89Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors.

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.

89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution.

Evaluation of [I]I- and [Lu]Lu-DTPA-A11 Minibody for Radioimmunotherapy in a Preclinical Model of PSCA-Expressing Prostate Cancer.

Purpose: Radioimmunotherapy uses tumor-specific antibodies to deliver therapeutic radionuclides, but hematological toxicity due to the long serum half-life of intact antibodies remains a challenge. We evaluated a smaller antibody fragment, the minibody, with faster kinetics and a potentially improved therapeutic index.

Procedures: The anti-prostate stem cell antigen (PSCA) minibody (A11 Mb) was radiolabeled with iodine-124 ([I]I-A11 Mb) or conjugated with deferoxamine (DFO) and labeled with zirconium-89 ([Zr]Zr-DFO-A11 Mb) for surrogate immunoPET to profile pharmacokinetics in a human prostate cancer xenograft model.

Tri-functional platform for construction of modular antibody fragments for F-PET or NIRF molecular imaging.

Positron emission tomography (PET) molecular imaging is a powerful tool for interrogating physiological and biochemical processes to understand the biology of disease and advance therapeutic developments. Near-infrared fluorescence (NIRF) optical imaging has become increasingly popular for intraoperative staging to enable cellular resolution imaging of tumor margins during surgical resection. In addition, engineered antibody fragments have emerged as promising molecular imaging agents given their exquisite target selectivity, rapid systemic clearance and site-selective chemical modification.

On-demand radiosynthesis of -succinimidyl-4-[F]fluorobenzoate ([F]SFB) on an electrowetting-on-dielectric microfluidic chip for F-labeling of protein.

An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric (EWOD) mechanism was developed for on-demand synthesis of -succinimidyl-4-[F]fluorobenzoate ([F]SFB), the most commonly used F-prosthetic group for biomolecule labeling. In order to facilitate the development of peptides, and proteins as new diagnostic and therapeutic agents, we have diversified the compact EWOD microfluidic platform to perform the three-step radiosynthesis of [F]SFB starting from the no carrier added [F]fluoride ion. In this report, we established an optimal microliter droplet reaction condition to obtain reliable yields and synthesized [F]SFB with sufficient radioactivity for subsequent conjugation to the anti-PSCA cys-diabody (A2cDb) and for small animal imaging.

[Zr]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.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for F-Immuno-PET and Fluorescence Imaging.

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. 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 F-labeled -cyclooctene (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 F-TCO.

Dual-Modality ImmunoPET/Fluorescence Imaging of Prostate Cancer with an Anti-PSCA Cys-Minibody.

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.

F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice.

Purpose: Metabolic imaging using [F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG.

Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models.

Purpose: The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery.

Experimental Design: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW.

Monovalent TNF receptor 1-selective antibody with improved affinity and neutralizing activity.

Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398.

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. 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.

Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis.

Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation.

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

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. 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.

ImmunoPET Imaging of Murine CD4 T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging.

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.

An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy.

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).

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.

Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody.

Introduction: This work describes the development and characterization of two antibody fragments that specifically target the α(v)β(6) integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound α(v)β(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively.

A fully human scFv phage display library for rapid antibody fragment reformatting.

Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source of fully human antibodies for scientific and clinical applications. Frequently, scFvs form the basis of larger, bivalent formats to increase valency and avidity. A small and versatile bivalent antibody fragment is the diabody, a cross-paired scFv dimer (∼55 kDa).

Immuno-PET of Murine T Cell Reconstitution Postadoptive Stem Cell Transplantation Using Anti-CD4 and Anti-CD8 Cys-Diabodies.

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.

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.

Anti-MET immunoPET for non-small cell lung cancer using novel fully human antibody fragments.

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.

Enhanced immunoPET of ALCAM-positive colorectal carcinoma using site-specific ⁶⁴Cu-DOTA conjugation.

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.

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.

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.