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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

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.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD/SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

Multistep targeting can improve the therapeutic index of antibody-based targeting, particularly relevant to pediatric tumors where acute toxicity and late effects of treatment are major concerns. Neuroblastoma is uniquely suited for such investigations because of its abundance of surface ganglioside GD2.5F11scFv (scFv = single-chain variable fragment) was constructed from the variable regions of the heavy (V(H)) and kappa-light (V(L)) chain complementary DNA (cDNA) of anti-GD2 IgM hybridoma 5F11 and ligated to full-length streptavidin cDNA for expression in Escherichia coli. Purified 5F11-scFv-streptavidin (5F11-scFv-SA) was a homotetramer and showed comparable avidity to 5F11 IgM and a 30-fold improvement over monomeric scFv. Biodistribution of 5F11-scFv-SA was studied in nude mice xenografted with neuroblastoma LAN-1. Twenty-four hours after intravenous injection of 300-900 microg 5F11-scFv-SA, 150-450 microg of a thiogalactoside-containing clearing agent, (Gal-NAc)(16)-alpha-S-C(5)H(10)-NH-LC-N-Me-biotin (molecular weight, 8652), were administered intravenously, followed by approximately 2.5 microg (1.85-3.7 MBq) (111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin ((111)In-DOTA-biotin) intravenously 4 h later and clocked as time 0.Tumor uptake (percentage of injected dose per gram [%ID/g]) at 2 h was 7 %ID/g and decayed with a half-life of 72 h, whereas blood %ID/g rapidly decreased to1/500 of that of tumor after the first 24 h. The tumor-to-nontumor (T/NT) ratio at 72 h was high (median, 106; range, 3.4 [kidney] to 1660 [blood]). When the area under the radioactivity curve was computed, the T/NT organ ratio was favorable (4.8 for kidney and 162 for blood). When human and murine tumors were surveyed, the T/NT ratio of (111)In-DOTA-biotin uptake correlated with their levels of GD2 expression as assayed by flow cytometry. Biotinylated polypeptides (bovine serum albumin and vasointestinal peptides) achieved selective tumor targeting when the multistep strategy was applied.Improvement in the T/NT ratio using pretargeting strategy may increase the efficacy and safety of scFv-based approaches in cancer therapy. Additionally, since biotinylated polypeptides can be rendered tumor selective, a large repertoire of agents can potentially be explored.