Inclusion of 4-1BB Costimulation Enhances Selectivity and Functionality of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand-based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ-containing CARs (IL13ζ), second-generation 4-1BB (CD137)-containing or CD28-containing CARs (IL13-BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro coculture assays at high tumor burden establish that second-generation IL13-BBζ or IL13-28ζ outperform first-generation IL13ζ and third-generation IL13-28BBζ CAR designs, with IL13-BBζ providing superior CAR proliferation and in vivo antitumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and antitumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior antitumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition. Significance: This study reveals how modulating CAR design outside the antigen targeting domain improves selective tumor recognition. Specifically, this work shows improved specificity, persistence, and efficacy of 4-1BB-based IL13-ligand CARs. Human clinical trials evaluating IL13-41BB-CAR T cells are ongoing, supporting the clinical significance of these findings.

Resection followed by stereotactic radiosurgery to resection cavity for intracranial metastases.

PURPOSE: In patients who undergo resection of central nervous system metastases, whole brain radiotherapy (WBRT) is added to reduce the rates of recurrence and neurologic death. However, the risk of late neurotoxicity has led many patients to decline WBRT. We offered adjuvant stereotactic radiosurgery (SRS) or stereotactic radiotherapy (SRT) as an alternative to select patients with resected brain metastases. METHODS AND MATERIALS: We performed a retrospective review of patients who underwent brain metastasis resection followed by SRS/SRT. WBRT was administered only as salvage treatment. Patients had one to four brain metastases. The dose was 15-18 Gy for SRS and 22-27.5 Gy in four to six fractions for SRT. Target margins were typically expanded by 1 mm for rigid immobilization and 3 mm for mask immobilization. SRS/SRT involved the use of linear accelerator radiosurgery using the IMRT 21EX or Helical Tomotherapy unit. RESULTS: Between December 1999 and January 2007, 30 patients diagnosed with intracranial metastases were treated with resection followed by SRS or SRT to the resection cavity. Of the 30 patients, 4 (13.3%) developed recurrence in the resection cavity, and 19 (63%) developed recurrences in new intracranial sites. The actuarial 12-month survival rate was 82% for local recurrence-free survival, 31% for freedom from new brain metastases, 67% for neurologic deficit-free survival, and 51% for overall survival. Salvage WBRT was performed in 14 (47%) of the 30 patients. CONCLUSION: Our results suggest that for patients with newly diagnosed brain metastases treated with surgical resection, postoperative SRS/SRT to the resection cavity is a feasible option. WBRT can be reserved as salvage treatment with acceptable neurologic deficit-free survival.

Quantitative analysis of diffusion tensor orientation: theoretical framework.

Diffusion-tensor MRI (DT-MRI) yields information about the magnitude, anisotropy, and orientation of water diffusion of brain tissues. Although white matter tractography and eigenvector color maps provide visually appealing displays of white matter tract organization, they do not easily lend themselves to quantitative and statistical analysis. In this study, a set of visual and quantitative tools for the investigation of tensor orientations in the human brain was developed. Visual tools included rose diagrams, which are spherical coordinate histograms of the major eigenvector directions, and 3D scatterplots of the major eigenvector angles. A scatter matrix of major eigenvector directions was used to describe the distribution of major eigenvectors in a defined anatomic region. A measure of eigenvector dispersion was developed to describe the degree of eigenvector coherence in the selected region. These tools were used to evaluate directional organization and the interhemispheric symmetry of DT-MRI data in five healthy human brains and two patients with infiltrative diseases of the white matter tracts. In normal anatomical white matter tracts, a high degree of directional coherence and interhemispheric symmetry was observed. The infiltrative diseases appeared to alter the eigenvector properties of affected white matter tracts, showing decreased eigenvector coherence and interhemispheric symmetry. This novel approach distills the rich, 3D information available from the diffusion tensor into a form that lends itself to quantitative analysis and statistical hypothesis testing.

Continuous 28-day iododeoxyuridine infusion and hyperfractionated accelerated radiotherapy for malignant glioma: a phase I clinical study.

PURPOSE: To investigate the maximal tolerated dose of a continuous 28-day iododeoxyuridine (IUdr) infusion combined with hyperfractionated accelerated radiotherapy (HART); to analyze the percentage of IUdr-thymidine replacement in peripheral granulocytes as a surrogate marker for IUdr incorporation into tumor cells; to measure the steady-state serum IUdr levels; and to assess the feasibility of continuous IUdr infusion and HART in the management of malignant glioma. METHODS AND MATERIALS: Patients were required to have biopsy-proven malignant glioma. Patients received 100 (n = 4), 200 (n = 3), 300 (n = 3), 400 (n = 6), 500 (n = 4), 625 (n = 5), or 781 (n = 6) mg/m(2)/d of IUdr by continuous infusion for 28 days. HART was started 7 days after IUdr initiation. The total dose was 70 Gy (1.2 Gy b.i.d. for 25 days with a 10-Gy boost [2.0 Gy for 5 Saturdays]). Weekly assays were performed to determine the percentage of IUdr-DNA replacement in granulocytes and serum IUdr levels using standard high performance liquid chromatography methods. Standard Phase I toxicity methods were used. RESULTS: Between June 1994 and August 1999, 31 patients were enrolled. No patient had Grade 3 or worse HART toxicity. Grade 3 or greater IUdr toxicity predominantly included neutropenia (n = 3), thrombocytopenia (n = 3), and elevated liver function studies (n = 3). The maximal tolerated dose was 625 mg/m(2)/d. Thymidine replacement in the peripheral granulocytes peaked at 3 weeks and increased with the dose (maximal thymidine replacement 4.9%). The steady-state plasma IUdr level increased with the dose (maximum, 1.5 microM). CONCLUSION: In our study, continuous long-term IUdr i.v. infusion had a maximal tolerated dose of 625 mg/m(2)/d. Granulocyte incorporation data verified the concept that prolonged IUdr infusion results in IUdr-DNA replacement that corresponds to a high degree of cell labeling. IUdr steady-state plasma levels increased with increasing dose and attained levels needed for clinical radiosensitization. Continuous IUdr infusion and HART were both feasible and well tolerated.