Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte-Associated Protein Blockade.

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models.Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses.Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy.Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190-202. ©2017 AACR.

Evaluation of (89)Zr-pertuzumab in Breast cancer xenografts.

Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2-specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with (89)Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of (89)Zr-pertuzumab in HER2-expressing BT-474 and HER2-nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2-expressing cells. In vivo evaluation of (89)Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. (89)Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of (89)Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that (89)Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.

Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor.

UNLABELLED: Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand's affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a (64)Cu-labeled affinity peptide tag, (64)Cu-L19K-(5-fluoro-2,4-dinitrobenzene) ((64)Cu-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of (64)Cu-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, (64)Cu-L19K-(2,4-dinitrophenyl) ((64)Cu-L19K-DNP) and (64)Cu-L19K. METHODS: The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with (64)Cu with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. (64)Cu-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of (64)Cu-L19K-FDNB, (64)Cu-L19K-DNP, and the native (64)Cu-L19K were compared in HCT-116 xenografts. Blocking studies of (64)Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. RESULTS: In vitro binding studies confirmed the covalent and irreversible binding of (64)Cu-L19K-FDNB to VEGF, whereas (64)Cu-L19K-DNP and (64)Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with (64)Cu-L19K-FDNB than with (64)Cu-L19K-DNP and (64)Cu-L19K, with mean standardized uptake values of 0.62 ± 0.05, 0.18 ± 0.06, and 0.34 ± 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 ± 0.05, 0.32 ± 0.14, and 0.30 ± 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with (64)Cu-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection. CONCLUSION: In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.

Investigation of a vitamin B12 conjugate as a PET imaging probe.

Nutrient demand is a fundamental characteristic of rapidly proliferating cells. Vitamin B12 is vital for cell proliferation; thus neoplastic cells have an increased demand for this essential nutrient. In this study we exploited the vitamin B12 uptake pathway to probe the nutritional demand of proliferating cells with a radiolabeled B12 derivative in various preclinical tumor models. We describe the synthesis and biological evaluations of copper-64-labeled B12 -ethylenediamine-benzyl-1,4,7-triazacyclononane-N,N',N''-triacetic acid (B12 -en-Bn-NOTA-(64) Cu), the first example of a B12 derivative for positron emission tomography (PET) imaging. Small-animal imaging and pharmacological evaluation show high tumor uptake ranging from 2.20 to 4.84% ID g(-1) at 6 h post-administration. Competition studies with excess native B12 resulted in a 95% decrease in tumor accumulation, indicating the specificity of this radiopharmaceutical for B12 endocytotic transport proteins. These results show that a vitamin B12 PET radiopharmaceutical has potential utility for non-invasive imaging of enhanced nutrient demand in proliferating cells.

Imaging the L-type amino acid transporter-1 (LAT1) with Zr-89 immunoPET.

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [(89)Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [(18)F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [(89)Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

Imaging of CD47 expression in xenograft and allograft tumor models.

CD47 functions as a marker of "self" by inhibiting phagocytosis of autologous cells. CD47 has been shown to be overexpressed by various tumor types as a means of escaping the antitumor immune response. The goal of this research was to investigate the utility of CD47 imaging using positron emission tomography (PET) in both human xenograft and murine allograft tumor models. Anti-CD47 antibodies were conjugated with p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89Zr. We employed xenograft and allograft small-animal models of cancer in biodistribution and PET imaging studies to investigate the specificity and PET imaging robustness of CD47. Ab-Df-Bz-NCS conjugates were labeled with 89Zr with specific activity of 0.9 to 1.6 µCi/µg. Biodistribution studies in the xenograft and allograft model showed similar specific tumor uptake of the antihuman and antimouse CD47 antibodies. However, the tracer retention in the liver, spleen, and kidneys was significantly higher in the allograft-bearing animals, suggesting uptake mediated by the CD47 normally expressed throughout the reticular endothelial system. CD47, a marker of "self," was evaluated as a diagnostic PET biomarker in xenograft and allograft cancer animal models. CD47 imaging is feasible, warranting further studies and immunoPET tracer development.

Pyrophosphate-bridged complexes with picomolar toxicity.

Recently, we have observed the emergence of a new series of pyrophosphate-bridged coordination complexes. Such complexes have been prepared by overcoming the ready hydrolysis of the pyrophosphate moiety. To date, no exploration has been conducted on the cytotoxicity of such complexes. Three pyrophosphate-bridged complexes, namely {[Ni(phen)(2)](2)(mu-P(2)O(7))}.27H(2)O, {[Cu(phen)(H(2)O)](2)(mu-P(2)O(7))}.8H(2)O and {[Co(phen)(2)](2)(mu-P(2)O(7))}.6MeOH, (where phen is 1,10'-phenanthroline) were chosen for their comparative structural similarities and suitable aqueous solubility. Cytotoxicity studies in the adriamycin-resistant ovarian cancer cell line A2780/AD demonstrated highly significant efficacy, with values as low as 160pM for the cobalt complex at 72h. The underlying mechanism for such exceptional toxicity is investigated focusing on DNA interactions, topoisomerase I enzyme inhibition and oxidative stress (followed by intracellular glutathione levels). The role of hydrolysis in uptake and toxicity is also explored (followed by electronic absorption spectroscopy, (31)P NMR, and confocal microscopy) and the complexes are compared to cisplatin controls. Overall a clear picture of the extraordinary toxicity emerged. The results demonstrate a new class of prodrugs with significant potential for future development for the treatment of drug-resistant cancer cell lines.