BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized. METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition. RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice. CONCLUSION: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Synthesis, characterization, and imaging of radiopaque bismuth beads for image-guided transarterial embolization.

Current therapy for hypervascular cancers, e.g., hepatocellular carcinoma, includes occlusion of the tumor blood supply by arterial infusion of embolic microspheres (beads) suspended in iodine-based contrast under fluoroscopic guidance. Available radiopaque, imageable beads use iodine as the radiopacifier and cannot be differentiated from contrast. This study aimed to synthesize and characterize imageable beads using bismuth as the radiopacifier that could be distinguished from iodine contrast based upon the difference in the binding energy of k-shell electrons (k-edge). Radiodense bismuth beads were successfully synthesized some with uniform bismuth distribution across the beads. The beads were spherical and could be infused through clinical microcatheters. The bismuth beads could be imaged with clinical dual-energy computed tomography (CT), where iodine-based contrast could be distinguished from the microspheres. The ability to separate iodine from bismuth may enhance the diagnostic information acquired on follow-up CT, identifying the distribution of the embolic beads separately from the contrast. Furthermore, with sequential use of iodine- and bismuth-based beads, the two radiopaque beads could be spatially distinguished on imaging, which may enable the development of dual drug delivery and dual tracking.

Exploration of a F(ab')2 Fragment as the Targeting Agent of α-Radiation Therapy: A Comparison of the Therapeutic Benefit of Intraperitoneal and Intravenous Administered Radioimmunotherapy.

Refinement of treatment regimens enlisting targeted α-radiation therapy (TAT) is an ongoing effort. Among the variables to consider are the target molecule, radionuclide, dosage, and administration route. The panitumumab F(ab')2 fragment targeting epidermal growth factor receptor tolerated modification with the TCMC chelate as well as radiolabeling with 203Pb or 212Pb. Good specific activity was attained when the immunoconjugate was labeled with 212Pb (9.6 ± 1.4 mCi/mg). Targeting of LS-174T tumor xenografts with the 203Pb-panitumumab F(ab')2 demonstrated comparable amounts of uptake to the similarly radiolabeled panitumumab IgG. A dose escalation study was performed to determine an effective working dose for both intraperitoneal (i.p.) and intravenous (i.v.) injections of 212Pb-panitumumab F(ab')2. Therapeutic efficacy, with modest toxicity, was observed with 30 µCi given i.p. Results for the i.v. administration were not as definitive and the experiment was repeated with a higher dose range. From this study, 20 µCi given i.v. was selected as the effective working dose. A subsequent therapy study combined gemcitabine or paclitaxel with i.v. 212Pb-panitumumab F(ab')2, which increased the median survival (MS) of LS-174T tumor-bearing mice to 208 and 239 d, respectively. Meanwhile, the MS of mice treated with i.v. 212Pb-panitumumab F(ab')2 alone was 61 and 11 d for the untreated group of mice. In conclusion, the panitumumab F(ab')2 fragment whether given by i.p. or i.v. injection, is a viable candidate as a delivery vector for TAT of disseminated i.p. disease.

Comparative studies on the therapeutic benefit of targeted α-particle radiation therapy for the treatment of disseminated intraperitoneal disease.

Identification of the appropriate combination of radionuclide, target and targeting vehicle is critical for successful radioimmunotherapy. For the treatment of disseminated peritoneal diseases such as pancreatic or ovarian cancer, α-emitting radionuclides have been proposed for targeted radiation therapy. This laboratory has taken a systematic approach investigating targeted α-radiation therapy, allowing comparisons to now be made between 211At, 227Th, 213Bi and 212Pb. Herein, trastuzumab radiolabeled with 211At and 227Th was evaluated for therapeutic efficacy in the LS-174T i.p. tumor model. A dose escalation study was conducted with each radioimmunoconjugate (RIC). Therapeutic benefit was realized with 211At-trastuzumab with doses of 20, 30 and 40 µCi. At doses >40 µCi, toxicity was observed with greater weight loss and 2-fold higher decrease in the platelet counts. Following a second study comparing the effect of 20, 30 and 40 µCi of 211At-trastuzumab, 30 µCi was selected as the dose for future studies. A parallel study was performed evaluating 0.25, 0.5, 1.0, 2.0 and 5.0 µCi of 227Th-trastuzumab. The 0.5 and 1.0 µCi injected dose resulted in a therapeutic response; a lower degree of weight loss was experienced by the mice in the 0.5 µCi cohort. When the data is normalized for comparing 211At, 227Th, 213Bi and 212Pb, the choice of radionuclide for RIT is perhaps not entirely based on simple therapeutic efficacy, other factors may play a role in choosing the "right" radionuclide.

Targeted α-Particle Radiation Therapy of HER1-Positive Disseminated Intraperitoneal Disease: An Investigation of the Human Anti-EGFR Monoclonal Antibody, Panitumumab.

Identifying molecular targets and an appropriate targeting vehicle, i.e., monoclonal antibodies (mAb) and their various forms, for radioimmunotherapy (RIT) remains an active area of research. Panitumumab, a fully human and less immunogenic mAb that binds to the epidermal growth factor receptor (Erb1; HER1), was evaluated for targeted α-particle radiation therapy using 212Pb, an in vivo α generator. A single dose of 212Pb-panitumumab administered to athymic mice bearing LS-174T intraperitoneal (i.p.) tumor xenografts was found to have greater therapeutic efficacy when directly compared with 212Pb-trastuzumab, which binds to HER2. A dose escalation study determined a maximum effective working dose of 212Pb-panitumumab to be 20µCi with a median survival of 35 days versus 25 days for the untreated controls. Pretreatment of tumor-bearing mice with paclitaxel and gemcitabine 24hours prior to injection of 212Pb-pantiumumab at 10 or 20µCi resulted in the greatest enhanced therapeutic response at the higher dose with median survivals of 106 versus 192 days, respectively. The greatest therapeutic impact, however, was observed in the animals that were treated with topotecan 24hours prior to RIT and then again 24hours after RIT; the best response from this combination was also obtained with the lower 10-µCi dose of 212Pb-panitumumab (median survival >280 days). In summary, 212Pb-panitumumab is an excellent candidate for the treatment of HER1-positive disseminated i.p. disease. Furthermore, the potentiation of the therapeutic impact of 212Pb-pantiumumab by chemotherapeutics confirms and validates the importance of developing a multimodal therapy regimen.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

First-In-Human Study Demonstrating Tumor-Angiogenesis by PET/CT Imaging with (68)Ga-NODAGA-THERANOST, a High-Affinity Peptidomimetic for αvß3 Integrin Receptor Targeting.

UNLABELLED: (68)Ga-NODAGA-THERANOST™ is an αvß3 integrin antagonist and the first radiolabeled peptidomimetic to reach clinical development for targeting integrin receptors. In this first-in-human study, the feasibility of integrin receptor peptidomimetic positron emission tomography/computed tomography (PET/CT) imaging was confirmed in patients with non-small-cell lung cancer and breast cancer. METHODS: Patients underwent PET/CT imaging with (68)Ga NODAGA-THERANOST. PET images were analyzed qualitatively and quantitatively and compared to 2-deoxy-2-((18)F) fluoro-d-glucose ((18)F-FDG) findings. Images were obtained 60 minutes postinjection of 300-500 MBq of (68)Ga-NODAGA-THERANOST. RESULTS: (68)Ga-NODAGA-THERANOST revealed high tumor-to-background ratios (SUVmax=4.8) and uptake at neoangiogenesis sites. Reconstructed fused images distinguished cancers with high malignancy potential and enabled enhanced bone metastasis detection. (18)F-FDG-positive lung and lymph node metastases did not show uptake, indicating the absence of neovascularization. CONCLUSIONS: (68)Ga-NODAGA-THERANOST was found to be safe and effective, exhibiting in this study rapid blood clearance, stability, rapid renal excretion, favorable biodistribution and PK/PD, low irradiation burden (µSv/MBq/µg), and convenient radiolabeling. This radioligand might enable theranostics, that is, a combination of diagnostics followed by the appropriate therapeutics, namely antiangiogenic therapy, image-guided presurgical assessment, treatment response evaluation, prediction of pathologic response, neoadjuvant-peptidomimetic-radiochemotherapy, and personalized medicine strategies. Further clinical trials evaluating (68)Ga-NODAGA-THERANOST are warranted.

Synthesis and characterization of gadolinium-Peptidomimetic complex as an αvß3 integrin targeted MR contrast agent.

There is growing interest in small and rigid peptidomimetic αvß3 integrin antagonists that are readily synthesized and characterized and amenable to physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-ß-alanine (IAC) was successfully conjugated to DOTA, complexed with Gd(III) and radiolabeled with (153)Gd. Radioassay results demonstrated specificity of the labeled conjugate by blocking ∼95% binding with the addition of a 50-fold molar excess of cold IAC to the reaction solution. Relaxometry was used to support the hypothesis that the specificity of the Gd-peptidomimetic targeting αvß3 integrin would increase the contrast and therefore enhance the sensitivity of an MRI scan of αvß3 integrin positive tissues. Magnetic resonance imaging of cell pellets (M21 human melanoma) was also performed, and the images clearly show that cells reacted with Gd(III)-DOTA-IAC display a brighter image than cells without the Gd(III)-DOTA-IAC contrast agent. In addition, Gd(III)-DOTA-IAC and IAC, with IC50 of 300nM and 230nM, respectively, are 2.1 and 2.7 times more potent than c(RGDfK) whose IC50 is 625nM. This promising preliminary data fuels further investigation of DOTA-IAC conjugates for targeting tumor associated angiogenesis and αvß3 integrin positive tumors using magnetic resonance imaging.

Evaluation of cetuximab as a candidate for targeted α-particle radiation therapy of HER1-positive disseminated intraperitoneal disease.

Although the epidermal growth factor receptor (EGFR), also known as HER1, has been studied for over a decade, it continues to be a molecule of great interest and focus of investigators for development of targeted therapies. The marketed monoclonal antibody cetuximab binds to HER1, and thus might serve as the basis for creation of imaging or therapies that target this receptor. The potential of cetuximab as a vehicle for the delivery of α-particle radiation was investigated in an intraperitoneal tumor mouse model. The effective working dose of 10 µCi of (212)Pb-cetuximab was determined from a dose (10-50 µCi) escalation study. Toxicity, as indicated by the lack of animal weight loss, was not evident at the 10 µCi dose of (212)Pb-cetuximab. A subsequent study demonstrated (212)Pb-cetuximab had a therapeutic efficacy similar to that of (212)Pb-trastuzumab (p = 0.588). Gemcitabine given 24 h prior to (212)Pb-cetuximab increased the median survival from 174 d to 283 d, but carboplatin suppressed the effectiveness of (212)Pb-cetuximab. Notably, concurrent treatment of tumor-bearing mice with (212)Pb-labeled cetuximab and trastuzumab provided therapeutic benefit that was greater than either antibody alone. In conclusion, cetuximab proved to be an effective vehicle for targeting HER1-expressing tumors with α-radiation for the treatment of disseminated intraperitoneal disease. These studies provide further evidence that the multimodality therapy regimens may have greater efficacy and benefit in the treatment of cancer patients.

In vitro and in vivo analysis of indocyanine green-labeled panitumumab for optical imaging-a cautionary tale.

Indocyanine green (IC-Green), the only FDA approved near-infrared (NIR) fluorophore for clinical use, is attractive to researchers for the development of targeted optical imaging agents by modification of its structure and conjugation to monoclonal antibodies (mAbs) or their fragments. IC-Green derivative, ICG-sulfo-OSu (ICG-sOSu), is frequently used for antibody conjugation. However, ICG-sOSu is amphiphilic and readily facilitates aggregation of mAbs that is not easily separable from the desired immunoconjugates. Complications originating from this behavior are frequently overlooked by researchers. This study examined detailed chemical and biological characteristics of an ICG-sOSu-labeled mAb, panitumumab, and provided a clinically applicable strategy to deliver a pure conjugation product. Size-exclusion high-performance liquid chromatography (SE-HPLC) analysis of conjugation reactions, performed at molar reaction ratios of ICG-sOSu: mAb of 5, 10, or 20, resulted in isolable desired ICG-sOSu-panitumumab conjugation product in 72%, 53%, and 19% yields, respectively, with the remainder consisting of high molecular weight aggregates (>150 kDa) 14%, 30%, and 51%, respectively. The HPLC-purified ICG-sOSu-panitumumab products were analyzed by native and SDS polyacrylamide gel electrophoresis (PAGE) followed by optical imaging. Results indicated that the interaction between ICG-sOSu and panitumumab was due to both covalent and noncovalent binding of the ICG-sOSu to the protein. Noncovalently bound dye in the ICG-sOSu-panitumumab conjugate products was removed by extraction with ethyl acetate to further purify the HPLC-isolated conjugation products. With conserved immunoreactivity, excellent target-specific uptake of the doubly purified bioconjugates was observed with minimal liver retention in athymic nude mice bearing HER1-expressing tumor xenografts. In summary, the preparation of well-defined bioconjugate products labeled with commercial ICG-sOSu dye is not a simple process and control of the conjugation reaction ratio and conditions is crucial. Furthermore, absolute purification and characterization of the products is necessitated prior to in vivo optical imaging. Use of validated and characterized dye conjugate products should facilitate the development of clinically viable and reproducible IC-Green derivative and other NIR dye mAb conjugates for optical imaging applications.

Gene expression profiling upon (212) Pb-TCMC-trastuzumab treatment in the LS-174T i.p. xenograft model.

Recent studies have demonstrated that therapy with (212) Pb-TCMC-trastuzumab resulted in (1) induction of apoptosis, (2) G2/M arrest, and (3) blockage of double-strand DNA damage repair in LS-174T i.p. (intraperitoneal) xenografts. To further understand the molecular basis of the cell killing efficacy of (212) Pb-TCMC-trastuzumab, gene expression profiling was performed with LS-174T xenografts 24 h after exposure to (212) Pb-TCMC-trastuzumab. DNA damage response genes (84) were screened using a quantitative real-time polymerase chain reaction array (qRT-PCR array). Differentially regulated genes were identified following exposure to (212) Pb-TCMC-trastuzumab. These included genes involved in apoptosis (ABL, GADD45α, GADD45γ, PCBP4, and p73), cell cycle (ATM, DDIT3, GADD45α, GTSE1, MKK6, PCBP4, and SESN1), and damaged DNA binding (DDB) and repair (ATM and BTG2). The stressful growth arrest conditions provoked by (212) Pb-TCMC-trastuzumab were found to induce genes involved in apoptosis and cell cycle arrest in the G2/M phase. The expression of genes involved in DDB and single-strand DNA breaks was also enhanced by (212) Pb-TCMC-trastuzumab while no modulation of genes involved in double-strand break repair was apparent. Furthermore, the p73/GADD45 signaling pathway mediated by p38 kinase signaling may be involved in the cellular response, as evidenced by the enhanced expression of genes and proteins of this pathway. These results further support the previously described cell killing mechanism by (212) Pb-TCMC-trastuzumab in the same LS-174T i.p. xenograft. Insight into these mechanisms could lead to improved strategies for rational application of radioimmunotherapy using α-particle emitters.

(111)In- and (203)Pb-Labeled Cyclic RGD Peptide Conjugate as an α(v)ß(3) Integrin-Binding Radiotracer.

Methodology for site-specific modification and chelate conjugation of a cyclic RGD (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting α(v)ß(3) integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the (111)In-labeled peptide conjugate with α(v)ß(3) integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a mono-valent modified RGD peptide.

Synthesis and characterization of αvß3-targeting peptidomimetic chelate conjugates for PET and SPECT imaging.

There is growing interest in small peptidomimetic α(v)ß(3) integrin antagonists that are readily synthesized and characterized and can be easily handled using physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-ß-alanine (IAC) was successfully conjugated to 1-(1-carboxy-3-carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane (NODA-GA(tBu)(3)) and 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)(4)) and radiolabeled with (111)In, (67)Ga and (203)Pb. Results of a radioimmunoassay demonstrated binding to purified α(v)ß(3) integrin when 1-4equiv of integrin were added to the reaction. Based on this promising result, investigations are moving forward to evaluate the NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogenesis and α(v)ß(3) integrin positive tumors to define their PET and SPECT imaging qualities as well as their potential for delivery of therapeutic radionuclides.

Evaluation of 99mTc-labeled cyclic RGD dimers: impact of cyclic RGD peptides and 99mTc chelates on biological properties.

The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (∼111 GBq/µmol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ∼ RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the α(v)ß(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor α(v)ß(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin α(v)ß(3)-positive tumors.

Preparation of cystamine core dendrimer and antibody-dendrimer conjugates for MRI angiography.

Herein we report the preparation along with the in vivo and in vitro MRI characterization of two generation four and five cystamine core dendrimers loaded with thirty and fifty-eight derivatized Gd-DOTA (G4SS30, G5SS58) respectively. Likewise the development and characterization of two half-dendrimers conjugated to the F(ab')(2) fragment of the monoclonal antibody (mAb) panitumumab functionalized with a maleimide conjugation functional group site (Ab-(G4S15)(4), Ab-(G5S29)(4)) are also described. The in vitro molar relaxivity of the Ab-(G4S15)(4) conjugate, measured at pH 7.4, 22 °C, and 3T showed a moderate increase in relaxivity as compared to Magnevist (6.7 vs 4.0 mM(-1) s(-1)) while the Ab-(G5S29)(4) conjugate was 2-fold higher (9.1 vs 4.0 mM(-1) s(-1)). The data showed that only a high injection dose (0.050 mmol Gd(3+)/kg) produced a detectable contrast enhanced contrast for the Ab-(G4S15)(4) conjugate while a lower dose (0.035 mmol Gd(3+)/kg) was sufficient for the Ab-(G5S29)(4) conjugate. The antibody-SMCC conjugate was purified by a Sephadex G-100 column, and the antibody-dendrimer-based agents were purified by spin filtration using a Centricon filter (50,000 MCO). The protein assay coupled with cysteine and Ellman's assay indicated an antibody to dendrimer ratio of 1:4. The in vivo blood clearance half-lives of the four agents measured at the jugular vein were ~12-22 min.

An overview of targeted alpha therapy.

The effectiveness of targeted α-therapy (TAT) can be explained by the properties of α-particles. Alpha particles are helium nuclei and are ~8,000 times larger than ß(-)-particles (electrons). When emitted from radionuclides that decay via an α-decay pathway, they release enormous amounts of energy over a very short distance. Typically, the range of α-particles in tissue is 50-100 µm and they have high linear energy transfer (LET) with a mean energy deposition of 100 keV/µm, providing a more specific tumor cell killing ability without damage to the surrounding normal tissues than ß(-)-emitters. Due to these properties, the majority of pre-clinical and clinical trials have demonstrated that α-emitters such as (225)Ac, (211)At, (212)Bi, (213)Bi, (212)Pb, (223)Ra, and (227)Th are ideal for the treatment of smaller tumor burdens, micrometastatic disease, and disseminated disease. Even though these α-emitters have favorable properties, the development of TAT has been limited by high costs, unresolved chemistry, and limited availability of the radionuclides. To overcome these limitations, more potent isotopes, additional sources, and more efficient isotope production methods should be addressed. Furthermore, better chelation and labeling methods with the improvements of isotope delivery, targeting vehicles, molecular targets, and identification of appropriate clinical applications are still required.

Evaluation of In-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability.

PURPOSE: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG(4)-c(RGDfK): PEG(4) = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD(2) (PEG(4)-E[c(RGDfK)](2), 2P-RGD(4) (E{PEG(4)-E[c(RGDfK)](2)}(2), 2P4G-RGD(4) (E{PEG(4)-E[G(3)-c(RGDfK)](2)}(2): G(3) = Gly-Gly-Gly) and 6P-RGD(4) (E{PEG(4)-E[PEG(4)-c(RGDfK)](2)}(2)) in binding to integrin α(v)ß(3), and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding (111)In radiotracers. METHODS: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD(2), DOTA-2P-RGD(4), DOTA-2P4G-RGD(4), DOTA-6P-RGD(4)), and their (111)In complexes were prepared. The integrin α(v)ß(3) binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against (125)I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts. RESULTS: The integrin α(v)ß(3) binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD(4) (IC(50) = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD(4) (IC(50) = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD(4) (IC(50) = 0.5 ± 0.1 nM) > DOTA-3P-RGD(2) (DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2): IC(50) = 1.5 ± 0.2 nM) > DOTA-P-RGD(2) (IC(50) = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC(50) = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC(50) = 49.9 ± 5.5 nM) >> DOTA-6P-RGK(4) (IC(50) = 437 ± 35 nM). The fact that DOTA-6P-RGK(4) had much lower integrin α(v)ß(3) binding affinity than DOTA-6P-RGD(4) suggests that the binding of DOTA-6P-RGD(4) to integrin α(v)ß(3) is RGD-specific. This conclusion is consistent with the lower tumor uptake for (111)In(DOTA-6P-RGK(4)) than that for (111)In(DOTA-6P-RGD(4)). It was also found that the G(3) and PEG(4) linkers between RGD motifs have a significant impact on the integrin α(v)ß(3)-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of (111)In-labeled cyclic RGD peptides. CONCLUSION: On the basis of their integrin α(v)ß(3) binding affinity and tumor uptake of their corresponding (111)In radiotracers, it was conclude that 2P-RGD(4), 2P4G-RGD(4) and 6P-RGD(4) are most likely bivalent in binding to integrin α(v)ß(3), and extra RGD motifs might contribute to the long tumor retention times of (111)In(DOTA-2P-RGD(4)),( 111)In(DOTA-2P4G-RGD(4)) and (111)In(DOTA-6P-RGD(4)) than that of (111)In(DOTA-3P-RGD(3)) at 72 h p.i. Among the (111)In-labeled cyclic RGD tetramers evaluated in the glioma model, (111)In(DOTA-2P4G-RGD(4)) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that (90)Y(DOTA-2P4G-RGD(4)) and (177)Lu(DOTA-2P4G-RGD(4)) might have the potential for targeted radiotherapy of integrin α(v)ß(3)-positive tumors.

Evaluation of 99mTc-labeled cyclic RGD peptide with a PEG4 linker for thrombosis imaging: comparison with DMP444.

DMP444 is a (99m)Tc-labeled cyclic RGD peptide, which has been evaluated in preclinical canine deep vein thrombosis (DVT) and pulmonary embolism (PE) models, and in patients with DVT and PE by SPECT (single photon emission computed tomography). Clinical data indicated that DMP444 is useful for imaging DVT, but it had limited utility for imaging PE in patients. To understand its clinical findings, we prepared a new radiotracer P4-DMP444 by replacing the lipophilic 6-aminocaproic acid (CA) in DMP444 with a highly water-soluble PEG(4) (15-amino-4,7,10,13-tetraoxapentadecanoic acid) linker. The objective of this study was to explore the impact of PEG(4) on biological properties (biodistribution, excretion kinetics, and capability to image thrombi) of (99m)Tc radiotracer. We also used canine DVT and PE models to perform imaging studies with/without the heparin pretreatment. These studies were specifically designed to explore the impact of heparin treatment on thrombosis uptake of P4-DMP444. It was found that replacing the CA linker with PEG(4) could enhance the radiotracer clearance kinetics from blood and normal organs in both rats and dogs. The fact that P4-DMP444 and DMP444 share very similar thrombosis uptake in both DVT and PE models suggests that the PEG(4) linker has little effect on GPIIb/IIIa binding affinity of cyclic RGD peptide. Even though P4-DMP444 had less accumulation than DMP444 in the blood, heart, lungs, and muscle over the 2 h study period in both rats and dogs, the difference in PE/lung and DVT/muscle ratios is marginal, suggesting that one PEG(4) linker is not sufficient to dramatically change the contrast between thrombus and background. It is very important to note that the heparin treatment of dogs with DVT and PE resulted in dramatic decrease in accumulation of P4-DMP444 in fresh thrombi. On the basis of these results, we believe that DMP444 and P4-DMP444 are excellent radiotracers for imaging both DVT and PE, and should be used in patients without antithrombosis treatment at the time of imaging.

64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

99mTc-labeled cyclic RGD peptides for noninvasive monitoring of tumor integrin αVß3 expression.

This report describes the biologic evaluations of [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2: 6-hydrazinonicotinyl; 3P-RGD2  =  PEG4-E[PEG4-c(RGDfK)]2; PEG4  =  15-amino-4,7,10,13-tetraoxapentadecanoic acid; and TPPTS  =  trisodium triphenylphosphine-3,3',3''-trisulfonate), [99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)] (99mTc-3G-RGD2: 3G-RGD2  =  G3-E[G3-c(RGDfK)]2 and G3  =  Gly-Gly-Gly), and 99mTcO(MAG2-3G-RGD2) (MAG2  =  mercaptoacetylglycylglycyl) as radiotracers for noninvasive imaging of tumor integrin αvß3 expression in five xenografted tumor-bearing models. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG, MDA-MB-435, A549, HT29, or PC-3 tumor xenografts. Immunochemistry was performed using the cultured primary tumor cells and xenografted tumor tissues. It was found that the radiotracer tumor uptake followed the trend U87MG > MDA-MB-435 ≈ HT29 ≈ A549 > PC-3. The total integrin ß3 expression levels followed the general trend: U87MG > MDA-MB-435 ≈ A549∼HT29 > PC-3. There is a linear relationship between the radiotracer injected dose per gram tumor uptake and the total integrin ß3 expression levels. On the basis of these, it was concluded that radiotracer tumor uptake is contributed by integrin αvß3 expressed on tumor cells and activated endothelial cells of the tumor neovasculature. 99mTc-3P-RGD2 has the capability to monitor integrin αvß3 expression in a noninvasive fashion.