⁸⁹Zr-radiopharmaceuticals to study whole-body distribution and response to antibody-based cancer immunotherapies

Immunotherapy has improved the survival of patients with advanced stages of various cancers, however, not all patients respond. Clinicians could benefit from increased knowledge about the pharmacokinetics of new immunotherapeutic drugs and factors that play a role in the development of a tumor response. The aim of the studies described in this thesis is to investigate whether molecular imaging with positron emission tomography (PET) can support this in two ways, namely 1) in the development of new drugs and 2) in optimizing treatment with immunotherapy. By radiolabeling an immunotherapeutic drug with a suitable isotope, such as zirconium-89 (89Zr), tumor uptake and whole-body distribution can be non-invasively imaged. In this thesis, we describe the development of several 89Zr-labeled antibodies, also called 89Zr radiopharmaceuticals. We demonstrate how PET imaging with these 89Zr-radiopharmaceuticals can provide insight into the pharmacokinetics of various immunotherapeutic antibodies that are still in (clinical) development or have already been approved for treatment of patients with certain cancers. We also show in a limited number of patients that tumor response to treatment with immune checkpoint inhibitors may be predicted by tumor uptake on the 89Zr-PET scan. In the future, PET imaging with 89Zr-radiopharmaceuticals could support the selection of patients who potentially benefit from immunotherapy treatment, and thereby clinical decision-making

(89)Zr-Onartuzumab PET imaging of c-MET receptor dynamics

PURPOSE: c-MET and its ligand hepatocyte growth factor are often dysregulated in human cancers. Dynamic changes in c-MET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of c-MET dynamics could therefore potentially guide c-MET-directed therapies. We investigated the feasibility of (89)Zr-labelled one-armed c-MET antibody onartuzumab PET for detecting relevant changes in c-MET levels induced by c-MET-mediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib resistance or heat shock protein-90 (HSP90) inhibitor NVP-AUY-922 treatment in human non-small-cell lung cancer (NSCLC) xenografts. METHODS: In vitro membrane c-MET levels were determined by flow cytometry. HCC827ErlRes, an erlotinib-resistant clone with c-MET upregulation, was generated from the exon-19 EGFR-mutant human NSCLC cell line HCC827. Mice bearing HCC827 and HCC827ErlRes tumours in opposite flanks underwent (89)Zr-onartuzumab PET scans. The HCC827-xenografted mice underwent (89)Zr-onartuzumab PET scans before treatment and while receiving biweekly intraperitoneal injections of 100 mg/kg NVP-AUY-922 or vehicle. Ex vivo, tumour c-MET immunohistochemistry was correlated with the imaging results. RESULTS: In vitro, membrane c-MET was upregulated in HCC827ErlRes tumours by 213 ± 44% in relation to the level in HCC827 tumours, while c-MET was downregulated by 69 ± 9% in HCC827 tumours following treatment with NVP-AUY-922. In vivo, (89)Zr-onartuzumab uptake was 26% higher (P < 0.05) in erlotinib-resistant HCC827ErlRes than in HCC827 xenografts, while HCC827 tumour uptake was 33% lower (P < 0.001) following NVP-AUY-922 treatment. CONCLUSION: The results show that (89)Zr-onartuzumab PET effectively discriminates relevant changes in c-MET levels and could potentially be used clinically to monitor c-MET status

Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs

Background To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 (Zr-89) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice. Methods Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post(89)Zr-pembrolizumab (10 mu g, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 mu g) unlabeled pembrolizumab or(89)Zr-IgG(4)control (10 mu g, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1. Results PET imaging and biodistribution studies showed high(89)Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of(89)Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative. Conclusion Zr-89-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of(89)Zr-pembrolizumab whole-body distribution in patients

Probody therapeutic design of 89Zr-CX-072 promotes accumulation in PD-L1 expressing tumors compared to normal murine lymphoid tissue

PURPOSE: Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti-PD-L1-mediated toxicities. To study its targeting of PD-L1-expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 (89Zr). EXPERIMENTAL DESIGN: 89Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis. RESULTS: PET imaging revealed 89Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89Zr-PbCtrl. Tumor tissue autoradiography showed high 89Zr-CX-072 uptake in high PD-L1-expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89Zr-CX-075, which lacks the Probody design. CONCLUSIONS: 89Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491)

Preclinical PET imaging of bispecific antibody ERY974 targeting CD3 and glypican 3 reveals that tumor uptake correlates to T cell infiltrate

BACKGROUND: Bispecific antibodies redirecting T cells to the tumor obtain increasing interest as potential cancer immunotherapy. ERY974, a full-length bispecific antibody targeting CD3ε on T cells and glypican 3 (GPC3) on tumors, has been in clinical development However, information on the influence of T cells on biodistribution of bispecific antibodies, like ERY974, is scarce. Here, we report the biodistribution and tumor targeting of zirconium-89 (89Zr) labeled ERY974 in mouse models using immuno-positron emission tomography (PET) imaging. METHODS: To study both the role of GPC3 and CD3 on the biodistribution of [89Zr]Zr-N-suc-Df-ERY974, 89Zr-labeled control antibodies targeting CD3 and non-mammalian protein keyhole limpet hemocyanin (KLH) or KLH only were used. GPC3 dependent tumor targeting of [89Zr]Zr-N-suc-Df-ERY974 was tested in xenograft models with different levels of GPC3 expression. In addition, CD3 influence on biodistribution of [89Zr]Zr-N-suc-Df-ERY974 was evaluated by comparing biodistribution between tumor-bearing immunodeficient mice and mice reconstituted with human immune cells using microPET imaging and ex vivo biodistribution. Ex vivo autoradiography was used to study deep tissue distribution. RESULTS: In tumor-bearing immunodeficient mice, [89Zr]Zr-N-suc-Df-ERY974 tumor uptake was GPC3 dependent and specific over [89Zr]Zr-N-suc-Df-KLH/CD3 and [89Zr]Zr-N-suc-Df-KLH/KLH. In mice engrafted with human immune cells, [89Zr]Zr-N-suc-Df-ERY974 specific tumor uptake was higher than in immunodeficient mice. Ex vivo autoradiography demonstrated a preferential distribution of [89Zr]Zr-N-suc-Df-ERY974 to T cell rich tumor tissue. Next to tumor, highest specific [89Zr]Zr-N-suc-Df-ERY974 uptake was observed in spleen and lymph nodes. CONCLUSION: [89Zr]Zr-N-suc-Df-ERY974 can potentially be used to study ERY974 biodistribution in patients to support drug development

First-in-human study of the biodistribution and pharmacokinetics of ⁸⁹Zr-CX-072, a novel immunopet tracer based on an anti–PD-L1 probody

Purpose: CX-072, a PD-L1–targeting Probody therapeutic, is engineered to be activated by tumor proteases that remove a masking peptide. To study effects on biodistribution and pharmacokinetics, we performed 89Zr-CX-072 positron emission tomography (PET) imaging. Experimental Design: Patients received 1 mg, 37 MBq 89Zr-CX-072 plus 0, 4, or 9 mg unlabeled CX-072 and PET scans at days 2, 4, and 7. After that, treatment comprised 10 mg/kg CX-072 q2 weeks (n ¼ 7) þ 3 mg/kg ipilimumab q3w 4 (n ¼ 1). Normal organ tracer uptake was expressed as standardized uptake value (SUV)mean and tumor uptake as SUVmax. PD-L1 expression was measured immunohistochemically in archival tumor tissue. Results: Three of the eight patients included received 10-mg protein dose resulting in a blood pool mean SUVmean SD of 4.27 0.45 on day 4, indicating sufficient available tracer. Tumor uptake was highest at day 7, with a geometric mean SUVmax 5.89 (n ¼ 113) and present in all patients. The median follow-up was 12 weeks (4–76þ). One patient experienced stable disease and two patients a partial response. PD-L1 tumor expression was 90% in one patient and ≤1% in the other patients. Mean SUVmean SD day 4 at 10 mg in the spleen was 8.56 1.04, bone marrow 2.21 0.46, and liver 4.97 0.97. Four patients out of seven showed uptake in normal lymph nodes and Waldeyer’s ring. The tracer was intact in the serum or plasma. Conclusions: 89Zr-CX-072 showed tumor uptake, even in lesions with ≤1% PD-L1 expression, and modest uptake in normal lymphoid organs, with no unexpected uptake in other healthy tissues