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Purpose: The aims of the study were to develop and evaluate a novel residualizing peptide for labeling internalizing antibodies with 124 I to support clinical development using immuno-positron emission tomography (PET). Methods: The anti-epidermal growth factor receptor antibody ch806 was radiolabeled directly or indirectly with isotopes and various residualizing peptides. Azido-derivatized radiolabeled peptides were conjugated to dibenzylcyclooctyne-derivatized ch806 antibody via click chemistry. The radiochemical purities, antigen-expressing U87MG.de2-7 human glioblastoma cell-binding properties, and targeting of xenografts at 72 hours post injection of all radioconjugates were compared. Biodistribution of 124 I-PEG 4 -tptddYddtpt-ch806 and immuno-PET imaging were evaluated in tumor-bearing mice. Results: Biodistribution studies using xenografts at 72 hours post injection showed that 131 I-PEG 4 -tptddYddtpt-ch806 tumor uptake was similar to 111 In-CHX-A″-DTPA-ch806. 125 I-PEG 4 -tptddyddtpt-ch806 showed a lower tumor uptake value but higher than directly labeled 125 I-ch806. 124 I-PEG 4 -tptddYddtpt-ch806 was produced at 23% labeling efficiency, 98% radiochemical purity, 25.9 MBq/mg specific activity, and 64% cell binding in the presence of antigen excess. Tumor uptake for 124 I-PEG 4 -tptddYddtpt-ch806 was similar to 111 In-CHX-A″-DTPA-ch806. High-resolution immuno-PET/magnetic resonance imaging of tumors showed good correlation with biodistribution data. Conclusions: The mixed d / l -enantiomeric peptide, d Thr- d Pro- d Thr- dA sp -dA sp-Tyr -dA sp -dA sp- d Thr- d Pro- d Thr, is suitable for radiolabeling antibodies with radiohalogens such as 124 I for high-resolution immuno-PET imaging of tumors and for evaluation in early-phase clinical trials.

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

In tumors, hypoxia stimulates angiogenesis and correlates with treatment resistance and poor prognosis. We have previously demonstrated hypoxia in human renal cell carcinoma (RCC) via direct oxygen probe measurements. Carbonic anhydrase IX (CA IX) is a protein stimulated by hypoxia and involved in angiogenesis, and is a potential tumor target for imaging and therapies using cG250, a monoclonal antibody that recognizes CAIX. Our objectives were to characterize intratumoral hypoxia in a human RCC xenograft model using oxygen probe measurements; investigate if (124)I-cG250 targets RCC correlating uptake on noninvasive positron emission tomography-computerized tomography (PET-CT) against traditional biodistribution studies, and investigate CAIX expression in this RCC model.BALB/c nude mice had human RCC (SK-RC-52) subcutaneously xenografted with oxygen levels measured by probe. Positron emission tomography (PET/CT) and biodistribution studies ((124)I-cG250) were correlated with oxygen measurements. Immunohistochemistry and autoradiography were performed on selected tumors to confirm CAIX expression.Oxygen tension in normal tissue (muscle) was 35.08 ± 2.41 mmHg (mean ± 95% CI), significantly greater compared to xenograft SK-RC-52 tumors at 5.02 ± 1.12 mmHg. Biodistribution studies of (124)I-cG250 demonstrated isotope uptake in SK-RC-52 xenografts peaking at 23.45 ± 5.07% ID/g (mean ± SD) 48 hours after antibody injection, which was maintained for a further 2 days (19.43 ± 4.31 and 10.64 ± 5.64 % ID/g, respectively). PET studies demonstrated excellent localization of (124)I-cG250 in tumor, and a significant correlation between SUVmean, SUVmax, and %/ID (124)I-cG250. CAIX expression was present in all groups studied but there was no significant correlation between it and any oxygen parameter studied.Intratumoral hypoxia does exist within a human RCC xenograft model using invasive oxygen probe measurements. (124)I-cG250 targets RCC with correlation between uptake on noninvasive PET-CT studies and traditional biodistribution studies opening the possibility of using PET/CT in future studies. Finally, CAIX expression was not related to hypoxia in this model, supporting the hypothesis that cell lines may subvert known hypoxia mechanisms in hypoxic environments.

Radioimmunotherapy (RIT) of solid tumor is often limited in efficacy because of restrictions in achieved tumor dose. In an effort to overcome this, the combination of RIT with other therapeutic modalities was investigated in an animal model of breast carcinoma. The rationale for this combined-modality RIT (CMRIT) was to increase the therapeutic efficacy of RIT through the use of paclitaxel to arrest cells in the radiosensitive G(2)/M phase of the cell cycle.In this study, the biodistribution and therapeutic efficacy of (90)Y-radiolabeled humanized anti-Lewis Y hu3S193 monoclonal antibody ((90)Y-hu3S193) RIT in combination with paclitaxel chemotherapy was explored in a Lewis Y-expressing MCF-7 tumor xenografted BALB/c nude mouse model of breast cancer.Biodistribution studies demonstrated excellent tumor targeting and limited normal tissue uptake by (90)Y-hu3S193. A therapeutic study with established tumors assessed (90)Y-hu3S193 as a single agent and demonstrated significant antitumor effects in all animals receiving a single intravenous 1.85 or 3.70 MBq dose of this treatment compared with phosphate-buffered saline placebo controls (P = 0.0008 vs. P0.0001). Complete responses were observed in all animals in the 3.70 MBq study arm for the duration of the study. Single-dose (90)Y-hu3S193 plus paclitaxel (600 microg) CMRIT displayed improved efficacy over single-modality therapies, with a significant difference (P0.0001) between the mean percentage change in tumor volume in mice receiving 0.46 MBq (90)Y-hu3S193 alone and when combined with 600 mug paclitaxel.The significant efficacy of (90)Y-hu3S193 and paclitaxel CMRIT at low radiation doses in this model of breast carcinoma indicates its therapeutic potential and warrants further investigation into this promising therapeutic approach.