Molecular imaging applications of antibody-based immunotherapeutics to understand cancer drug distribution

With the landscape-transforming arrival of cancer immunotherapy, such as drugs that block immune checkpoints, durable responses are observed for several different cancer types. Regrettably, only part of the patients initially respond, and once responded, resistance to immune checkpoint blockade may occur. Therefore, new treatment options are explored to enhance the immune system. These approaches include engaging T cells or inhibit immunosuppressive cell types like tumor-associated macrophages (TAMs). T-cell activating drugs include bispecific T cell engagers (BiTEs) and T cell-redirecting antibodies, both redirect T cells to a predefined tumor target for subsequent infiltration and cytotoxicity. Another approach are drugs that inhibit TAMs, resulting in an enhanced tumor immune response. Limited information is available regarding the pharmacological behavior of these new molecular entities. Radiolabeling these types of drugs with positron emission tomography (PET) isotopes allows molecular imaging using PET to assess whole-body drug distribution and tumor targeting. Ex vivo, techniques like tissue autoradiography, radioactive gel electrophoresis of plasma or tissue lysate, and ex vivo biodistribution complement PET imaging. The research described in this thesis aims to gain insight with molecular imaging in the pharmacological behavior of antibody-based immunotherapeutics using molecular imaging. We show the tumor targeting properties of BiTES in both the preclinical as clinical setting. In addition we preclinically studied the distribution of a T cell-redirecting antibody and a TAM-directed antibody. Overall, molecular imaging of new radiolabeled cancer drugs could provide information to support drug development

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

Assessing the role of tumour-associated macrophage subsets in breast cancer subtypes using digital image analysis

Purpose: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. Methods: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. Results: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. Conclusion: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series

Molecular Imaging in Cancer Drug Development

Developing new oncology drugs has increased since the improved understanding of cancer's complex biology. The oncology field has become the top therapeutic research area for new drugs. However, only a limited number of drugs entering clinical trials will be approved for use as standard of care for cancer patients. Molecular imaging is increasingly perceived as a tool to support go/no-go decisions early during drug development. It encompasses a wide range of techniques including radiolabeling a compound of interest followed by visualization with single photon emission computed tomography or positron emission tomography. Radiolabeling can be performed using a variety of radionuclides that are preferably matched to the compound based on size and half-life. Imaging can provide information on drug behavior in vivo, whole body drug target visualization, and heterogeneity in drug target expression. This review focuses on current applications of molecular imaging in the development of small molecules, antibodies, and anti-hormonal anticancer drugs

Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N-succinyl-desferrioxamine (N-suc-DFO) and subsequently radiolabeled with zirconium-89 ((89)Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and (89)Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [(89)Zr]Zr-DFO-N-suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [(89)Zr]Zr-DFO-N-suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [(89)Zr]Zr-DFO-N-suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did (89)Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that (89)Zr was localized to macrophages within lymphoid tissues. Following [(89)Zr]Zr-DFO-N-suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm(2). We hypothesize that intratumoral macrophage depletion by [(89)Zr]Zr-DFO-N-suc-CSF1R-mAb precluded tumor uptake higher than (89)Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development

Tumor-associated macrophages in breast cancer:Innocent bystander or important player?

Tumor-associated macrophages (TAMs) are important tumor-promoting cells in the breast tumor micro environment. Preclinically TAMs stimulate breast tumor progression, including tumor cell growth, invasion and metastasis. TAMs also induce resistance to multiple types of treatment in breast cancer models. The underlying mechanisms include: induction and maintenance of tumor-promoting phenotype in TAMs, inhibition of CD8 + T cell function, degradation of extracellular matrix, stimulation of angiogenesis and inhibition of phagocytosis. Several studies reported that high TAM infiltration of breast tumors is correlated with a worse patient prognosis. Based on these findings, macrophage-targeted treatment strategies have been developed and are currently being evaluated in clinical breast cancer trials. These strategies include: inhibition of macrophage recruitment, re polarization of TAMs to an antitumor phenotype, and enhancement of macrophage-mediated tumor cell killing or phagocytosis. This review summarizes the functional aspects of TAMs and the rationale and current evidence for TAMs as a therapeutic target in breast cancer

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U- 13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified. </p

Molecular Imaging of Radiolabeled Bispecific T-cell Engager 89Zr-AMG211 Targeting CEA-positive Tumors

BACKGROUND: AMG 211, a bispecific T‑cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T‑cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor targeting properties. EXPERIMENTAL DESIGN: 89Zr‑AMG211 was administered to mice bearing CEA‑positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA‑negative HL‑60 promyelocytic leukemia xenografts. Biodistribution studies with 2‑10 µg 89Zr‑AMG211 supplemented with unlabeled AMG 211 up to 500 µg protein dose were performed. A BiTE® that does not bind CEA, 89Zr‑Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo. Intratumoral distribution was studied with IRDye800CW‑AMG211. Moreover, 89Zr‑AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199 Results: 89Zr‑AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with 2 μg dose, and the lowest tumor uptake was observed with 500 μg dose. After 24 hours, higher uptake of 10 μg 89Zr‑AMG211 occurred in CEA‑positive xenografts, compared to CEA‑negative xenografts. Although the blood half‑life of 89Zr‑AMG211 was ~1 hour, tumor retention persisted for at least 24 hours. 89Zr‑Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr‑AMG211. 800CW‑AMG211 was specifically localized in CEA‑expressing viable tumor tissue. GMP‑manufactured 89Zr‑AMG211 fulfilled release specifications. CONCLUSIONS: 89Zr‑AMG211 showed dose‑dependent CEA‑specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior