111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model

L

Development of Radiolabeled Antibody-Targeted Gemini Nanoparticles for Melanoma

L

A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system

Abstract Background Advances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70–120 kDa) and bi- or tri-valency. Results We described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv. Conclusion We used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens

Zirconium-89 Labeled Gene Delivery Nanoparticles as Theranostic Agents for Melanoma

KAL

Development and preliminary evaluation of 68Ga-labeled pamoic acid derivative for early non-invasive detection of necrosis by PET

Objectives: Recently, we reported that the 99mTc-tricarbonyl complex of N,N'-bis(diethylenetriamino pentaacetato)-4,4'-methylene bis(2-hydroxy-3-naphthoic hydrazide) (bis-DTPA-pamoate) shows high avidity for necrotic tissue in different animal models of necrosis. In the present study we have developed a 68Ga-bis-DTPA-pamoate and explored its suitability for non-invasive in vivo detection of necrosis by µPET. Methods: Bis-DTPA-pamoate was labeled with 68Ga3+ (68Ga/68Ge generator) and the complex was purified using a SPE column. Biodistribution was studied in normal NMRI mice at 30 min and 4 h post injection (p.i.). Necrosis avidity was evaluated in vivo in rats with reperfused hepatic infarction, ethanol induced liver and muscular necrosis after injection of 28 MBq 68Ga-bis-DTPA-pamoate by dynamic µPET imaging and ex vivo by autoradiography and gamma counting in correlation with histochemical staining (TTC and H&E) techniques. Results: Radiolabeling yield of 68Ga-bis-DTPA-pamoate was >90%. In normal mice, the tracer agent was excreted via the hepatobiliary (liver activity was 3.4 and 2.8 %ID/g at 30 min and 4 h p.i., respectively) and renal pathways (kidneys activity was 13.8 and 8.4 %ID/g at 30 min and 4 h p.i., respectively). In all three models of necrosis, using 68Ga-bis-DTPA-pamoate, necrotic tissue was unequivocally delineated in vivo by µPET at 30 min p.i. Specificity for necrotic tissue was also confirmed ex vivo by autoradiography with infarct:viable tissue activity ratio of 10:1 (90 min p.i.) and gamma counting. TTC and H&E staining confirmed the presence of necrotic tissue. Conclusions: The study demonstrates the feasibility of imaging necrosis with a novel PET imaging agent.status: publishe

Non-invasive detection and quantification of acute myocardial infarction in rabbits using mono-[123I]iodohypericin microSPECT

AIMS: Mono-[(123)I]iodohypericin ([(123)I]MIH) has been reported to have high avidity for necrosis. In the present study, by using rabbit models of acute myocardial infarction, we explored the suitability of [(123)I]MIH micro single photon emission computed tomography (microSPECT) for non-invasive visualization of myocardial infarcts in comparison with [(13)N]ammonia micro positron emission tomography (microPET) imaging, postmortem histomorphometry, and [(123)I]MIH autoradiography. METHODS AND RESULTS: Fourteen rabbits were divided into four groups. The left circumflex coronary artery was permanently occluded in group A (n = 3), reperfused by releasing the ligature after 15 min in group B (n = 3) or 90 min in group C (n = 6), or not occluded in group D (n = 2). Animals received [(13)N]ammonia microPET perfusion imaging 18 h after infarct induction followed by microSPECT imaging at 2-3.5, 9-11, and 22-24 h post injection (p.i.) of [(123)I]MIH. The cardiac images were assembled into polar maps for assessment of tracer uptake. Animals were sacrificed and the excised heart was sliced for autoradiography, triphenyl tetrazolium chloride, and haematoxylin-eosin staining. Using [(123)I]MIH microSPECT, infarcts were well delineated at 9 h p.i. Mean microSPECT infarct size was 38.8 and 32.7% of left ventricular area for groups A and C, respectively, whereas group B showed low uptake of [(123)I]MIH. Highest mean infarct/viable tissue activity ratio of 61/1 was obtained by autoradiography in group C animals at 24 h p.i. CONCLUSION: The study indicates the suitability of [(123)I]MIH for in vivo visualization of myocardial infarcts.status: publishe

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

Antibodies that recognize cancer biomarkers, such as MUC16, can be used as vehicles to deliver contrast agents (imaging) or cytotoxic payloads (therapy) to the site of tumors. MUC16 is overexpressed in 80% of epithelial ovarian cancer (EOC) and 65% of pancreatic ductal adenocarcinomas (PDAC), where effective ‘theranostic’ probes are much needed. This work aims to develop fully human antibodies against MUC16 and evaluate them as potential immuno-PET imaging probes for detecting ovarian and pancreatic cancers. We developed a fully human monoclonal antibody, M16Ab, against MUC16 using phage display. M16Ab was conjugated with p-SCN-Bn-DFO and radiolabeled with 89Zr. 89Zr-DFO-M16Ab was then evaluated for binding specificity and affinity using flow cytometry. In vivo evaluation of 89Zr-DFO-M16Ab was performed by microPET/CT imaging at different time points at 24–120 h post injection (p.i.) and ex vivo biodistribution studies in mice bearing MUC16-expressing OVCAR3, SKOV3 (ovarian) and SW1990 (pancreatic) xenografts. 89Zr-DFO-M16Ab bound specifically to MUC16-expressing cancer cells with an EC50 of 10nM. 89Zr-DFO-M16Ab was stable in serum and showed specific uptake and retention in tumor xenografts even after 120 h p.i. (microPET/CT) with tumor-to-blood ratios > 43 for the SW1990 xenograft. Specific tumor uptake was observed for SW1990/OVCAR3 xenografts but not in MUC16-negative SKOV3 xenografts. Pharmacokinetic study shows a relatively short distribution (t1/2α) and elimination half-life (t1/2ß) of 4.4 h and 99 h, respectively. In summary, 89Zr-DFO-M16Ab is an effective non-invasive imaging probe for ovarian and pancreatic cancers and shows promise for further development of theranostic radiopharmaceuticals