(I-333) Characterization of decellularized porcine brain-derived hydrogels for intratumoral administration of cellular immunotherapies in post-resection glioblastoma

The treatment of glioblastoma (GBM) includes surgical resection, radiation, and chemotherapy, with minimal improvement to survival and prognosis over the past decade. GBM has also shown remarkable resistance to peripherally administered cellular immunotherapies such as chimeric antigen receptor (CAR) CAR-T cells, largely due to the obstacles facing successful CAR-T cell trafficking into these heterogeneous and suppressive brain tumors. Intracranial administration has been proven safe and effective in the delivery of other anti-cancer drugs in GBM, such as the Gliadel chemotherapy-loaded wafers that have been placed into the post-resection space for sustained release into the local tissue. GBM recurrence is consistently local, suggesting that regional therapy delivery would serve to enhance the efficacy of cellular immunotherapies in the hard-to-penetrate intratumoral space.

            Biologic scaffolds composed of extracellular matrix (ECM) have been employed as repair devices in preclinical and clinical settings, but the use of these scaffolds for central nervous system applications is currently limited. An injectable ECM-derived hydrogel capable of in vitro polymerization and conformation to irregular lesion spaces can aid in the opportunistic delivery of cellular therapies into the cavity resulting from GBM resection surgeries. Local delivery will provide additional anti-tumor activity to the nearby surgical margins where invaded tumor cells ultimately lead to recurrence.

To create biocompatible scaffolds for cellular implantation that mimic the local environment, commercially procured healthy porcine brain tissue was decellularized and solubilized to create temperature-responsive decellularized ECM (dECM) solutions. The pre-gel solution forms a hydrogel network upon placement in physiologic body temperatures. We confirm extent of decellularization using DNA quantification of processed tissue and quantify major protein composition of solubilized pre-gel product using both protein quantification kits and mass spectroscopy. dECM hydrogels processed for SEM reveal porous networked structures composed majorly of collagen fibrils. Gelation kinetics and resulting storage modulus are measured using turbidometry and rheology, with an average gelation time of 15 + 8 min and storage modulus ranging from 100-250 Pa. We have developed a mouse model of GBM resection and recurrence to evaluate the efficacy of hydrogel-delivered CAR T cell treatment after resection surgery, using intravital imaging to monitor tumor growth and recurrence until endpoint where tissues are collected and processed for immunohistochemical analysis.

Extent of decellularization is consistent across batches, reducing DNA content to < 25 ng per mg of brain tissue while retaining > 50 mg collagen protein and > 20 mg glycosaminoglycans per mg of brain tissue processed. Our group has previously demonstrated that CAR T cell therapy targeting epidermal growth factor variant III (EGFRvIII) produces anti-tumor activity against recurrent GBM. Cell viability in dECM hydrogels is observed by encapsulation of healthy donor T cells and transduced cells containing EGFRvIII-targeting CAR scFv, all cells were observed to be >75% viable over 4d in vitro using Transwell assays. Released CAR T cells from dECM hydrogels over time were found to be potently cytotoxic in tumor-killing assays using EGFRvIII+ U87MG and patient-derived EGFRvIII+ GBM cell lines at 1:1 E:T using impedance cytotoxicity assays. CAR T cells released from dECM hydrogel carriers performed comparably to freshly made, non-encapsulated CAR T cells and performed better than CAR T cells of the same donor released from Matrigel or Collagen scaffolds at the same timepoints.

These brain-mimetic injectable scaffolds will support an immediate clinical need in the form of augmentation of delivery, expansion, and actuation of CAR T cell function within the solid tumor microenvironment and without the challenges of peripheral delivery to a specialized organ system.