mass effect. The tumor measured 8.8 cm3 with a pituitary height-to-brain area (P/B) ratio of 1.86 cm-1 [pituitary height (cm) ×10/brain area (cm2)]. To reduce tumor volume and neurological signs, 166HoMS were administered in the tumor center by transsphenoidal CT-guided needle injections. Results: Two manual CT-guided injections were performed containing 0.6 ml of 166HoMS suspension in total. A total of 1097 MBq was delivered, resulting in a calculated average tumor dose of 1866 Gy. At 138 days after treatment, the tumor volume measured 5.3 cm3 with a P/B ratio of 1.41 cm-1, revealing a total tumor volume reduction of 40%. Debulking surgery was performed five months after 166HoMS treatment due to recurrent neurological signs. The patient was euthanized two weeks later at request of the owners. Histopathological analysis indicated a pituitary adenoma at time of treatment, with more malignant characteristics during debulking surgery. Conclusion: The 40% tumor volume reduction without evident severe periprocedural side effects demonstrated the feasibility of intracranial intratumoral 166HoMS treatment in this single dog.

Toll-Like Receptor 4 Expression on Lymphoma Cells Is Critical for Therapeutic Activity of Intratumoral Therapy With Synthetic TLR4 Agonist Glucopyranosyl Lipid A.

Intratumoral (IT) injections of Glucopyranosyl lipid A (G100), a synthetic toll-like receptor 4 (TLR4) agonist formulated in a stable emulsion, resulted in T-cell inflammation of the tumor microenvironment (TME) and complete cure of 60% of mice with large established A20 lymphomas. Strong abscopal effects on un-injected lesions were observed in a bilateral tumor model and surviving mice resisted a secondary tumor challenge. Depletion of CD8 T-cells, but not CD4 or NK cells, abrogated the anti-tumor effect. Unexpectedly, TLR4 knock-out rendered A20 tumors completely non-responsive to G100. In vitro studies showed that GLA has direct effect on A20 cells, but not on A20 cells deficient for TLR4. As shown by genotyping and phenotyping analysis, G100 strongly activated antigen presentation functions in A20 cells in vitro and in vivo and induced their apoptosis in a dose dependent manner. Similarly, the TLR4 positive human mantle cell lymphoma line Mino showed in vitro activation with G100 that was blocked with an anti-TLR4 antibody. In the A20 model, direct activation of B-lymphoma cells with G100 is sufficient to induce protective CD8 T-cell responses and TLR4 expressing human B-cell lymphomas may be amenable to this therapy as well.

Targeting IL-13Rα2 for effective treatment of malignant peripheral nerve sheath tumors in mouse models.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that harbor a high potential for metastasis and have a devastating prognosis. Combination chemoradiation aids in tumor control and decreases tumor recurrence but causes deleterious side effects and does not extend long-term survival. An effective treatment with limited toxicity and enhanced efficacy is critical for patients suffering from MPNSTs.METHODSThe authors recently identified that interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed on MPNSTs and could serve as a precision-based target for delivery of chemotherapeutic agents. In the work reported here, a recombinant fusion molecule consisting of a mutant human IL-13 targeting moiety and a point mutant variant of Pseudomonas exotoxin A (IL-13.E13 K-PE4E) was utilized to treat MPNST in vitro in cell culture and in an in vivo murine model.RESULTSIL-13.E13 K-PE4E had a potent cytotoxic effect on MPNST cells in vitro. Furthermore, intratumoral administration of IL-13.E13 K-PE4E to orthotopically implanted MPNSTs decreased tumor burden 6-fold and 11-fold in late-stage and early-stage MPNST models, respectively. IL-13.E13 K-PE4E treatment also increased survival by 23 days in the early-stage MPNST model.CONCLUSIONSThe current MPNST treatment paradigm consists of 3 prongs: surgery, chemotherapy, and radiation, none of which, either singly or in combination, are curative or extend survival to a clinically meaningful degree. The results presented here provide the possibility of intratumoral therapy with a potent and highly tumor-specific cytotoxin as a fourth treatment prong with the potential to yield improved outcomes in patients with MPNSTs.