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Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

Preclinical and clinical studies have indicated that somatostatin receptor (sst)-expressing tumors demonstrate higher uptake of radiolabeled sst antagonists than of sst agonists. In 4 consecutive patients with advanced neuroendocrine tumors, we evaluated whether treatment with 177 Lu-labeled sst antagonists is feasible. Methods: After injection of approximately 1 GBq of 177 Lu-DOTA-[Cpa-c(DCys-Aph(Hor)-DAph(Cbm)-Lys-Thr-Cys)-DTyr-NH 2 ] ( 177 Lu-DOTA-JR11) and 177 Lu-DOTATATE, 3-dimensional voxel dosimetry analysis based on SPECT/CT was performed. A higher tumor-to-organ dose ratio for 177 Lu-DOTA-JR11 than for 177 Lu-DOTATATE was the prerequisite for treatment with 177 Lu-DOTA-JR11. Results: Reversible minor adverse effects of 177 Lu-DOTA-JR11 were observed. 177 Lu-DOTA-JR11 showed a 1.7-10.6 times higher tumor dose than 177 Lu-DOTATATE. At the same time, the tumor-to-kidney and tumor-to-bone marrow dose ratio was 1.1-7.2 times higher. All 4 patients were treated with 177 Lu-DOTA-JR11, resulting in partial remission in 2 patients, stable disease in 1 patient, and mixed response in the other patient. Conclusion: Treatment of neuroendocrine tumors with radiolabeled sst antagonists is clinically feasible and may have a significant impact on peptide receptor radionuclide therapy. The purpose of this pilot study was to evaluate the feasibility of peptide receptor radionuclide therapy with the novel sst antagonist 177 Lu-DOTA-JR11. Before treatment, tumor and organ doses of 177 Lu-DOTATATE and 177 Lu-DOTA-JR11 were compared in the same patient after injection of a nontherapeutic test dose. A higher tumor-toorgan dose ratio for 177 Lu-DOTA-JR11 than for 177 Lu-DOTATATE was the prerequisite for treatment with 177 Lu-DOTA-JR11. MATERIALS AND METHODS PatientsFour consecutive patients with progressive neuroendocrine tumors and limited treatment options due to chronic grade 2 or 3 kidney disease were prospectively recruited. Patient characteristics are summarized in Table 1. Exclusion criteria were concurrent antitumor treatment (octreotide [Sandostatin; Novartis Pharmaceuticals] depot less than 4 wk before test injection and treatment), preexisting grade 3 and 4 hematologic toxicity, and pregnancy or breastfeeding. The institutional review board approved this study, and all subjects gave written informed consent in accordance with the Declaration of Helsinki.Radiochemistry DOTA-JR11 was synthesized as previously described (5). DOTA-TATE was received from piChem (Austria). For the preparation of 177 Lu-DOTA-JR11 and 177 Lu-DOTATATE, the corresponding peptide conjugate was dissolved in 500 mL of ascorbate buffer, pH 5.0, and 177 LuCl 3 was added. The solution was incubated at 95°C for 30 min. Quality control was performed by analytic reverse-phase high-performance liquid chromatography on a Phenomenex Jupiter C18 4-mm, 250 · 4.6 mm column (eluents, A 5 0.1% trifluoroacetic acid in water and B 5 acetonitrile; gradient, 0-25 min, 95%-50% A; flow, 0.75 mL/min). The labeling yield of 177 Lu-DOTATATE and 177 Lu-DOTA-JR11 was mor...

Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.