Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy.

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization.

Nanoparticles (NPs) were widely used in drugs/probes delivery for improved disease diagnosis and/or treatment. Targeted delivery to cancer cells is a highly attractive application of NPs. However, few studies have been performed on the targeting mechanisms of these ligand-modified delivery systems. Additional studies are needed to understand the transport of nanoparticles in the cancer site, the interactions between nanoparticles and cancer cells, the intracellular trafficking of nanoparticles within the cancer cells and the subcellular destiny and potential toxicity. Interleukin 13 (IL-13) peptide can specifically bind IL-13Rα2, a receptor that is highly expressed on glioma cells but is expressed at low levels on other normal cells. It was shown that the nanoparticels modification with the IL-13 peptide could improve glioma treatment by selectively increasing cellular uptake, facilitating cell internalization, altering the uptake pathway and increasing glioma localization.

Direct intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma: a report by the Cintredekin Besudotox Intraparenchymal Study Group.

PURPOSE: Glioblastoma multiforme (GBM) is a devastating brain tumor with a median survival of 6 months after recurrence. Cintredekin besudotox (CB) is a recombinant protein consisting of interleukin-13 (IL-13) and a truncated form of Pseudomonas exotoxin (PE38QQR). Convection-enhanced delivery (CED) is a locoregional-administration method leading to high-tissue concentrations with large volume of distributions. We assessed the use of intracerebral CED to deliver CB in patients with recurrent malignant glioma (MG). PATIENTS AND METHODS: Three phase I clinical studies evaluated intracerebral CED of CB along with tumor resection. The main objectives were to assess the tolerability of various concentrations and infusion durations; tissue distribution; and methods for optimizing delivery. All patients underwent tumor resection followed by a single intraparenchymal infusion (in addition to the intraparenchymal one following resection), with a portion of patients who had a preresection intratumoral infusion. RESULTS: A total of 51 patients with MG were treated including 46 patients with GBM. The maximum tolerated intraparenchymal concentration was 0.5 microg/mL and tumor necrosis was observed at this concentration. Infusion durations of up to 6 days were well tolerated. Postoperative catheter placement appears to be important for optimal drug distribution. CB- and procedure-related adverse events were primarily limited to the CNS. Overall median survival for GBM patients is 42.7 weeks and 55.6 weeks for patients with optimally positioned catheters with patient follow-up extending beyond 5 years. CONCLUSION: CB appears to have a favorable risk-benefit profile. CED is a complex delivery method requiring catheter placement via a second procedure to achieve accurate catheter positioning, better drug distribution, and better outcome.

Induction of hyperintense signal on T2-weighted MR images correlates with infusion distribution from intracerebral convection-enhanced delivery of a tumor-targeted cytotoxin.

OBJECTIVE: Convection-enhanced delivery is a promising approach to intracerebral drug delivery in which a fluid pressure gradient is used to infuse therapeutic macromolecules through an indwelling catheter into the interstitial spaces of the brain. Our purpose was to test the hypothesis that hyperintense signal changes on T2-weighted images produced by such infusions can be used to track drug distribution. SUBJECTS AND METHODS: Seven adults with recurrent malignant glioma underwent concurrent intracerebral infusions of the tumor-targeted cytotoxin cintredekin besudotox and 123I-labeled human serum albumin. The agents were administered through a total of 18 catheters among the seven patients. Adequacy of distribution of drug was determined by evidence of distribution of 123I-labeled human serum albumin on SPECT images coregistered with MR images. Qualitative analysis was performed by three blinded observers. Quantitative analysis also was performed. RESULTS: Infusions into 12 catheters produced intraparenchymal distribution as seen on SPECT images, but infusions into six catheters did not. At qualitative assessment of signal changes on MR images, reviewers correctly predicted which catheters would produce extraparenchymal distribution and which catheters would produce parenchymal distribution. Of the 12 infusions that produced intraparenchymal distribution, four catheters had been placed in regions of relatively normal signal intensity and produced regions of newly increased signal intensity, the volume of which highly correlated with the volume and geometry of distribution on SPECT (r2 = 0.9502). Eight infusions that produced intraparenchymal distribution were performed in regions of preexisting hyperintense signal. In these brains, additional signal changes were always produced, but quantitative correlations between areas of newly increased signal intensity and the volume and geometry of distribution on SPECT could not be established. CONCLUSION: Convection-enhanced infusions frequently do not provide intraparenchymal drug distribution, and these failures can be identified with MRI soon after infusion. When infusions are performed into regions of normal signal intensity, development of hyperintense signal change strongly correlates with the volume and geometry of distribution of infusate.

Interleukin-13 receptor-targeted nanovesicles are a potential therapy for glioblastoma multiforme.

The difficulties associated with treatment of malignant brain tumors are well documented. For example, local infiltration of high-grade astrocytomas prevents the complete resection of all malignant cells. It is, therefore, critical to develop delivery systems for chemotherapeutic agents that ablate individual cancer cells without causing diffuse damage to surrounding brain tissue. Here, we describe sterically stable human interleukin-13 (IL-13)-conjugated liposomes, which efficiently bind to the brain cancer cells that overexpress the IL-13 receptor alpha2 protein. The conjugated liposomes bind to glioblastoma multiforme tissue specimens but not to normal cortex. Conjugating the liposomes with human IL-13 allows for specific binding to glioma cells and uptake of the liposomes via endocytosis. Delivering doxorubicin to glioma cells by IL-13-conjugated liposomes results in enhanced cytotoxicity and increased accumulation and retention of drug in the glioma cells compared with delivery of free drug. The therapeutic potential and targeting efficacy of the IL-13-conjugated liposomes carrying doxorubicin was tested in vivo using a s.c. glioma tumor mouse model. Animals receiving i.p. injections of IL-13-conjugated liposomes carrying doxorubicin for 7 weeks had a mean tumor volume of 37 mm3 compared with a mean volume of 192 mm3 in animals injected with nontargeted liposomes. These results strongly suggest that IL-13-conjugated liposomes carrying cytotoxic agents are a feasible approach for creating a nanovesicle drug delivery system for brain tumor therapy.

Convection-enhanced and local delivery of targeted cytotoxins in the treatment of malignant gliomas.

Despite advances in our knowledge about the genesis, molecular biology, and natural history of malignant gliomas and the use of a multi-disciplinary approach to their treatment, patients harboring this diagnosis continue to face a grim prognosis. At the time of diagnosis, patients typically undergo surgery for the establishment of a histologic diagnosis, the reduction of tumor burden, and the relief of mass effect, with the maintenance of the patient's neurological function in mind. This is followed by the administration of adjuvant therapeutics, including radiation therapy and chemotherapy. Many investigational agents with laboratory evidence of efficacy against malignant gliomas have not met their promise in the clinical setting, largely due to the barriers that they must overcome to reach the tumor at a therapeutically meaningful concentration for a durable period of time. The relevant aspects of the blood-brain barrier, blood-tumor barrier, and blood-cerebrospinal fluid barrier, as they pertain to the delivery of agents to the tumor, will be discussed along with the strategies devised to circumvent them. This discussion will be followed by a description of agents currently in preclinical and clinical development, many of which are the result of intense ongoing research into the molecular biology of gliomas.

Real-time, image-guided, convection-enhanced delivery of interleukin 13 bound to pseudomonas exotoxin.

PURPOSE: To determine if the tumor-targeted cytotoxin interleukin 13 bound to Pseudomonas exotoxin (IL13-PE) could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution in real-time using a surrogate magnetic resonance imaging tracer, we used convection-enhanced delivery to perfuse rat and primate brainstems with IL13-PE and gadolinium-bound albumin (Gd-albumin). EXPERIMENTAL DESIGN: Thirty rats underwent convective brainstem perfusion of IL13-PE (0.25, 0.5, or 10 microg/mL) or vehicle. Twelve primates underwent convective brainstem perfusion of either IL13-PE (0.25, 0.5, or 10 microg/mL; n = 8), co-infusion of 125I-IL13-PE and Gd-albumin (n = 2), or co-infusion of IL13-PE (0.5 microg/mL) and Gd-albumin (n = 2). The animals were permitted to survive for up to 28 days before sacrifice and histologic assessment. RESULTS: Rats showed no evidence of toxicity at all doses. Primates showed no toxicity at 0.25 or 0.5 microg/mL but showed clinical and histologic toxicity at 10 microg/mL. Quantitative autoradiography confirmed that Gd-albumin precisely tracked IL13-PE anatomic distribution and accurately showed the volume of distribution. CONCLUSIONS: IL13-PE can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and over clinically relevant volumes using convection-enhanced delivery. Moreover, the distribution of IL13-PE can be accurately tracked by co-infusion of Gd-albumin using real-time magnetic resonance imaging.

Immunotoxin pharmacokinetics: a comparison of the anti-glioblastoma bi-specific fusion protein (DTAT13) to DTAT and DTIL13.

DTAT13, a novel recombinant bispecific immunotoxin (IT) consisting of truncated diphtheria toxin, an amino-terminal (AT) fragment of the urokinase-type plasminogen activator (uPa), and a fragment of human IL-13 was assembled in order to target receptors on glioblastoma multiforme (GBM) and its associated neovasculature. Previous in vitro studies confirmed the efficacy of DTAT13 against various GBM cell lines expressing both IL-13 receptor or uPA receptor, and previous in vivo testing demonstrated the efficacy of DTAT13 in significantly inhibiting a range of xenograft tumors and showed that DTAT13 was 160- and 8-fold less toxic to the parental fusion IT, DTAT and DTIL13, respectively. To further understand the properties of DTAT13, pharmacokinetic/biodistribution experiments were performed. Binding analysis revealed that the IL-13 domain functioned independently of the uPA domain and that the K (d) for each binding domain was essentially the same as that of DTIL13 and DTAT. Flow cytometry studies indicated that DTAT13 bound better than DTAT or DTIL13. Analysis of the rate of protein synthesis inhibition in U87 MG cells by DTAT13 compared to DTAT revealed a faster rate of inhibition with DTAT13 compared to DTAT. The rate of protein synthesis inhibition of DTAT13 was identical to that of DTIL13 in U373 MG cells. Intracranial biodistribution studies revealed that DTAT13 was able to cross to the contralateral hemisphere unlike DTIL13 but similar to DTAT. These studies show that DTAT13 has properties encompassing those of both DTIL13 and DTAT and warrants further consideration for clinical development.

Distribution kinetics of targeted cytotoxin in glioma by bolus or convection-enhanced delivery in a murine model.

OBJECT: Interleukin-13 receptor (IL-13R)-targeted cytotoxin (IL-13-PE38) displays a potent antitumor activity against a variety of human tumors including glioblastoma multiforme (GBM) and, thus, this agent is being tested in the clinical trial for the treatment of recurrent GBM. In this study, the authors determined the safety and distribution kinetics of IL-13 cytotoxin when infused intracranially by a bolus injection and by convection-enhanced delivery (CED) in an athymic nude mouse model of GBM. METHODS: For the safety studies, athymic nude mice were given intracranial infusions of IL-13 cytotoxin into normal parenchyma by either a bolus injection or a 7-day-long CED. Toxicity was assessed by performing a histological examination of the mouse brains. For the drug distribution studies, nude mice with intracranially implanted U251 GBM tumors were given an intratumor bolus or a CED infusion of IL-13 cytotoxin. Brain tumor samples obtained between 0.25 and 72 hours after the infusion were assessed for drug distribution kinetics by performing immunohistochemical and Western blot analyses. Based on the histological changes in the tumor and brain, the maximum tolerated dose of intracranial IL-13 cytotoxin infusion in nude mice was determined to be 4 microg when delivered by a bolus injection and 10 microg when CED was used. Drug distribution reached the maximum level 1 hour after the bolus injection and the volume of distribution was determined to be 19.3 +/- 5.8 mm3. Interleukin-13 cytotoxin was barely detectable 6 hours after the injection. Interestingly, when delivered by bolus injections IL-13 cytotoxin exhibited superior distribution in larger rather than smaller tumors. Convection-enhanced delivery was superior for drug distribution in the U251 tumors because when CED was used the drug remained in the tumors 6 hours after the infusion. CONCLUSIONS: These studies provide confirmation of a previous hypothesis that CED of IL-13 cytotoxin is superior to bolus injections not only for the safety of the normal brain but also for maintaining drug levels for a prolonged period in infused brain tumors. These findings are highly relevant and important for the optimal clinical development of IL-13 cytotoxin or any other targeted antitumor agent for GBM therapy, in which multiple routes of delivery of an agent are being contemplated.

The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats.

PURPOSE: Interleukin (IL)-13 is a strong immunomodulatory cytokine that inhibits macrophages from secreting proinflammatory mediators. This study was conducted to investigate the effect of intraocular injection of IL-13 on the development of endotoxin-induced uveitis (EIU) in the Lewis rat. METHODS: One injection into the anterior chamber of recombinant human IL-13 (6 ng in 10 microl saline) was performed either simultaneously with a single injection of lipopolysaccharide (LPS) from Salmonella typhimurium into the footpad or 6 hours before the IL-13 injection. EIU was evaluated by slit lamp examination at 6, 16, and 24 hours after LPS injection. Counts of inflammatory cells were performed on cryostat sections after specific immunostaining. Anterior chamber paracentesis was performed, and kinetic analysis of the IL-13 injected in the anterior chamber was performed by ELISA. Cytokine and chemokine gene expression in the iris-ciliary body and the retina was evaluated by reverse transcription-polymerase chain reaction. RESULTS: A significant inhibition of ocular inflammation was observed in IL-13-treated rats at 16 and 24 hours after LPS injection. Unilateral injection of IL-13 inhibited EIU only in the injected eye. High levels of IL-13 were detected in the aqueous humor at 2 hours after local IL-13 injection to remain high up to 18 hours. In contrast, IL-13 was not detected in the corresponding sera. Quantitative analysis of inflammatory cells in ocular tissues showed a significant decrease in OX-42(+) cells (microglia, activated macrophages, dendritic cells, and polymorphonuclear leukocytes) and ED1(+) cells (monocytes-macrophages and dendritic cells) in treated rats. A decreased expression of TNF-alpha, IL-1 beta, IL-6, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 mRNAs was observed in the iris-ciliary body and the retina from IL-13-treated rats, whereas IFN-gamma was upregulated in the iris-ciliary body. CONCLUSIONS: Injection of IL-13 into the anterior chamber may inhibit the ocular inflammation induced by LPS injection by reducing intraocular cytokine and chemokine mRNA expression in ocular tissues.