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Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >105-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, and IL2) when GD2(+) tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells. Cancer Immunol Res; 3(3); 266–77. ©2014 AACR.

Supplemental Figure S1: Tumor free survival in humanized DKO mice. Supplemental Figure S2: T cell survival in the sc tumors. Supplemental Figure S3: Efficacy of hu3F8-BsAb vs. 5HLDS(15)BA(Y) in humanized DKO mice. Supplemental Figure S4: Cytokine release from different BsAb formats.

Supplemental Table S1: Affinities relative to hu3F8-IgG1. Supplemental Table S2: Affinities to both human and mouse FcR(n). Supplemental Figure S1: Kinetic analysis of in vitro binding of antibodies to CD16A(158V) by SPR. Supplemental Figure S2: Blood clearance of the antibodies in nude mice.

Purpose: The norepinephrine transporter (NET) is a critical regulator of catecholamine uptake in normal physiology and is expressed in neuroendocrine tumors like neuroblastoma. Although the norepinephrine analog, meta-iodobenzylguanidine (MIBG), is an established substrate for NET, 123I/131I-MIBG has several clinical limitations for diagnostic imaging. In the current studies, we evaluated meta-[18F]-fluorobenzylguanidine ([18F]-MFBG) and compared it with 123I-MIBG for imaging NET-expressing neuroblastomas.Experimental Design: NET expression levels in neuroblastoma cell lines were determined by Western blot and 123I-MIBG uptake assays. Five neuroblastoma cell lines and two xenografts (SK-N-BE(2)C and LAN1) expressing different levels of NET were used for comparative in vitro and in vivo uptake studies.Results: The uptake of [18F]-MFBG in cells was specific and proportional to the expression level of NET. Although [18F]-MFBG had a 3-fold lower affinity for NET and an approximately 2-fold lower cell uptake in vitro compared with that of 123I-MIBG, the in vivo imaging and tissue radioactivity concentration measurements demonstrated higher [18F]-MFBG xenograft uptake and tumor-to-normal organ ratios at 1 and 4 hours after injection. A comparison of 4 hours [18F]-MFBG PET (positron emission tomography) imaging with 24 hours 123I-MIBG SPECT (single-photon emission computed tomography) imaging showed an approximately 3-fold higher tumor uptake of [18F]-MFBG, but slightly lower tumor-to-background ratios in mice.Conclusions: [18F]-MFBG is a promising radiopharmaceutical for specifically imaging NET-expressing neuroblastomas, with fast pharmacokinetics and whole-body clearance. [18F]-MFBG PET imaging shows higher sensitivity, better detection of small lesions with low NET expression, allows same day scintigraphy with a shorter image acquisition time, and has the potential for lower patient radiation exposure compared with 131I/123I-MIBG. Clin Cancer Res; 20(8); 2182–91. ©2014 AACR.

MATERIALS AND METHODS Table S1. Biodistribution of [18F]-MFBG in neuroblastoma xenografts. Table S2. Biodistribution of 123I-MIBG in neuroblastoma xenografts.

PDF file - 85KB, Table S1. Biodistribution of 18F-MFBG in neuroblastoma xenografts. Table S2. Biodistribution of 123I-MIBG in neuroblastoma xenografts.

PURPOSE: Radioimmunotherapy (RIT) delivered through the cerebrospinal fluid (CSF) has been shown to be a safe and promising treatment for leptomeningeal metastases. Pharmacokinetic models for intra-Ommaya anti-GD2 monoclonal antibody (131)I-3F8 have been proposed to improve therapeutic effect while minimizing radiation toxicity. In this study, we now apply pharmacokinetic modeling to intra-Ommaya (131)I-omburtamab (8H9), an anti-B7-H3 antibody which has shown promise in RIT of leptomeningeal metastases. METHODS: Serial CSF samples were collected and radioassayed from 61 patients undergoing a total of 177 intra-Ommaya administrations of (131)I-omburtamab for leptomeningeal malignancy. A two-compartment pharmacokinetic model with 12 differential equations was constructed and fitted to the radioactivity measurements of CSF samples collected from patients. The model was used to improve anti-tumor dose while reducing off-target toxicity. Mathematical endpoints were (a) the area under the concentration curve (AUC) of the tumor-bound antibody, AUC[C(IAR)(t)], (b) the AUC of the unbound “harmful” antibody, AUC[C(IA)(t)], and (c) the therapeutic index, AUC[C(IAR)(t)]÷AUC[C(IA)(t)]. RESULTS: The model fit CSF radioactivity data well (mean R=96.4%). The median immunoreactivity of (131)I-omburtamab matched literature values at 69.1%. Off-target toxicity (AUC[C(IA)(t)]) was predicted to increase more quickly than AUC[C(IAR)(t)] as a function of (131)I-omburtamab dose, but the balance of therapeutic index and AUC[C(IAR)(t)] remained favorable over a broad range of administered doses (0.48–1.40mg or 881–2592MBq). While anti-tumor dose and therapeutic index increased with antigen density, the optimal administered dose did not. Dose fractionization into two separate injections increased therapeutic index by 38%, and splitting into 5 injections by 82%. Increasing antibody immunoreactivity to 100% only increased therapeutic index by 17.5%. CONCLUSION: The 2-compartmental pharmacokinetic model when applied to intra-Ommaya (131)I-omburtamab yielded both intuitive and non-intuitive therapeutic predictions. The potential advantage of further dose fractionization warrants clinical validation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT00089245