The monoclonal antibody, cetuximab, binds to epidermal growth-factor receptor and thus provides an opportunity to create both imaging and therapies that target this receptor. The potential of cetuximab as a radioimmunoconjugate, using the acyclic bifunctional chelator, CHX-A″-DTPA, was investigated. The pharmacokinetic behavior in the blood was determined in mice with and without tumors. Tumor targeting and scintigraphic imaging were evaluated in mice bearing xenografts of LS-174T (colorectal), SHAW (pancreatic), SKOV3 (ovarian), DU145 (prostate), and HT-29 (colorectal). Excellent tumor targeting was observed in each of the models with peak tumor uptakes of 59.8 ± 18.1, 22.5 ± 4.7, 33.3 ± 5.7, 18.2 ± 7.8, and 41.7 ± 10.8 injected dose per gram (%ID/g) at 48–72 hours, respectively. In contrast, the highest tumor %ID/g obtained in mice bearing melanoma (A375) xenografts was 6.3 ± 1.1 at 72 hours. The biodistribution of 111In-cetuximab was also evaluated in nontumor-bearing mice. The highest %ID/g was observed in the liver (9.3 ± 1.3 at 24 hours) and the salivary glands (8.1 ± 2.8 at 72 hours). Scintigraphy showed excellent tumor targeting at 24 hours. Blood pool was evident, as expected, but cleared over time. At 168 hours, the tumor was clearly discernible with negligible background.
Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (αvβ3). We have pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with RGD-cyclopeptides. An orthogonal oxime-ligation strategy was applied to chemoselectively effect conjugation of the PAMAM dendrimers with RGD-cyclopeptides for targeting αvβ3. Fluorescent dyes for optical imaging and chelates for gadolinium-based magnetic resonance (MR) imaging were subsequently appended to create robust multimodal macromolecular imaging agents. Fluorescence microscopy revealed selective binding of the resulting RGD peptide-bearing dendrimer with empty chelates to αvβ3-expressing cells, but somewhat reduced selectivity was observed following Gd(III) complexation. The expected incomplete saturation of chelates with Gd(III) ions permitted radiometal complexation, and an in vivo tissue distribution of the resulting agent in M21 melanoma tumor-bearing mice showed mostly renal and reticuloendothelial accumulation, with the tumor:blood ratio peaking (3.30 ± 0.03) at 2 h postinjection.
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.
The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.
The studies described herein assess the potential of combining platinum-based chemotherapy with high-linear energy transfer (LET) α-particle-targeted radiation therapy using trastuzumab as the delivery vehicle. An initial study explored the combination of cisplatin with (213)Bi-trastuzumab in the LS-174T i.p. xenograft model. This initial study determined the administration sequence of cisplatin and (213)Bi-trastuzumab. Cisplatin coinjected with (213)Bi-trastuzumab increased the median survival (MS) to 90 days versus 65 days for (213)Bi-trastuzumab alone. Toxicity was observed with a weight loss of 17.6% in some of the combined treatment groups. Carboplatin proved to be better tolerated. Maximal therapeutic benefit, that is, a 5.1-fold increase in MS, was obtained in the group injected with (213)Bi-trastuzumab, followed by carboplatin 24 hours later. This was further improved by administration of multiple weekly doses of carboplatin. The MS achieved with administration of 3 doses of carboplatin was 180 days versus 60 days with (213)Bi-trastuzumab alone. The combination of carboplatin with (212)Pb radioimmunotherapy was also evaluated. The therapeutic efficacy of (212)Pb-trastuzumab (58-day MS) increased when the mice were pretreated with carboplatin 24 hours prior (157-day MS). These results again demonstrate the necessity of empirically determining the administration sequence when combining therapeutic modalities.
In this paper, we report a new method to prepare and characterize a contrast agent based on a fourth-generation (G4) polyamidoamine (PAMAM) dendrimer conjugated to the gadolinium complex of the bifunctional diethylenetriamine pentaacetic acid derivative (1B4M-DTPA). The method involves preforming the metal-ligand chelate in alcohol prior to conjugation to the dendrimer. The dendrimer-based agent was purified by a Sephadex G-25 column and characterized by elemental analysis. The analysis and SE-HPLC data gave a chelate to dendrimer ratio of 30:1 suggesting conjugation at approximately every other amine terminal on the dendrimer. Molar relaxivity of the agent measured at pH 7.4 displayed a higher value than that of the analogous G4 dendrimer based agent prepared by the postmetal incorporation method (r(1) = 26.9 vs 13.9 mM(-1) s(-1) at 3 T and 22 degrees C). This is hypothesized to be due to the higher hydrophobicity of this conjugate and the lack of available charged carboxylate groups from noncomplexed free ligands that might coordinate to the metal and thus also reduce water exchange sites. Additionally, the distribution populations of compounds that result from the postmetal incorporation route are eliminated from the current product simplifying characterization as quality control issues pertaining to the production of such agents for clinical use as MR contrast agents. In vivo imaging in mice showed a reasonably fast clearance (t(1/2) = 24 min) suggesting a viable agent for use in clinical application.
1223 Background: Mesothelin (MSLN) targeted thorium-227 (MSLN-TTC; [227Th]Th-3,2-HOPO-MSLN-mAb) has demonstrated in vivo efficacy in MSLN positive tumors1,2. This MSLN antibody (MSLN-mAb, BAY 861903) based TTC is currently being evaluated in Phase I clinical trial (NCT03507452). Due to the low gamma emission with low abundance of measurable photons in the decay chain of thorium-227, tumor imaging using thorium-227 based conjugate is technically challenging3,4. Besides, forming a complex with thorium-227, the 3-hydroxypyridin-2-one (3,2-HOPO) chelator can also form complex with zirconium-89.,6. This approach thus would allow for radiolabeling the 3,2-HOPO-MSLN-mAb either with therapeutic or imaging radionuclide. Thus, the 3,2-HOPOH-MSLN-mAb was labeled with zirconium-89 to produce [89Zr]Zr-3,2-HOPO-MSLN-mAb. In parallel, the MSLN-mAb was conjugated to deferoxamine (DFO; [89Zr]Zr-DFO-MSLN-mAb), one of the widely used chelator of zirconium-89. Both the zirconium-89 conjugates were compared in vitro and in vivo using MSLN positive tumor xenograft mouse models.
Methods: 3,2-HOPO-MSLN-mAb (BAY 2287409) and DFO-MSLN-mAb conjugates were labeled with zirconium-89 to yield [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb respectively. Radiochemical purity (RCP) was determined by size exclusion HPLC. The zirconium-89 conjugates were evaluated in vitro (binding affinity) and in vivo for biodistribution and PET imaging of HT29-MSLN and patient-derived (PDXs, NCI-Meso21 and NCI-Meso16) tumor xenografts. After injecting (i.v) tumor bearing mice (Athymic, nu/nu, female) with the radioactive conjugates, biodistribution and imaging was performed on days 1, 3, and 6 for HT29-MSLN xenografts and on day 3 for PDXs. Tissue associated radioactivity was determined by gamma counter and used to calculate % injected dose/g (%ID/g), tissue:blood (T:B), and tissue:muscle (T:M) ratios.
Results: The RCP of [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb was 52-76% (n=20) and 90-92% (n=8) respectively. [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb exhibited a low nanomolar binding affinity (Kd=0.16-2.3 nM) for MSLN. Pharmacokinetics over the time-course was similar for both the zirconium-89 conjugates except for blood, tumor, and femur. [89Zr]Zr-DFO-MSLN-mAb showed higher HT29-MSLN tumor uptake (28-33 %ID/g) at all time-points compared to [89Zr]Zr-3,2-HOPO-MSLN-mAb (7-11 %ID/g). Similarly, on day 3, PDX tumor accumulation of [89Zr]Zr-DFO-MSLN-mAb (15.88 -19.49%ID/g) was higher than [89Zr]Zr-3,2-HOPO-MSLN-mAb (7.95-13.07%ID/g). T:B and T:M ratios were also lower for [89Zr]Zr-3,2-HOPO-MSLN-mAb than the zirconium-89 DFO conjugate. However, femur uptake of [89Zr]Zr-3,2-HOPO-MSLN-mAb (6.74%ID/g) was ~2-fold higher compared to [89Zr]Zr-DFO-MSLN-mAb (3.57%ID/g) at day 1 and then increased to ~3-4-fold over 6 days. At all times, PET imaging results paralleled the biodistribution pattern of both the zirconium-89 conjugates.
Conclusions: In vitro, both conjugates exhibited a high binding affinity for MSLN. In vivo, [89Zr]Zr-DFO-MSLN-mAb showed higher tumor uptake and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb. As [89Zr]Zr-3,2-HOPO-MSLN-mAb uses the same chelator as [227Th]Th-3,2-HOPO-MSLN-mAb), the same 3,2-HOPO-MSLN-mAb conjugate could be better at studying organ distribution and lesion uptake of the MSLN-TTC, with the caveat that detection of MSLN positive tumors in the lower extremity might be more difficult if high femur uptake is also seen in humans.
12 Objectives: Prostate Specific Membrane Antigen (PSMA) is a transmembrane protein that is found at low levels on normal prostate cells but is increasingly expressed in prostate cancer (PCa) during disease progression. Hence, development of PSMA-targeted PET imaging agents has proven successful in identifying PCa and monitoring PSMA expression levels during treatment. Standard treatment for PCa patients is androgen deprivation therapy (ADT), which inhibits the ability of Androgen Receptor (AR) to induce PCa growth. However, ADT has shown variable effects in PET imaging studies on PSMA expression in cancerous lesions. Thus, there is need for defining the genomic and phenotypic properties that impact PSMA detection, especially relative to ADT. ADT has been found to increase PSMA in vitro in LNCaP cells, but cell lines do not model the intratumoral heterogeneity associated with PCa in patients. With the development of the LuCaP PDX platform and in vitro organoid culture methods designed to preserve the genomic integrity of the PDX, the role of AR regulation in PSMA expression can be explored as it relates to disease diversity in a variety of patient-derived models. Therefore, LuCaP PDX organoid cultures provide a clinically relevant ADT model in vitro where PSMA expression could be determined with a PSMA-targeted probe. Towards this end, PSMA PET imaging agent [18F]DCFPyL was used initially to characterize PSMA in vitro in LuCaP PDXs and organoid model systems. Following this, an in vitro ADT protocol was established using LuCaP141 organoids (AR+) in which changes in PSMA were studied with [18F]DCFPyL over the treatment time course.
Methods: [18F]DCFPyL was synthesized following our recently published method1. Representative LuCaP PDXs were chosen for study based on PSMA gene expression, ADT response, and AR mutational status, using PC3 cells that express PSMA as a positive control. For the organoid models, PDX or PC3 tissues were processed into a single cell suspension, cultured and harvested at 7 days. In vitro binding studies using [18F]DCFPyL were done to assess the binding affinity (Kd) and PSMA protein levels (Bmax) with organoid, LuCAP PDX, and PC3 membrane preparations. In LuCaP141 (ADT-responsive), organoids were compared in androgen-replete and androgen-deprived (no dihydrotestosterone [DHT], plus enzalutamide [MDV3100]) states.
Results: Levels of PSMA detected by [18F]DCFPyL in PDX tumor and PDX-derived organoids were remarkably similar, ranging from 0.01-4.66 femtomoles PSMA per microgram of protein. LuCaP77 demonstrated the highest amount in both the PDX and organoid models and LuCaP167 demonstrated the lowest amount detected in the PDX and undetectable PSMA in organoids. For the most part, the PSMA level of each in vivo LuCaP PDX tumor was comparable to its cultured organoid counterpart. PSMA levels were slightly higher in PDX vs. organoids for those models expressing low levels of PSMA, probably due to the contributions of other cell types (tumor stroma) present in the PDX but not the organoids (Figure 1A). PSMA in LuCaP141 organoids increased gradually following ADT, becoming significant at day 7 (Figure 1B). These data demonstrate the fidelity and utility of PDX-derived organoids, which unlike PDX tumors, are facile for biochemical and genetic manipulations.
Conclusions: [18F]DCFPyL was used to characterize PSMA in the LuCaP series, showing similarity between in vivo PDX and in vitro organoid models. Furthermore, AR was shown to negatively regulate PSMA expression in LuCaP141 organoids, validating in this model that PSMA changes in treatment response can be exploited in therapeutic monitoring. The use of multiple patient-derived genomically-heterogeneous models coupled with molecular correlates of PSMA response to ADT is anticipated to provide insights into the heterogeneity of PSMA detection in patients.
Reference: 1 Basuli, F.et al. J Label Compd Radiopharm 60, 168-175, (2017).
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