[89Zr]A2cDb Immuno-PET of Prostate Cancer in a Human Prostate Stem Cell Antigen Knock-in (hPSCA KI) Syngeneic Model.

PURPOSE: A great challenge in the diagnosis and treatment of prostate cancer is distinguishing between indolent or local disease and aggressive or metastatic disease. Antibody-based positron emission tomography (immuno-PET) as a cancer-specific imaging modality could improve diagnosis of primary disease, aid the detection of metastases to regional lymph nodes as well as to distant sites (e.g., bone), and monitor response to therapy. PROCEDURE: In search for a more physiologically relevant disease model, a human prostate stem cell antigen knock-in (hPSCA KI) mouse model was generated. The use of a syngeneic prostate cancer cell line transduced to express human PSCA (RM-9-hPSCA) enabled the evaluation of anti-PSCA immuno-PET in immunocompetent mice and in the context of normal tissue expression of PSCA. Two PSCA-specific humanized antibody fragments, A11 minibody and A2 cys-diabody, were radiolabeled with positron emitters iodine-124 and zirconium-89, respectively ([124I]A11 Mb and [89Zr]A2cDb), and used for immuno-PET in wild-type, hPSCA KI and tumor-bearing mice. RESULTS: The hPSCA KI mice express PSCA at low levels in the normal prostate, bladder and stomach, reproducing the expression pattern seen in humans. [124I]A11 Mb immuno-PET detected increased levels of PSCA expression in the stomach, and because I-124 is non-residualizing, very little activity was seen in organs of clearance (liver, kidney, spleen). However, due to the longer half-life of the 80 kDa protein, blood activity (and thus urine activity) at 20 h postinjection remains high. The smaller 50 kDa [89Zr]A2cDb cleared faster, resulting in lower blood and background activity, despite the use of a residualizing radiometal. Importantly, [89Zr]A2cDb immuno-PET showed antigen-specific targeting of PSCA-expressing tumors and minimal nonspecific uptake in PSCA-negative controls. CONCLUSION: Tracer biodistribution was not significantly impacted by normal tissue expression of PSCA. [89Zr]A2cDb immuno-PET yielded high tumor-to-blood ratio at early time points. Rapid renal clearance of the 50 kDa tracer resulted in an unobstructed view of the pelvic region at 20 h postinjection that would allow the detection of cancer in the prostate.

Dual-Modality Immuno-PET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using an Anti-Prostate Stem Cell Antigen Cys-Diabody.

Pancreatic cancer has a high mortality rate due to late diagnosis and the tendency to invade surrounding tissues and metastasize at an early stage. A molecular imaging agent that enables both presurgery antigen-specific PET (immuno-PET) and intraoperative near-infrared fluorescence (NIRF) guidance might benefit diagnosis of pancreatic cancer, staging, and surgical resection, which remains the only curative treatment. Methods: We developed a dual-labeled probe based on A2 cys-diabody (A2cDb) targeting the cell-surface prostate stem cell antigen (PSCA), which is expressed in most pancreatic cancers. Maleimide-IRDye800CW was site-specifically conjugated to the C-terminal cys-tag (A2cDb-800) without impairing integrity or affinity (half-maximal binding, 4.3 nM). Direct radioiodination with 124I (124I-A2cDb-800) yielded a specific activity of 159 ± 48 MBq/mg with a radiochemical purity exceeding 99% and 65% ± 4.5% immunoreactivity (n = 3). In vivo specificity for PSCA-expressing tumor cells and biodistribution of the dual-modality tracer were evaluated in a prostate cancer xenograft model and compared with single-labeled 124I-A2cDb. Patient-derived pancreatic ductal adenocarcinoma xenografts (PDX-PDACs) were grown subcutaneously in NSG mice and screened for PSCA expression by immuno-PET. Small-animal PET/CT scans of PDX-PDAC-bearing mice were obtained using the dual-modality 124I-A2cDb-800 followed by postmortem NIRF imaging with the skin removed. Tumors and organs were analyzed ex vivo to compare the relative fluorescent signals without obstruction by other organs. Results: Specific uptake in PSCA-positive tumors and low nonspecific background activity resulted in high-contrast immuno-PET images. Concurrent with the PET studies, fluorescent signal was observed in the PSCA-positive tumors of mice injected with the dual-tracer 124I-A2cDb-800, with low background uptake or autofluorescence in the surrounding tissue. Ex vivo biodistribution confirmed comparable tumor uptake of both 124I-A2cDb-800 and 124I-A2cDb. Conclusion: Dual-modality imaging using the anti-PSCA cys-diabody resulted in high-contrast immuno-PET/NIRF images of PDX-PDACs, suggesting that this imaging agent might offer both noninvasive whole-body imaging to localize PSCA-positive pancreatic cancer and fluorescence image-guided identification of tumor margins during surgery.

ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo.

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivoResults:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR.

Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time.

PURPOSE: The inability to visualize cancer during prostatectomy contributes to positive margins, cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. The goal of this study was to develop a probe for image-guided prostate cancer surgery. EXPERIMENTAL DESIGN: An antibody fragment (cys-diabody, cDb) against prostate stem cell antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the probe to PSCA was confirmed by gel electrophoresis, size exclusion, and flow cytometry, respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the biodistribution and in vivo kinetics, whereas an invasive intramuscular model was utilized to explore the performance of Cy5-cDb-mediated fluorescence guidance in representative surgical scenarios. Finally, a prospective, randomized study comparing surgical resection with and without fluorescent guidance was performed to determine whether this probe could reduce the incidence of positive margins. RESULTS: Cy5-cDb demonstrated excellent purity, stability, and specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA(+) and negative (-) dual xenografts, the mean fluorescence ratio of PSCA(+/-) tumors was 4.4:1. In surgical resection experiments, residual tumors <1 mm that were missed on white light surgery were identified and resected using fluorescence guidance, which reduced the incidence of positive surgical margins (0/8) compared with white light surgery alone (7/7). CONCLUSIONS: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer xenografts in mice, and facilitates more complete tumor removal than conventional white light surgery alone.

Applications of immunoPET: using 124I-anti-PSCA A11 minibody for imaging disease progression and response to therapy in mouse xenograft models of prostate cancer.

PURPOSE: Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to (18)F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation. EXPERIMENTAL DESIGN: Osteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with serial (124)I-anti-PSCA A11 minibody immunoPET and (18)F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation. RESULTS: A11 minibody demonstrated improved sensitivity and specificity over (18)F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo. CONCLUSIONS: A11 minibody immunoPET has the potential to improve the sensitivity and specificity of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the development of castration resistance.

Improved modeling of in vivo kinetics of slowly diffusing radiotracers for tumor imaging.

UNLABELLED: Large-molecule tracers, such as labeled antibodies, have shown success in immuno-PET for imaging of specific cell surface biomarkers. However, previous work has shown that localization of such tracers shows high levels of heterogeneity in target tissues, due to both the slow diffusion and the high affinity of these compounds. In this work, we investigate the effects of subvoxel spatial heterogeneity on measured time-activity curves in PET imaging and the effects of ignoring diffusion limitation on parameter estimates from kinetic modeling. METHODS: Partial differential equations (PDE) were built to model a radially symmetric reaction-diffusion equation describing the activity of immuno-PET tracers. The effects of slower diffusion on measured time-activity curves and parameter estimates were measured in silico, and a modified Levenberg-Marquardt algorithm with Bayesian priors was developed to accurately estimate parameters from diffusion-limited data. This algorithm was applied to immuno-PET data of mice implanted with prostate stem cell antigen-overexpressing tumors and injected with (124)I-labeled A11 anti-prostate stem cell antigen minibody. RESULTS: Slow diffusion of tracers in linear binding models resulted in heterogeneous localization in silico but no measurable differences in time-activity curves. For more realistic saturable binding models, measured time-activity curves were strongly dependent on diffusion rates of the tracers. Fitting diffusion-limited data with regular compartmental models led to parameter estimate bias in an excess of 1,000% of true values, while the new model and fitting protocol could accurately measure kinetics in silico. In vivo imaging data were also fit well by the new PDE model, with estimates of the dissociation constant (Kd) and receptor density close to in vitro measurements and with order of magnitude differences from a regular compartmental model ignoring tracer diffusion limitation. CONCLUSION: Heterogeneous localization of large, high-affinity compounds can lead to large differences in measured time-activity curves in immuno-PET imaging, and ignoring diffusion limitations can lead to large errors in kinetic parameter estimates. Modeling of these systems with PDE models with Bayesian priors is necessary for quantitative in vivo measurements of kinetics of slow-diffusion tracers.

A mutated anti-CA19-9 scFv-Fc for positron emission tomography of human pancreatic cancer xenografts.

PURPOSE: Intact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts. PROCEDURES: The anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g). RESULTS: Biochemical characterization was consistent with the creation of a 105 kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7 nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver. CONCLUSIONS: We successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.

Quantitative immunoPET of prostate cancer xenografts with 89Zr- and 124I-labeled anti-PSCA A11 minibody.

UNLABELLED: Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. METHODS: A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient-based partial-volume correction, and the results were compared with ex vivo biodistribution. RESULTS: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing (124)I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing (89)Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the (124)I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor-to-soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 (124)I-A11 and 4.87 ± 0.52 (89)Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. CONCLUSION: Both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the (124)I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor-to-soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both (124)I- and (89)Zr-labeled A11 minibody.

Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.

The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.

Endocytosis and intracellular trafficking properties of transferrin-conjugated block copolypeptide vesicles.

Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles.

Advances in immuno-positron emission tomography: antibodies for molecular imaging in oncology.

Identification of cancer cell-surface biomarkers and advances in antibody engineering have led to a sharp increase in the development of therapeutic antibodies. These same advances have led to a new generation of radiolabeled antibodies and antibody fragments that can be used as cancer-specific imaging agents, allowing quantitative imaging of cell-surface protein expression in vivo. Immuno-positron emission tomography (immunoPET) imaging with intact antibodies has shown success clinically in diagnosing and staging cancer. Engineered antibody fragments, such as diabodies, minibodies, and single-chain Fv (scFv) -Fc, have been successfully employed for immunoPET imaging of cancer cell-surface biomarkers in preclinical models and are poised to bring same-day imaging into clinical development. ImmunoPET can potentially provide a noninvasive approach for obtaining target-specific information useful for titrating doses for radioimmunotherapy, for patient risk stratification and selection of targeted therapies, for evaluating response to therapy, and for predicting adverse effects, thus contributing to the ongoing development of personalized cancer treatment.