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Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions., (©2024 American Association for Cancer Research.)

We report the development of a hydrophilic 18 F-labeled a-TCO derivative [ 18 F]3 (log  P = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [ 18 F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

Dramatic, but uneven, progress in the development of immunotherapies for cancer has created a need for better diagnostic technologies including innovative non-invasive imaging approaches. This review discusses challenges and opportunities for molecular imaging in immuno-oncology and focuses on the unique role that antibodies can fill. ImmunoPET has been implemented for detection of immune cell subsets, activation and inhibitory biomarkers, tracking adoptively transferred cellular therapeutics, and many additional applications in preclinical models. Parallel progress in radionuclide availability and infrastructure supporting biopharmaceutical manufacturing has accelerated clinical translation. ImmunoPET is poised to provide key information on prognosis, patient selection, and monitoring immune responses to therapy in cancer and beyond., (© 2021. World Molecular Imaging Society.)

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18 F-labeled trans -cyclooctene ( 18 F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18 F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18 F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18 F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Imaging plays a central role in evaluating responses to therapy in neuro-oncology patients. The advancing clinical use of immunotherapies has demonstrated that treatment-related inflammatory responses mimic tumor growth via conventional imaging, thus spurring the development of new imaging approaches to adequately distinguish between pseudoprogression and progressive disease. To this end, an increasing number of advanced imaging techniques are being evaluated in preclinical and clinical studies. These novel molecular imaging approaches will serve to complement conventional response assessments during immunotherapy. The goal of these techniques is to provide definitive metrics of tumor response at earlier time points to inform treatment decisions, which has the potential to improve patient outcomes. This review summarizes the available immunotherapy regimens, clinical response criteria, current state-of-the-art imaging approaches, and groundbreaking strategies for future implementation to evaluate the anti-tumor and immune responses to immunotherapy in neuro-oncology applications., Competing Interests: Competing Interests: Anna Wu is a founder, board member, and consultant to ImaginAb, Inc., and a consultant to Avidity Biosciences. James Markert has received funding from a structured buyout of Catherex, Inc., and holds <8% equity interest in Aettis, Inc. Both companies had/have interests in oncolytic virotherapy.

Purpose: Metabolic imaging using [ 18 F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 ( 18 F, t 1/2 110 min), enabling same-day imaging., Methods: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[ 18 F]-fluorobenzoate ([ 18 F]SFB), or thiol-reactive N-[2-(4-[ 18 F]-fluorobenzamido)ethyl]maleimide ([ 18 F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [ 18 F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [ 18 F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution., Results: The GAcDb was successfully 18 F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([ 18 F]FB-GAcDb and [ 18 F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [ 18 F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [ 18 F]FDG PET but with added specificity for the therapeutic target., Conclusions: [ 18 F]FB-GAcDb and [ 18 F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [ 18 F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

Inadequate diagnostic methods for prostate cancer lead to over- and undertreatment, and the inability to intraoperatively visualize positive margins may limit the success of surgical resection. Prostate cancer visualization could be improved by combining the complementary modalities of immuno-positron emission tomography (immunoPET) for preoperative disease detection, and fluorescence imaging-guided surgery (FIGS) for real-time intraoperative tumor margin identification. Here, we report on the evaluation of dual-labeled humanized anti-prostate stem cell antigen (PSCA) cys-minibody (A11 cMb) for immunoPET/fluorescence imaging in subcutaneous and orthotopic prostate cancer models. Methods: A11 cMb was site-specifically conjugated with the near-infrared fluorophore Cy5.5 and radiolabeled with 124 I or 89 Zr. 124 I-A11 cMb-Cy5.5 was used for successive immunoPET/fluorescence imaging of prostate cancer xenografts expressing high or moderate levels of PSCA (22Rv1-PSCA and PC3-PSCA). 89 Zr-A11 cMb-Cy5.5 dual-modality imaging was evaluated in an orthotopic model. Ex vivo biodistribution at 24 h was used to confirm the uptake values, and tumors were visualized by post-mortem fluorescence imaging. Results: A11 cMb-Cy5.5 retained low nanomolar affinity for PSCA-positive cells. Conjugation conditions were established (dye-to-protein ratio of 0.7:1) that did not affect the biodistribution, pharmacokinetics, or clearance of A11 cMb. ImmunoPET using dual-labeled 124 I-A11 cMb-Cy5.5 showed specific targeting to both 22Rv1-PSCA and PC3-PSCA s.c. xenografts in nude mice. Ex vivo biodistribution confirmed specific uptake to PSCA-expressing tumors with 22Rv1-PSCA:22Rv1 and PC3-PSCA:PC3 ratios of 13:1 and 5.6:1, respectively. Consistent with the immunoPET, fluorescence imaging showed a strong signal from both 22Rv1-PSCA and PC3-PSCA tumors compared with non-PSCA expressing tumors. In an orthotopic model, 89 Zr-A11 cMb-Cy5.5 immunoPET was able to detect intraprostatically implanted 22Rv1-PSCA cells. Importantly, fluorescence imaging clearly distinguished the prostate tumor from surrounding seminal vesicles. Conclusion: Dual-labeled A11 cMb specifically visualized PSCA-positive tumor by successive immunoPET/fluorescence, which can potentially be translated for preoperative whole-body prostate cancer detection and intraoperative surgical guidance in patients., Competing Interests: Competing Interests: A. Wu has an ownership interest in, and is a board member and consultant for ImaginAb, Inc.

Purpose: Noninvasive and quantitative tracking of CD8 + T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64 Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64 Cu was developed. The accumulation of 64 Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains ( n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8 + T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped ( n = 6-8/group studied with imaging and IHC or flow cytometry). Results: We demonstrate the ability of immunoPET to detect small differences in CD8 + T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2 -driven model of mammary adenocarcinoma (NDL), 64 Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8 + T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols. Conclusions: 64 Cu-169cDb imaging can spatially map the distribution of CD8 + T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8 + T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976-87. ©2018 AACR ., (©2018 American Association for Cancer Research.)

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 ( 89 Zr) versus non-residualizing ( 124 I) 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 vivo Results: 124 I-GAcDb and 124 I-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 89 Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124 I-labeled tracers. Pairwise comparison of 89 Zr- and 124 I-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 ., (©2017 American Association for Cancer Research.)

Purpose: Molecular imaging of CD4 + T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4 + T cell viability, proliferation, CD4 expression, and function., Procedures: The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89 Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed., Results: For immunoPET imaging, the lowest protein dose of 2 μg of 89 Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4 + T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4 + T cells., Conclusions: Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4 + T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes.

The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic., (©2015 American Association for Cancer Research.)

Unlabelled: The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole-body T lymphocyte dynamics noninvasively in vivo, we generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for (89)Zr-immuno-PET detection of helper and cytotoxic T cell populations., Methods: Anti-CD4 and -CD8 cDbs were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cDbs were site-specifically conjugated to maleimide-desferrioxamine for (89)Zr radiolabeling and subsequent small-animal PET/CT acquisition and ex vivo biodistribution in both wild-type mice and a model of hematopoietic stem cell (HSC) transplantation., Results: Immuno-PET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild-type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 wk after HSC transplantation using the (89)Zr-radiolabeled anti-CD4 and -CD8 cDbs., Conclusion: These newly developed anti-CD4 and -CD8 immuno-PET reagents represent a powerful resource to monitor T cell expansion, localization, and novel engraftment protocols. Future potential applications of T cell-targeted immuno-PET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

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., (©2014 American Association for Cancer Research.)

Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability. ALCAM constructs with linker lengths of eight, five and three amino acids were produced to make true non-covalent site-specifically modified cDbs. Characterization by gel electrophoresis, size exclusion chromatography, flow cytometry and mass spectrometry of the various constructs was performed. To demonstrate the increased utility of targeting multiple malignancies expressing ALCAM, we compare the targeting of the site-specific versus non-site-specific conjugated cDbs to the human colorectal cancer xenograft LS174T. Interestingly, the conjugation strategy not only affects tumor targeting but also hepatic and renal uptake/clearance., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

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.

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.

Background: Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer., Methods: The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images., Results: Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro., Conclusions: Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment., (Published by Elsevier Inc.)

Purpose: As imaging of the cell surface tetraspan protein epithelial membrane protein-2 (EMP2) expression in malignant tumors may provide important prognostic and predictive diagnostic information, the goal of this study is to determine if antibody fragments to EMP2 may be useful for imaging EMP2 positive tumors., Procedures: The normal tissue distribution of EMP2 protein expression was evaluated by immunohistochemistry and found to be discretely expressed in both mouse and human tissues. To detect EMP2 in tumors, a recombinant human anti-EMP2 minibody (scFv-hinge-C(H)3 dimer; 80 kDa) was designed to recognize a common epitope in mice and humans and characterized. In human tumor cell lines, the antibody binding induced EMP2 internalization and degradation, prompting the need for a residualizing imaging strategy. Following conjugation to DOTA (1,4,7,10-tetraazacyclododecane-N,N',N',N'″-tetraacetic acid), the minibody was radiolabeled with (64)Cu (t (1/2) = 12.7 h) and evaluated in mice as a positron emission tomography (PET) imaging agent for human EMP2-expressing endometrial tumor xenografts., Results: The residualizing agent, (64)Cu-DOTA anti-EMP2 minibody, achieved high uptake in endometrial cancer xenografts overexpressing EMP2 (10.2 ± 2.6, percent injected dose per gram (%ID/g) ± SD) with moderate uptake in wild-type HEC1A tumors (6.0 ± 0.1). In both cases, precise tumor delineation was observed from the PET images. In contrast, low uptake was observed with anti-EMP2 minibodies in EMP2-negative tumors (1.9 ± 0.5)., Conclusions: This new immune-PET agent may be useful for preclinical assessment of anti-EMP2 targeting in vivo. It may also have value for imaging of tumor localization and therapeutic response in patients with EMP2-positive malignancies.

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.

Combining the specificity of tumor-targeting antibodies with the sensitivity and quantification offered by positron emission tomography (PET) provides tremendous opportunities for molecular characterization of tumors in vivo. Until recently, significant challenges have been faced when attempting to combine antibodies which show long biological half-lives and positron-emitting radionuclides with comparably short physical half-lives, in particular (18)F (half-life, 109 min). A fast and simple microwave-assisted method of generating N-succinimidyl-4-[(18)F]fluorobenzoate has been developed and employed for radiolabeling a small, rapidly targeting HER2-specific engineered antibody fragment, the cys-diabody. Using this tracer, HER2-positive tumor xenografts in mice were detected at 1-4 h post-injection by microPET. This confirms the rapid kinetics of [(18)F]fluorobenzoyl cys-diabody localization, and demonstrates the feasibility of same-day immunoPET imaging. This approach can be broadly applied to antibodies targeting cell surface biomarkers for molecular imaging of tumors and should be highly translatable for clinical use.

Purpose: The purpose of this study was to generate and evaluate a positron emission tomography (PET) radiotracer targeting activated leukocyte cell adhesion molecule (ALCAM)., Procedures: A human anti-ALCAM single chain variable fragment was reformatted to produce a covalent dimer, termed a cys-diabody (CysDb). Purified CysDb was characterized by gel electrophoresis and size exclusion chromatography, and immunoreactivity was assessed by flow cytometry and immunofluorescence. Targeting and imaging of ALCAM-positive tumors using (64)Cu-DOTA-CysDb were evaluated in mice bearing human pancreatic adenocarcinoma xenografts (HPAF-II or BxPC-3)., Results: CysDb binds specifically to ALCAM-positive cells in vitro with an apparent affinity in the range of 1-3 nM. MicroPET images at 4 h showed specific targeting of positive tumors in vivo, a finding confirmed by biodistribution analysis, with positive/negative tumor ratios of 1.9 ± 0.6 and 2.4 ± 0.6, and positive tumor/blood ratios of 2.5 ± 0.9 and 2.9 ± 0.6 (HPAF-II and BxPC-3, respectively)., Conclusions: Successful imaging with (64)Cu-DOTA-CysDb in animal models suggests further investigation of ALCAM as an imaging biomarker is warranted.

Background: Intact antibodies are poor imaging agents due to a long serum half-life (10-20 d) preventing adequate contrast between the tumor and surrounding blood. Smaller engineered antibody fragments overcome this problem by exhibiting shorter serum half-lives (4-20 h).The diabody (55 kDa) is the smallest antibody fragment, which retains the bivalency of the intact antibody. Our goal was to develop and characterize the anti-CA19-9 diabody fragment and determine its ability to provide antigen specific imaging of pancreas cancer., Methods: The diabody DNA construct was created by isolation of the variable region genes of the intact anti-CA19-9 antibody. Diabody expression was carried out in NS0 cells and purified using HPLC from supernatant. Specific antigen binding was confirmed with flow cytometry and immunofluorescence. Radiolabeled diabody was injected into mice harboring an antigen positive xenograft (BxPC3 or Capan-2) and a negative xenograft (MiaPaca-2). MicroCT and MicroPET were performed at successive time intervals after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the microPET images., Results: Immunofluorescence and flow cytometry showed specific binding of the anti-CA19-9 diabody. Pancreas xenograft imaging of BxPC3/MiaPaca-2 and Capan-2/MiaPaca-2 models with the anti-CA19-9 diabody demonstrated an average tumor:blood ratio of 5.0 and 2.0, respectively, and an average positive:negative tumor ratio of 11 and 6, respectively. With respect to the tumor:blood ratio, these data indicate five times and two times more radioactivity in the tumor than in the blood yielding adequate contrast between tumor tissue and background (i.e., blood) to create the representative microPET images., Conclusions: We successfully engineered a functional diabody against CA19-9, a tumor antigen present on the vast majority of pancreas cancers. Additionally, we demonstrate high contrast antigen specific microPET imaging of pancreas cancer in xenograft models., (Published by Elsevier Inc.)

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.

Purpose: Prostate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies., Methods: Yeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors., Results: The A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo., Conclusion: Of the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study.

The identification of tumor tissue biomarkers has led to the production, validation, and Food and Drug Administration-approval of a number of antibody-based targeted therapeutics in the past two decades. As a result of the significant role that these immunotherapeutics play in the management of cancer, and the potential utility of complementary imaging agents, immunoPET imaging has generated considerable interest. This update discusses the important factors to consider when designing a PET (positron emission tomography) imaging agent from the molecular target to the biological targeting molecule and radionuclide combination and also reviews recent preclinical and clinical findings in the immunoPET field. Although there are a variety of radionuclides that are currently utilized in PET studies, this update focuses on four of the positron emitters commonly used in labeling proteins: iodine-124, zirconium-89, copper-64, and fluorine-18. Notable advances in the preclinical setting include the continued development of immunoPET probes to predict the biodistribution of related radioimmunotherapeutics, the success of nontraditional radionuclide and antibody fragment combinations, the broader use of zirconium-89, and the recent emergence of (18)F-labeled diabodies for same-day imaging. Antibody-based PET probes constitute a valuable class of molecular imaging agents, and the progress made preclinically should expedite the transition of these targeted diagnostics to clinical applications.

Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V(L)-V(H) orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with (124)I for PET imaging, and biodistribution of (131)I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both (124)I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of (131)I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.

Unlabelled: The CD20 cell surface antigen is expressed at high levels by over 90% of B-cell non-Hodgkin lymphomas (NHL) and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific PET imaging of NHL, we sought to develop PET agents targeting CD20., Methods: Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-C(H)3 dimer; 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with (124)I, and the minibody was additionally labeled with (64)Cu (radiometal) after conjugation to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal-labeled minibody were evaluated in mice as small-animal PET imaging agents for the in vivo imaging of human CD20-expressing lymphomas., Results: Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, the tumor uptake levels and blood activities differed, resulting in different levels of contrast in the images. The better candidate was the minibody, with superior uptake (2-fold higher than that obtained with scFv-Fc) in CD20-positive tumors and low uptake in CD20-negative tumors. Ratios of CD20-positive tumors to CD20-negative tumors at 21 h were 7.0 +/- 3.1 (mean +/- SD) and 3.9 +/- 0.7 for the minibody and scFv-Fc, respectively. The ratio achieved with the (64)Cu-DOTA-minibody at 19 h was about 5-fold lower because of higher residual background activity in CD20-negative tumors., Conclusion: A radioiodinated minibody and a radioiodinated scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for imaging CD20-positive lymphomas in preclinical models and in humans with NHL.

The completion of the human genome, coupled with parallel major research efforts in proteomics and systems biology, has led to a flood of information on the roles of individual genes and proteins in normal physiologic processes and their disruptions in disease. In practical terms, this information has opened the door to increasingly targeted therapies as specific molecular markers are identified and validated. The ongoing transition from empiric to molecular medicine has engendered a need for corresponding molecular diagnostics, including noninvasive molecular imaging. Convergence of knowledge regarding key biomarkers that define normal biologic processes and disease with protein and imaging technology makes this an opportune time to revisit the combination of antibodies and PET, or immuno-PET.

Unlabelled: Reporter gene imaging has great potential for many clinical applications including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene-reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immunogenicity of the reporter gene. The objective of the present study was to evaluate a new approach for reporter gene imaging based on cell surface expression of antibody fragments that can irreversibly bind to radiometal chelates., Methods: We developed a new reporter gene, designated 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) antibody reporter 1 (DAbR1), which consists of the single-chain Fv (scFv) fragment of the anti-Y-DOTA antibody 2D12.5/G54C fused to the human T cell CD4 transmembrane domain. The corresponding reporter probe is yttrium-(S)-2-(4-acrylamidobenzyl)-DOTA (*Y-AABD), a DOTA complex that binds irreversibly to a cysteine residue in the 2D12.5/G54C antibody. U-87 glioma cells were stably transfected with a DAbR1 expression vector. Binding of *Y-AABD to transfected and wild-type cells was studied in vitro and in vivo., Results: Flow cytometry revealed high expression of the DAbR1 protein on the cell surface of tumor cells. Uptake of 90Y-AABD in DAbR1-expressing human U-87 glioma xenografts was 6.2 (+/-1.3) percentage injected dose per gram (%ID/g) at 1 h and 4.9 (+/-0.62) %ID/g at 24 h after injection. The corresponding tumor-to-plasma ratios were 45:1 and 428:1, respectively. Uptake by U-87 tumors without the DAbR1 gene was 0.16 (+/-0.02) %ID/g at 1 h and 0.05 (+/-0.03) %ID/g at 24 h. PET images in mice with 86Y-AABD demonstrated intense uptake in DAbR1-positive tumors and low background activity in the liver., Conclusion: These findings indicate that cell surface expression of radiometal chelate binding antibodies such as 2D12.5/G54C is a promising strategy for reporter gene imaging.

Many considerations, involving understanding and selection of multiple experimental parameters, are required to perform MicroPET studies properly. The large number of these parameters/variables and their complicated interdependence make their optimal choice nontrivial. We have a developed kinetic imaging system (KIS), an integrated software system, to assist the planning, design, and data analysis of MicroPET studies. The system serves multiple functions-education, virtual experimentation, experimental design, and image analysis of simulated/experimental data-and consists of four main functional modules--"Dictionary," "Virtual Experimentation," "Image Analysis," and "Model Fitting." The "Dictionary" module provides didactic information on tracer kinetics, pharmacokinetic, MicroPET imaging, and relevant biological/pharmacological information. The "Virtual Experimentation" module allows users to examine via computer simulations the effect of biochemical/pharmacokinetic parameters on tissue tracer kinetics. It generates dynamic MicroPET images based on the user's assignment of kinetics or kinetic parameters to different tissue organs in a 3-D digital mouse phantom. Experimental parameters can be adjusted to investigate the design options of a MicroPET experiment. The "Image Analysis" module is a full-fledged image display/manipulation program. The "Model Fitting" module provides model-fitting capability for measured/simulated tissue kinetics. The system can be run either through the Web or as a stand-alone process. With KIS, radiotracer characteristics, administration method, dose level, imaging sequence, and image resolution-to-noise tradeoff can be evaluated using virtual experimentation. KIS is designed for biology/pharmaceutical scientists to make learning and applying tracer kinetics fun and easy.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

Using gene modification of hematopoietic stem cells (HSC) to create persistent generation of multilineage immune effectors to target cancer cells directly is proposed. Gene-modified human HSC have been used to introduce genes to correct, prevent, or treat diseases. Concerns regarding malignant transformation, abnormal hematopoiesis, and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. Truncated epidermal growth factor receptor (EGFRt) was tested as a suicide gene system co-delivered with anti-CD19 chimeric antigen receptor (CAR) to human HSC. Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. An antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used, incubating target cells with leukocytes and monoclonal antibody cetuximab to determine the percentage of surviving cells. In vivo, gene-modified HSC were engrafted into NSG mice with subsequent treatment with intraperitoneal cetuximab. Persistence of gene-modified cells was assessed by flow cytometry, droplet digital polymerase chain reaction (ddPCR), and positron emission tomography (PET) imaging using 89Zr-Cetuximab. Cytotoxicity was significantly increased (p = 0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1 μg/mL compared to EGFRt+ cells without cetuximab and non-transduced cells with or without cetuximab, at all effector:target ratios. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p = 0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging confirmed ablation with a decrease of an average of 82.5% after cetuximab treatment. These results give proof of principle for CAR-modified HSC regulated by a suicide gene. Further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers, should also be evaluated.

PurposeMetabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging.MethodsGAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution.ResultsThe GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target.Conclusions[18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue.Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6-8/group studied with imaging and IHC or flow cytometry).Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols.Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976-87. ©2018 AACR.

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.

MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers, including non-small cell lung cancer (NSCLC). As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment-based immunopositron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were reformatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 to 5.1 nmol/L. The candidate with the highest affinity, H2, was radiolabeled with (89)Zr for immunoPET studies targeting NSCLC xenografts: low MET-expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET overexpression. ImmunoPET at as early as 4 hours after injection produced high-contrast images, and ex vivo biodistribution analysis at 20 hours after injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (P < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer), produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, whereas no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications.

Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability. ALCAM constructs with linker lengths of eight, five and three amino acids were produced to make true non-covalent site-specifically modified cDbs. Characterization by gel electrophoresis, size exclusion chromatography, flow cytometry and mass spectrometry of the various constructs was performed. To demonstrate the increased utility of targeting multiple malignancies expressing ALCAM, we compare the targeting of the site-specific versus non-site-specific conjugated cDbs to the human colorectal cancer xenograft LS174T. Interestingly, the conjugation strategy not only affects tumor targeting but also hepatic and renal uptake/clearance.

PurposeThe objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.ProceduresThe transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers.ResultsSurface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.ConclusionsThe in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.