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Immuno-positron emission tomography (immuno-PET) is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies (Abs). Methods L1 cell adhesion molecule (L1CAM)-targeting HuE71 immunoglobulin G1 (IgG1) and immunoglobulin G4 (IgG4) Abs bearing identical variable heavy and light chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with Zirconium-89 (89Zr) and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results In addition to showing uptake in L1CAM-expressing SKOV3 tumors like its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor (FcR) affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated FcR binding did not show lymphoid uptake. The use of IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline (S228P) in the hinge region of the IgG4 Ab to mitigate in vivo fragment antigen-binding (Fab) arm exchange. Conclusion Our findings highlight the influence of Fc-modifications and the choice of IgG subclass on the in vivo biodistribution of Abs and the potential outcomes thereof.

Introduction Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. Methods Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [89Zr]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). Results In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [89Zr]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. Conclusion Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharmacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation.

Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer-related death in women. Over the past decade, medical imaging has played an increasingly valuable role in the diagnosis, staging, and treatment planning of the disease. In this "Focus on Molecular Imaging" review, we seek to provide a brief yet informative survey of the current state of the molecular imaging of ovarian cancer. The article is divided into sections according to modality, covering recent advances in the MR, PET, SPECT, ultrasound, and optical imaging of ovarian cancer. Although primary emphasis is given to clinical studies, preclinical investigations that are particularly innovative and promising are discussed as well. Ultimately, we are hopeful that the combination of technologic innovations, novel imaging probes, and further integration of imaging into clinical protocols will lead to significant improvements in the survival rate for ovarian cancer.

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors co-localized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed–monoclonal antibodies that block Galectin-3–mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.

Background: T cell-mediated immunotherapy has shown great promise in the treatment of human cancers. Bispecific antibodies (BsAbs) that engage CD3 on T cells and a tumor antigen on target cells can induce polyclonal T cell-mediated cytotoxicity against both leukemia (expressing CD19) and solid tumors (expressing HER-2 or GD2), unrestricted by MHC. The L1 cell adhesion molecule (L1CAM) is a glycoprotein consisting of 6 Ig-like domains and 5 fibronectin-like repeats in the ectodomain. In normal tissues, L1CAM expression is restricted to neurons, renal tubules, and skin at low levels and plays an important role in brain development, including neuronal migration and differentiation. Interestingly, L1CAM is also overexpressed on various pediatric cancers including neuroblastoma (NB), and stimulates tumor proliferation, migration, and metastasis. Chimeric IgG1 antibody, chCE7, targeting the 6th Ig-like domain of human L1CAM, had high affinity (KD = 0.63 nM) and when 131I-labeled, had superior sensitivity and specificity over 131I-MIBG in patients with metastatic NB. However, chCE7 lacked ADCC ability in preclinical studies and no major clinical responses have been reported for anti-L1CAM chimeric antigen receptor (CAR) modified T cells (NCT02311621). Methods: By CDR grafting, the murine antibody E71 (specific for 2nd Ig-like domain with KD = 2.23 nM) was humanized to huE71, and huE71-BsAb using the IgG(L)-scFv BsAb platform previously described (Xu et al., Can Immunol Res 2016) was generated and compared to huE72 (the humanized version of CE7)-BsAb. The BsAbs were produced in CHO-S cells and purified by protein A affinity chromatography. Size homogeneity was confirmed by SEC HPLC. Binding to L1CAM and CD3, or to tumors and T cells was measured by SPR (Biacore) or by FACS, respectively. In vitro T-cell activation and cytokine release were assessed by FACS and by ELISA, respectively. In vitro tumor cytotoxicity was measured by 4-hour 51Cr release at an E:T ratio of 10:1. In vivo antitumor effect was assessed in Balb/c-Rag2-/-IL-2R-c-KO (DKO) mice xenografted with subcutaneous (s.c.) NB cell lines or patient-derived xenografts (PDX). Tumor targeting was performed by using 89Zr-labeled huE71 and huE72 IgG1 in xenografted nude mice. Results: Both BsAbs showed a molecular size of 210kDa with >90% purity. When assessed by SPR for binding to the L1CAM, huE71-BsAb retained its parental affinity, while that of huE72-BsAb decreased by 8-fold (KD of 5.15nM and 5.28nM, respectively). As expected from the IgG(L)-scFv design, both BsAbs showed lower binding to CD3(+) T cells when compared with huOKT3 IgG1. Both BsAbs induced CD25 expression on CD4(+) or CD8(+) T cells after 96 hours of culture in the presence of L1CAM(+) IMR32 NB cell line, and CD8(+) T cells showed more activation in the presence of huE72-BsAb. Both BsAbs induced similar level of Th1 cytokine (e.g. TNFα) from PBMCs following 24 hours of culture in the presence of IMR32. Both BsAbs mediated T-cell cytotoxicity on L1CAM(+) cell lines with pM EC50s. In nude mice xenografted with s.c. L1CAM(+) cancer cell line, intravenous (i.v.) huE71 IgG1 showed higher tumor targeting efficiency than huE72 IgG1. In DKO mice xenografted with s.c. IMR32 (carrying luciferase reporter gene) and s.c. PBMCs, i.v. huE71-BsAb significantly suppressed tumor growth while huE72-BsAb had no antitumor effect. Similar results were observed in an s.c. L1CAM(+) NB PDX model when PBMCs were injected i.v. Conclusions: Despite near-identical in vitro binding characteristics, BsAbs targeting different epitopes on the same target have strikingly different antitumor properties in vivo. HuE71-BsAb is a potential T cell engaging immunotherapeutic for NB and other L1CAM(+) solid tumors. Citation Format: Maya Suzuki, Hong Xu, Hongfen Guo, Brandon Nemieboka, Zhihao Wu, Jason Lewis, Nai-Kong V. Cheung. Novel T-cell engaging antibodies against L1CAM for neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A30.

The CA-125 antigen is the most widely used serum biomarker to monitor epithelial ovarian carcinoma (EOC) and for the differential diagnosis of pelvic masses. A repeating glycopeptide, CA-125 is an epitope of the larger transmembrane mucin protein, MUC16, which is overexpressed in EOC. MUC16 has an average molecular weight of 3-5 million Da and includes a putative cleavage site proximal to a tandem repeat region. To date, the majority of antibodies against MUC16 have targeted the tandem repeat region of the protein, which is shed into circulation following cleavage. Using synthetic MUC16 peptides, high affinity antibodies (9C9, 19C11, 7B12) were raised against the portion of MUC16 retained by the cell. Imaging modalities currently used for EOC are generally viewed as inadequate and as such, the goal of this study is to evaluate these three antibodies to determine the best candidate to be used as a PET imaging agent for EOC. These antibodies were conjugated with desferrioxamine-isothiocyanate and radiolabelled with 89Zr. Using a MUC16-positive cell line (SKOV3-MUC16c114), in vitro studies were performed to confirm immunoreactivity and binding affinities of the radiolabeled antibodies. In addition, in vitro internalization studies were conducted which demonstrated similar uptake among the tracers. Subcutaneous EOC tumors were established in athymic, nude mice for the in vivo characterization of the MUC16 antibodies. Firstly, biodistribution data for 89Zr-9C9 revealed an accumulation of the tracer in the tumor tissue at 24 hours post-injection of 9.41 ± 1.98 %ID/g. Kidney uptake of this tracer, although washing out over time, was extremely high at 24 hours (37.02 ± 1.35 %ID/g). The liver and spleen uptake of the tracer at 24 hours post-injection were 9.74 ± 0.76 and 4.76 ± 0.55 %ID/g, respectively. For 89Zr-19C11, despite in vitro data that demonstrated active internalization, biodistribution data for 89Zr-19C11 revealed minimal tumor uptake in vivo 24 hours post-injection (3.12 ± 0.68 %ID/g). Kidney uptake of this tracer was 4.84 ± 0.98 %ID/g and liver and spleen uptake were high 24 hours post-injection (34.73 ± 4.39 and 9.25 ± 0.90 %ID/g, respectively) indicating potential aggregation or stability issues in vivo. Unlike the other tracers, serum stability data of 89Zr-19C11 demonstrated a purity of 74.43 ± 1.51% after 7 days, an uncharacteristically low stability for a 89Zr-labeled antibody (typically greater than 90%). For 89Zr-7B12, biodistribution data revealed modest tumor uptake 24 hours post-injection (8.54 ± 0.95 %ID/g), kidney uptake of 7.19 ± 0.75 %ID/g, and liver and spleen uptake of 6.24 ± 0.83 and 7.22 ± 1.26 %ID/g, respectively. These in vitro and in vivo studies point to 89Zr-7B12 as the leading candidate thus far. Further studies, including imaging, will be conducted at longer time points to evaluate in vivo tumor uptake of 89Zr-7B12 in addition to blocking studies to demonstrate the specificity of the tracer. Research funded, in part, by the Center to Reduce Cancer Health Disparities of the National Cancer Institute (3R01CA176671-02S1). Citation Format: Brandon E. Nemieboka, Dharmarao Thapi, Ariana Bitton, Jason S. Lewis. Preclinical evaluation of radiolabeled MUC16 antibodies for PET imaging of epithelial ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B25.