Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy

IntroductionIn spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium(¹⁸⁸Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy.MethodsMice bearing A2058 melanoma xenografts were treated with either 1.5 mCi ¹⁸⁸Re-6D2 antibody, saline, unlabeled 6D2 antibody or ¹⁸⁸Re-labeled non-specific IgM.ResultsOn Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P < 0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers — chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells.ConclusionsThese results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5 + and JARID1B + cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.     

Future prospects for SPECT imaging using the radiolanthanide terbium-155 — production and preclinical evaluation in tumor-bearing mice

IntroductionWe assessed the suitability of the radiolanthanide ¹⁵⁵Tb (t_1/2 = 5.32 days, E_γ = 87 keV (32%), 105 keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging.Methods¹⁵⁵Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~ 1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. ¹⁵⁵Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules – a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) – were radiolabeled with ¹⁵⁵Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner.ResultsThe total yield of the two-step separation process of ¹⁵⁵Tb was 86%. ¹⁵⁵Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The ¹⁵⁵Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (> 95%). ¹⁵⁵Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of ¹⁵⁵Tb-DOTATATE and ¹⁵⁵Tb-MD, respectively. The relatively long physical half-life of ¹⁵⁵Tb matched in particular the biological half-lives of ¹⁵⁵Tb-cm09 and ¹⁵⁵Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration.ConclusionsThe radiolanthanide ¹⁵⁵Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the β^--emitting radiolanthanides ¹⁷⁷Lu, ¹⁶¹Tb, ¹⁶⁶Ho, and the pseudo-radiolanthanide ⁹⁰Y.     

Tracer level radiochemistry to clinical dose preparation of 177Lu-labeled cyclic RGD peptide dimer

AimIntegrin α_vβ₃ plays a significant role in angiogenesis during tumor growth and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine(R)-glycine(G)-aspartic acid(D) tripeptide sequence. The over-expression of integrin α_vβ₃ during tumor growth and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Considering the advantages of ¹⁷⁷Lu for targeted radiotherapy and enhanced tumor targeting capability of cyclic RGD peptide dimer, an attempt has been made to optimize the protocol for the preparation of clinical dose of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ (E = Glutamic acid, f = phenyl alanine, K = lysine) as a potential agent for targeted tumor therapy.Methods¹⁷⁷Lu was produced by thermal neutron bombardment on enriched Lu₂O₃ (82% in ¹⁷⁶Lu) target at a flux of 1 × 10¹⁴ n/cm².s for 21 d. Therapeutic dose of ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ (7.4 GBq) was prepared by adding the aqueous solution of the ligand and ¹⁷⁷LuCl₃ to 0.1 M NH₄OAC buffer containing gentisic acid and incubating the reaction mixture at 90 °C for 30 min. The yield and radiochemical purity of the complex was determined by HPLC technique. Parameters, such as, ligand-to-metal ratio, pH of the reaction mixture, incubation time and temperature were varied using tracer quantity of ¹⁷⁷Lu (37 MBq) in order to arrive at the optimized protocol for the preparation of clinical dose. Biological behavior of the radiotracer prepared was studied in C57/BL6 mice bearing melanoma tumors.Results¹⁷⁷Lu was produced with a specific activity of 950 ± 50 GBq/mg (25.7 ± 1.4 Ci/mg) and radionuclidic purity of 99.98%. A careful optimization of several parameters showed that ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ could be prepared with adequately high radiochemical purity using a ligand-to-metal ratio ~ 2. Based on these studies therapeutic dose of the agent with 7.4 GBq of ¹⁷⁷Lu was formulated in ~ 63 GBq/μM specific activity with high yield (98.2 ± 0.7%), radiochemical purity and in vitro stability. Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors revealed specific accumulation of the radiolabeled conjugate in tumor (3.80 ± 0.55% ID/g at 30 min p.i.) with high tumor to blood and tumor to muscle ratios. However, the uptake of the radiotracer in the tumor was found to be reduced to 1.51 ± 0.32 %ID/g at 72 h p.i.ConclusionsThe present work successfully demonstrates the formulation of an optimized protocol for the preparation of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ for PRRT applications using ¹⁷⁷Lu produced by direct neutron activation in a medium flux research reactor.     

99mTc-labeled SWL specific peptide for targeting EphA2 receptor

IntroductionEphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, ^99mTc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated.MethodsThe SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by ^99mTc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with ^99mTc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution.ResultsImmunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. ^99mTc-HYNIC-SWL displayed high binding affinity with A549 cells (K_D = 2.6 ± 0.7 nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44 ± 0.12 %ID/g) than in OCM-1 tumors (0.43 ± 0.20 %ID/g) at 1 h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58 ± 0.20 %ID/g) was seen. These data suggest that ^99mTc-HYNIC-SWL specifically targets EphA2 in tumors.ConclusionsThe expression of EphA2 can be noninvasively investigated using ^99mTc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of ^99mTc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging.     

A systematic review of yttrium-90 radioembolization for unresectable liver metastases of melanoma

PurposeTo assess the effectiveness of yttrium-90 (⁹⁰Y) radioembolization in the treatment of unresectable liver metastases of melanoma.MethodsPubMed and EMBASE were systemically searched for all English language studies related to ⁹⁰Y radioembolization for unresectable liver metastases of melanoma, including clinical trials, observational studies, and abstracts from conferences, published between January 1991 and March 2016.ResultsA total of 12 reports (7 observational studies and 5 abstracts from conferences) involving 255 patients were included in the analysis. The primary sites of melanoma were cutaneous (n = 22; 8.6%), ocular (n = 197; 77.3%), rectal (n = 3; 1.2%), and unknown (n = 33; 12.9%). The median disease control rate at 3 months was 73.6% (range, 58.3%–88.9%). Among the 207 patients for whom tumor response at 3 months was reported, complete response was seen in 1.0% (2/207), partial response was seen in 19.3% (40/207), stable disease was seen in 46.9% (97/207), and progressive disease was seen in 32.9% (68/207). The median survival was 10 months (range, 7–13.4 months), and the median 1-year survival rate was 34.6% (range, 23%–80%). Complications of ⁹⁰Y radioembolization were reported in 13 cases. The most common side effects were fatigue (median, 36.1%), abdominal pain (median, 17.8%), and nausea (median, 15.0%).Conclusions⁹⁰Y radioembolization is a promising alternative therapy for the treatment of unresectable liver metastases of melanoma, with encouraging effects on disease control and survival. Some complications can occur, and side effects are frequent but mild.     

Zirconium-89 labeled panitumumab: a potential immuno-PET probe for HER1-expressing carcinomas

IntroductionAnti-HER1 monoclonal antibody (mAb), panitumumab (Vectibix) is a fully human mAb approved by the FDA for the treatment of epidermal growth factor receptor (EGFR, HER1)-expressing colorectal cancers. By combining the targeted specificity of panitumumab with the quantitative in vivo imaging capabilities of PET, we evaluated the potential of ⁸⁹Zr-DFO-panitumumab PET/CT imaging and performed non-invasive, in vivo imaging of HER1 expression and estimated human dosimetry.MethodsPanitumumab was radiolabeled with ⁸⁹Zr using a derivative of desferrioxamine (DFO-Bz-NCS) and with ¹¹¹In using CHX-A” DTPA as bifunctional chelators. Comparative biodistribution/dosimetry of both radiotracers was performed in non-tumor bearing athymic nude mice (n=2 females and n=2 males) over 1-week following i.v. injection of either using ⁸⁹Zr-DFO-panitumumab or ¹¹¹In-CHX-A”-DTPA-panitumumab. Micro-PET/CT imaging of female athymic nude mice bearing human breast cancer tumors (n=5 per tumor group) with variable HER1-expression very low (BT-474), moderate (MDA-MB-231), and very high (MDA-MB-468) was performed at over 1 week following i.v. injection of ⁸⁹Zr-DFO-panitumumab.ResultsRadiochemical yield and purity of ⁸⁹Zr-Panitumumab was > 70% and > 98% respectively with specific activity 150 ± 10 MBq/mg of panitumumab in a ~ 4 hr synthesis time. Biodistribution of ¹¹¹In-CHX-A” DTPA -panitumumab and ⁸⁹Zr-DFO-panitumumab in athymic non-tumor bearing nude mice displayed similar percent injected dose per gram of tissue with prominent accumulation of both tracers in the lymph nodes, a known clearance mechanism of panitumumab. Also exhibited was prolonged blood pool with no evidence of targeted accumulation in any organ. Human radiation dose estimates showed similar biodistributions with estimated human effective doses of 0.578 and 0.183 mSv/MBq for ⁸⁹Zr-DFO-panitumumab and ¹¹¹In-CHX-A”-DTPA–panitumumab, respectively. Given the potential quantitative and image quality advantages of PET, imaging of tumor bearing mice was only performed using ⁸⁹Zr-DFO-panitumumab. Immuno-PET imaging of ⁸⁹Zr-DFO-panitumumab in mice bearing breast cancer xenograft tumors with variable HER1 expression showed high tumor uptake (SUV > 7) in the MDA-MB-468 high HER1-expressing mice and a strong correlation between HER1-expression level and tumor uptake (R²= 0.857, P < .001).Conclusions⁸⁹Zr-DFO-panitumumab can prepared with high radiochemical purity and specific activity. ⁸⁹Zr-DFO-panitumumab microPET/CT showed uptake corresponding to HER-1 expression. Due to poor clearance, initial dosimetry estimates suggest that only a low dose ⁸⁹Zr-DFO-panitumumab shows favorable human dosimetry; however due to high tumor uptake, the use of ⁸⁹Zr-DFO-panitumumab is expected to be clinically feasible.     

A heterodimeric [RGD-Glu-[64Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors

IntroductionIn the present study, we describe a ⁶⁴Cu-radiolabeled heterodimeric peptide conjugate for dual α_vβ₃/GRPr (α_vβ₃ integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for α_vβ₃ integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting).MethodsRGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization–mass spectrometry (ESI-MS). Radiolabeling of the conjugate with ⁶⁴Cu produced [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥ 95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model.ResultsA competitive displacement receptor binding assay in human prostate PC-3 cells using ¹²⁵I-[Tyr⁴]BBN as the radioligand showed high binding affinity of [RGD-Glu-[^natCu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09 ± 0.34 nM). A similar assay in human, glioblastoma U87-MG cells using ¹²⁵I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvβ₃ integrin (518 ± 37.5 nM). In vivo studies of [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86 ± 1.01 %ID/g, 1 h post-intravenous injection (p.i.)) and prolonged retention (4.26 ± 1.23 %ID/g, 24 h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4 h p.i.ConclusionsThe favorable pharmacokinetics and enhanced tumor uptake of ⁶⁴Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy.     

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by 177Lu-labeled siRNA targeting IGF-1R

PurposeThe radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 (¹⁷⁷Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this ¹⁷⁷Lu-siRNA complex in the SW480 colon cancer cells were evaluated.MethodsIGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with ¹⁷⁷Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively.ResultsCombined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P < 0.05), but it was significant compared to the Luciferase siRNAs (P < 0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the ¹⁷⁷Lu-IGF-1R siRNA in comparison with either ¹⁷⁷Lu or IGF-1R siRNA (P < 0.001).ConclusionRadioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and ¹⁷⁷Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of ¹⁷⁷Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.     

68Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor expression

PurposeVascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis. In this study, we labeled VEGF₁₂₁ with ⁶⁸Ga using a hydrophilic chelating agent, NODAGA and evaluated the resulting ⁶⁸Ga-NODAGA-VEGF₁₂₁ for in vivo imaging of VEGF receptor (VEGFR) expression.MethodsNODAGA-VEGF₁₂₁ was prepared and its binding affinity for VEGFR2 was measured using ¹²⁵I-VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared by labeling NODAGA-VEGF₁₂₁ with ⁶⁸GaCl₃ followed by purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding studies of ⁶⁸Ga-NODAGA-VEGF₁₂₁ were performed at 37 °C for 4 h. MicroPET imaging followed by biodistribution studies were performed in U87MG tumor-bearing mice injected with ⁶⁸Ga-NODAGA-VEGF₁₂₁. Immunofluorescence staining of the tumor tissues was performed to verify VEGFR2 expression.ResultsBinding affinity of NODAGA-VEGF₁₂₁ for VEGFR2 was found to be comparable to that of VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared in 47.8% yield with specific activity of 3.4 GBq/mg. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was avidly taken up by HAECs with a time-dependent increase from 9.88 %ID at 1 h to 20.86 %ID at 4 h. MicroPET imaging of mice demonstrated high liver and spleen uptake with clear visualization of tumor at 1 h after injection. ROI analysis of tumors revealed 2.53 ± 0.11 %ID/g at 4 h after injection. In the blocking study, tumor uptake was inhibited by 29% at 4 h. Subsequent biodistribution studies demonstrated tumor uptake of 2.38 ± 0.15 %ID/g. Immunofluorescence staining of the tumor tissues displayed high level of VEGFR2 expression.ConclusionsThese results demonstrate that ⁶⁸Ga-NODAGA-VEGF₁₂₁ led to VEGFR-specific distribution in U87MG tumor-bearing mice. This study also suggests that altered physicochemical properties of VEGF₁₂₁ after radiolabeling may affect biodistribution of the radiolabeled VEGF₁₂₁.     

Synthesis and biological evaluation of N-(2-[18F]Fluoropropionyl)-L-methionine for tumor imaging

IntroductionN-position radiolabeled amino acids, such as N-(2-[¹⁸F]fluoropropionyl)-L-methionine ([¹⁸F]FPMET) as a derivative of L-methionine (MET), can potentially serve as a PET tracer for tumor imaging. In the current study, radiosynthesis and biological evaluation of [¹⁸F]FPMET as a new PET tumor agent are performed.Methods[¹⁸F]FPMET was synthesized by reacting 4-nitrophenyl 2-[¹⁸F]fluoropropionate ([¹⁸F]NFP) with MET. In vitro competitive inhibition and protein incorporation experiments were performed with Hepa1-6 hepatoma cell lines. The biodistribution of [¹⁸F]FPMET was determined in S180 fibrosarcoma-bearing mice. PET/CT studies of [¹⁸F]FPMET were conducted in S180 fibrosarcoma-bearing mice, A549 lung adenocarcinoma-bearing nude mice, and PC-3 prostate cancer-bearing nude mice.Results[¹⁸F]FPMET was synthesized in 72% ± 4% uncorrected radiochemical yield (n = 10) from [¹⁸F]NFP. In vitro experiments showed that [¹⁸F]FPMET was primarily transported through Na⁺-dependent system A, system ASC, and system B^0,+, and was not incorporated into protein. Biodistribution and PET/CT imaging studies indicated that [¹⁸F]FPMET could delineate S180 fibrosarcoma, A549 lung adenocarcinoma, and PC-3 prostate cancer.ConclusionAn efficient synthesis of N-position [¹⁸F]labeled amino acids with a classic [¹⁸F]NFP prosthetic group is developed. The results support that [¹⁸F]FPMET seems to be a potential tracer for tumor imaging with PET.     

Photodynamic therapeutic efficacy of symmetrical diiodinated squaraine in in vivo skin cancer models

BackgroundPhotodynamic therapy (PDT) has clinical approval for use as a minimally invasive therapeutic procedure that is able to exert selective cytotoxic activity toward malignant cells. The dye selected for our study, symmetrical diiodinated squaraine, is one of the newly developed photosensitizers. The study is designed to determine the efficacy of PDT mediated by symmetrical diiodinated squaraine in skin tumor induced Swiss albino mice.MethodsSkin tumor was induced in mice with dimethyl benzanthracene (DMBA) and croton oil. After squaraine administration to the tumor mice, photodynamic treatment of tumors was performed using a 1000 W halogen lamp corresponding to the light dose of 100 J/cm². The mice were euthanized and skin flaps and tumor tissues from the back of mice were excised for biochemical studies. The biochemical parameters analyzed include some relevant tumor markers for epithelial tissues, inflammatory markers and markers of apoptosis. The gene expression studies were done by RT-PCR.ResultsAfter two weeks of the treatment, there was significant inflammation. However at 90 days after PDT, the parameters reverted to near-normal values. All marker parameters of tumor progression brought back to normal levels by PDT. Increased caspase-3 activity in PDT treated group shows that cell death might have occurred by apoptosis. The gene expression profile confirms the results.ConclusionsThe results of the whole study show the therapeutic efficacy and apoptosis mediated tumor destruction by squaraine PDT.     

Detection of activated platelets in a mouse model of carotid artery thrombosis with 18F-labeled single-chain antibodies

IntroductionActivated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel ¹⁸F PET radiotracer based on this antibody.MethodsScFv_anti-LIBS and control antibody mut-scFv were reacted with N-succinimidyl-4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl₃ induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied.ResultsAfter incubation with increasing concentrations of ¹⁸F-scFv_anti-LIBS clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled ¹⁸F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6).ConclusionsOur results show that the novel antibody radiotracer ¹⁸F-scFv_anti-LIBS is useful for the sensitive detection of activated platelets and thrombosis.Advances in knowledge and implications for patient careWe describe the first ¹⁸F variant of a scFv_anti-LIBS against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.     

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

IntroductionWith a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors.Methods5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N^?-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N^α-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-d-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations.ResultsThe radiochemical yields for Iodogen and [¹³¹I]IB-Mal-d-GEEEK labeling were 83.6 ± 5.0% (n = 10) and 59.6 ± 9.4% (n = 15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-d-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65 ± 0.61% ID/g vs. 2.92 ± 0.24% ID/g at 8 h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios.ConclusionsTaken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-d-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²⁹I) and PET imaging (¹²⁴I) of patients with HER2-expressing tumors.     

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

ObjectivesWith the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with ¹¹C and evaluated using small-animal PET.Methods[¹¹C]MC113 was synthesized by reaction of O-desmethyl MC113 with [¹¹C]methyl triflate. Small-animal PET was performed with [¹¹C]MC113 in FVB wild-type and Mdr1a/b^(-/-) mice (n = 3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n = 5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15 mg/kg).ResultsBrain uptake of [¹¹C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC_0-60), was moderately but not significantly increased in Mdr1a/b^(-/-) compared with wild-type mice (mean ± SD AUC_0-60, Mdr1a/b^(-/-): 88 ± 7 min, wild-type: 62 ± 6 min, P = 0.100, Mann Whitney test). In the tumor model, AUC_0-60 values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC₅₀) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375 ± 60 nM for MC113 versus 8.5 ± 2.5 nM for tariquidar.Conclusion[¹¹C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [¹¹C]MC113 was only to a low extent effluxed by Pgp. However, [¹¹C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.     

High tumor uptake of 64Cu: Implications for molecular imaging of tumor characteristics with copper-based PET tracers

IntroductionThe use of copper-based positron emission tomography (PET) tracers in cancer studies is increasing. However, as copper has previously been found in high concentrations in human tumor tissue in vivo, instability of PET tracers could result in tumor accumulation of non-tracer-bound radioactive copper that may influence PET measurements. Here we determine the degree of ⁶⁴Cu uptake in five commonly used human cancer xenograft models in mice. Additionally, we compare copper accumulation in tumor tissue to gene expression of human copper transporter 1 (CTR1).MethodsSmall animal PET scans were performed on five different human cancer xenograft mice models 1 h and 22 h post injection (p.i.) of ⁶⁴CuCl₂. Regions of interest (ROIs) were drawn on tumor tissue and sections of various organs on all images. Quantitative real-time PCR (qPCR) gene expression measurements of CTR1 were performed on tumor samples obtained after the 22 h scan.ResultsA relatively high tumor uptake of ⁶⁴Cu was seen in four out of five tumor types and an increase in ⁶⁴Cu accumulation was seen in three out of five tumor types between 1 h and 22 h p.i. No relationship was found between tumor uptake of ⁶⁴Cu and gene expression of CTR1.ConclusionsThe relatively high, time- and tumor type dependent ⁶⁴Cu uptake demonstrated here in five different human cancer xenograft models in mice, emphasizes the importance of validating tracer uptake and indicates that high in vivo stability of copper-based PET tracers is of particular importance because non-tracer-bound copper can accumulate in tumor tissue to a level that could potentially lead to misinterpretation of PET data.     

Monitoring tumor response with [18F]FMAU in a sarcoma-bearing mouse model after liposomal vinorelbine treatment

ObjectivePrevious studies have shown that the accumulation level of FMAU in tumor is proportional to its proliferation rate. This study demonstrated that 2′-deoxy-2′-[¹⁸F]fluoro-β-d-arabinofuranosyluracil ([¹⁸F]FMAU) is a promising PET probe for noninvasively monitoring the therapeutic efficacy of 6% PEGylated liposomal vinorelbine (lipo-VNB) in a subcutaneous murine NG4TL4 sarcoma mouse model.MethodsFemale syngenic FVB/N mice were inoculated with NG4TL4 cells in the right flank. After tumor size reached 150 ± 50 mm³ (day 0), lipo-VNB (5 mg/kg) was intravenously administered on days 0, 3 and 6. To monitor the therapeutic efficacy of lipo-VNB, [¹⁸F]FMAU PET was employed to evaluate the proliferation rate of tumor, and it was compared with that observed from [¹⁸F]FDG/[¹⁸F]fluoroacetate PET. The expression of proliferating cell nuclear antigen (PCNA) in tumor during treatment was determined by semiquantitative analysis of immunohistochemical staining.ResultsA significant inhibition (p < 0.001) in tumor growth was observed on day 3 after a single dose treatment. The tumor-to-muscle ratio (T/M) derived from [¹⁸F]FMAU-PET images of lipo-VNB-treated group declined from 2.33 ± 0.16 to 1.26 ± 0.03 after three doses of treatment, while that of the control remained steady. The retarded proliferation rate of lipo-VNB-treated sarcoma was confirmed by PCNA immunohistochemistry staining. However, both [¹⁸F]FDG and [¹⁸F]fluoroacetate microPET imaging did not show significant difference in T/M between the therapeutic and the control groups throughout the entire experimental period.ConclusionLipo-VNB can effectively impede the growth of NG4TL4 sarcoma. [¹⁸F]FMAU PET is an appropriate modality for early monitoring of the tumor response during the treatment course of lipo-VNB.     

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model

IntroductionDysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using ¹⁸ F-FDG and ¹⁸ F-FLT PET.MethodsU-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 μg) were administered, and mice were evaluated with ¹⁸ F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 μg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using ¹⁸ F-FDG and ¹⁸ F-FLT PET.ResultsIn the dose response study, rilotumumab at doses of 300 and 500 μg was similarly effective against tumor growth. Treatment with 300 and 500 μg rilotumumab inhibited ¹⁸ F-FDG accumulation with significant decreases of ? 37% and ? 40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 μg rilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation with a maximum %ID/g of ? 41% and ? 64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed.ConclusionsRilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate ¹⁸ F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.     

Predicting biochemical tumor control after brachytherapy for clinically localized prostate cancer: The Memorial Sloan-Kettering Cancer Center experience

PurposeTo identify predictors of biochemical tumor control and present an updated prognostic nomogram for patients with clinically localized prostate cancer treated with brachytherapy.Methods and MaterialsOne thousand four hundred sixty-six patients with clinically localized prostate cancer were treated with brachytherapy alone or along with supplemental conformal radiotherapy. Nine hundred one patients (61%) were treated with Iodine-125 (¹²⁵I) monotherapy to a prescribed dose of 144 Gy, and 41 (4.5%) were treated with Palladium-103 (¹⁰³Pd) monotherapy to a prescribed dose of 125 Gy. In patients with higher risk features (n = 715), a combined modality approach was used, which comprised ¹²⁵I or ¹⁰³Pd seed implantation or Iridium-192 high–dose rate brachytherapy followed 1–2 months later by supplemental intensity-modulated image-guided radiotherapy to the prostate.ResultsThe 5-year prostate-specific antigen relapse-free survival (PSA-RFS) outcomes for favorable-, intermediate-, and high-risk patients were 98%, 95%, and 80%, respectively (p < 0.001). Multivariate Cox regression analysis identified Gleason score (p < 0.001) and pretreatment PSA (p = 0.04) as predictors for PSA tumor control. In this cohort of patients, the use of neoadjuvant and concurrent androgen deprivation therapy did not influence biochemical tumor control outcomes. In the subset of patients treated with ¹²⁵I monotherapy, D₉₀ > 140 Gy compared with lower doses was associated with improved PSA-RFS. A nomogram predicting PSA-RFS was developed based on these predictors and had a concordance index of 0.70.ConclusionsResults with brachytherapy for all treatment groups were excellent. D₉₀ higher than 140 Gy was associated with improved biochemical tumor control compared with lower doses. Androgen deprivation therapy use did not impact on tumor control outcomes in these patients.     

Synthesis and application of a novel cysteine-based DTPA-NCS for targeted radioimmunotherapy

IntroductionFor the development of safe and effective protein-based radiolabeled complexes such as radioimmunotherapy (RIT), the selection of the radionuclides and the chelating agents used for the radiolabeling of tumor-targeting molecules is a critical factor. We aim to synthesize a novel bifunctional chelating agent containing the isothiocyanate group for easy conjugation with antibodies having the characteristics of high stable chelation with therapeutic radionuclides.MethodsWe have synthesized the DTPA analogue retaining L-cysteine as a core ligand of the thiol group. The chelating power of cysteine-based DTPA-NCS (cys-DTPA-NCS) was compared with that of commercial ρ-SCN-Bn-DTPA. In an application, the cetuximab was radioimmunoconjugated with ¹⁷⁷Lu using cys-DTPA-NCS. The affinity was tested in a cell line overexpressing EGFR. A therapy study was conducted in nude mice with subcutaneous HT-29 xenografts.ResultsThe cys-DTPA-NCS presents an excellent ability to chelate as compared to the ρ-SCN-Bn-DTPA. For mean ratio chemical labeling yields of 95%, the result was 0.97. ¹⁷⁷Lu-cys-DTPA-NCS-cetuximab was prepared under ambient condition with a high radiolabeling yield and the radiochemical purity was sustained for at least 6 days. The IC₅₀ value of the ¹⁷⁷Lu-labeled cetuximab was 10 nM (95% confidence). The stability and therapeutic efficacy of the candidate radiopharmaceutical were verified.ConclusionThe new DTPA derivative, cys-DTPA-NCS, is a good bifunctional chelating agent that can be used for protein-based radiopharmaceutical using lanthanides such as ¹⁷⁷Lu and ⁹⁰Y. The prepared ¹⁷⁷Lu-cys-DTPA-NCS-cetuximab can be used for the diagnosis and treatment of human colorectal tumor.     

Comparison between two labeled agents in mice using a coinjection-ratio approach in contrast to a conventional group approach

IntroductionThe differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios.MethodsA model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rg^null mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 μg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 μg of ⁹⁰Y-cMORF and 2.5 μg of ^99mTc-cMORF. At 3 h post radioactivity injection, the distribution of ^99mTc was imaged on a SPECT/CT camera and then organs were excised and counted for ⁹⁰Y and ^99mTc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of ⁹⁰Y and ^99mTc data enabled a conventional group comparison. In a new effort, ⁹⁰Y/^99mTc ratios were calculated. Student’s t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis.ResultsThe Student’s t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the ⁹⁰Y/^99mTc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable.ConclusionsAlthough the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into “coinjection correctable” and “coinjection uncorrectable”.