University of Groningen Radiopharmaceuticals for translational imaging studies in the field of cancer immunotherapy van der Veen, Elly

University of Groningen

Radiopharmaceuticals for translational imaging studies in the field of cancer immunotherapy

van der Veen, Elly

DOI:

10.33612/diss.128579303

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2020

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van der Veen, E. (2020). Radiopharmaceuticals for translational imaging studies in the field of cancer

immunotherapy. University of Groningen. https://doi.org/10.33612/diss.128579303

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Chapter 1

General introduction

General introduction

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BACKGROUND

Cancer is a major burden of disease worldwide, with 18.1 million new cancer cases and 9.6 million cancer related deaths in 2018. It is expected that 24 million patients will be diagnosed with cancer in 2035.1-3 _{Standard treatment of tumors often comprises surgery,}

radiotherapy and systemic therapy. Systemic treatment consists of chemotherapy, hormonal therapy, targeted therapy and immunotherapy. The youngest development is cancer

immunotherapy, aimed to improve the tumor immune response.4 _{There are several types}

of immunotherapy, including adoptive cellular therapy, cytokine therapy, cancer vaccines and immune checkpoint inhibiting monoclonal antibodies. Especially this last group has shown remarkable anti-tumor effects, in a broad range of tumor types. Tumor cells can protect themselves from attack by immune cells, by activating so-called immune checkpoint pathways. These co-inhibitory pathways dampen the tumor immune response. Blocking these pathways by immune checkpoint inhibitors leads to a re-activation of the immune response and elimination of tumor cells. Several immune checkpoint inhibitors, targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein (PD-1)/programmed death-ligand 1 (PD-L1) have already been approved for clinical use for

many tumor types. Many other immunotherapeutics are in development.5

Although immune checkpoint inhibitors as single agents, or as combination of anti-PD-1 with anti-CTLA-4, have already shown impressive antitumor responses, even better antitumor effects might be achieved using combinational strategies with other immunotherapeutics, chemotherapeutic drugs, radiotherapy or targeted agents. Currently over 3000 ongoing clinical trials are evaluating immunotherapeutics, applied as single agent or in combinational strategies.5

Treatment with cancer immunotherapy has led to important difficulties in the clinic. Not every patient responds to therapy and serious toxicity including immune-related adverse events can occur. Moreover, the large amount of clinical studies ongoing, requires a huge number of patients and major financial investments. It is therefore clear that strategies to improve patient selection to accelerate immuno-oncology clinical development are urgently needed. However, the search for biomarkers in this field is challenging, as the tumor immune response is highly complex and dynamic. Many different cells and molecules are involved. Currently, Food and Drug Administration (FDA) approved biomarkers are based on PD-L1 measured with immunohistochemistry (IHC), and microsatellite instability-high and mismatch repair deficient status measurement by IHC and polymerase-chain-reaction-based assays. However, treatment can also be beneficial in tumors without these biomarkers.6 _{A potential explanation for these}

conflicting results is that these assays use one single biopsy, and therefore do not capture the heterogeneity across various tumor lesions in a patient and within a single lesion.

Chapter 1

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To obtain characteristics of all tumor lesions within the body of an individual patient, whole body molecular imaging modalities might be used. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) using radiolabeled drugs or other molecules, can provide non-invasive information about the biodistribution of immunomodulatory drugs in the body, heterogeneity of target expression, effects of immunotherapy on immune cells, and therapy effects on other cells in the tumor microenvironment. For these purposes different radiopharmaceuticals can be applied. Aim of this thesis is to develop new radiopharmaceuticals for molecular imaging in the field of cancer immunotherapy.

OUTLINE THESIS

In chapter 2 we provide an overview of the current preclinical and clinical molecular imaging approaches to support immunotherapy decision making. The MEDLINE (PubMed) data base was searched for articles that described molecular imaging techniques assessing drug targets and cellular effects potentially useful as biomarkers in cancer immunotherapy. The subsequent list was complemented with relevant articles not yet MEDLINE indexed and abstracts from recent conferences. The database ClinicalTrials.gov was searched for active trials in the area of molecular imaging and immunotherapy, and the monoclonal antibody (mAb) database IMGT/ mAB-DB (www.imgt.org) for therapeutic immune checkpoint modulating antibodies. Based on this search strategy we divided three main groups for imaging strategies. Firstly, imaging of tumor and immune cells by targeting immune checkpoint proteins. A second strategy is imaging of immune cells. A third imaging strategy comprises imaging of extracellular matrix components, including adhesion molecules, growth factors and cytokines.

We developed different radiopharmaceuticals targeting immune checkpoints. For this purpose, monoclonal antibodies targeting immune checkpoint proteins can be radiolabeled with zirconium-89 (89_{Zr). In chapter 3 we aimed to study the in vivo pharmacokinetics and}

whole-body distribution of the anti-PD-1 antibody 89_{Zr-pembrolizumab with PET in humanized}

mice. PET imaging and biodistribution studies have been performed in humanized mice and as a control in non-humanized immunodeficient mice, xenografted with the human A375M melanoma cells. PET imaging was performed on day 7 post 89_{Zr-pembrolizumab administration,}

followed by ex vivo biodistribution studies. Other humanized mice bearing A375M tumors received a co-injection of excess unlabeled pembrolizumab or 89_Zr-IgG

4 control.Tumor and

spleen tissue were studied with autoradiography and immunohistochemical analyses, including PD-1 staining. Finally, 89_{Zr-pembrolizumab production was converted to a Good Manufacturing}

General introduction

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Next to full antibodies also smaller molecules can be used as imaging agents to evaluate immune checkpoint protein expression levels. The PD-L1 targeting fluor-18 (18_{F)-labeled adnectin-based}

PET tracer 18_{F-BMS-986192 has been developed for this purpose. In chapter 4 we evaluated the}

usability of 18_{F-BMS-986192 PET imaging to detect different PD-L1 expression levels and}

therapy-induced changes of PD-L1 expression in tumors. First, in vitro binding assays were performed in human tumor cell lines with different total cellular and membrane PD-L1 protein expression levels. Subsequently, PET imaging was executed in immunodeficient mice xenografted with these cell lines. Furthermore, we performed modulation studies, by either upregulation or downregulation of PD-L1 expression levels. For PD-L1 tumor upregulation mice were treated with interferon gamma (IFNγ) intraperitoneally for 3 days. For downregulation mice received the mitogen activated protein kinase kinase (MEK1/2) inhibitor selumetinib by oral gavage for 24 hours. Thereafter 18_{F-BMS-986192 was administered intravenously, followed by PET imaging and}

ex vivo biodistribution studies. Tissues were collected to evaluate ex vivo tracer biodistribution

and to perform flow cytometric, Western blot, and immunohistochemical tumor analyses. Another important molecular imaging strategy is targeting immune cells specifically. Previously the PET tracer N-(4-18_{F-fluorobenzoyl)-interleukin-2 (}18_{F-FB-IL2) was developed preclinically for}

this purpose. This radiopharmaceutical targets T-cells by binding to the IL2-receptor. In chapter 5 the complex manufacturing method of clinical-grade 18_{F-FB-IL2 is described. The preclinical}

synthesis of 18_{F-FB-IL2 was the starting point for translation to an automated procedure following}

GMP regulations, resulting in a radiopharmaceutical suitable for clinical use. To overcome several challenges, major adaptations in the production process were executed. These challenges comprised upscaling, characterization, formulation and automation. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier (IMPD).

Although successfully applied in both preclinical and clinical studies the production of this

18_{F-FB-IL2 tracer is complex and time-consuming. Therefore, new strategies for IL2 radiolabeling}

have been investigated. In chapter 6 we describe the development of two novel interleukin-2 PET radioligands: 18_{F-AlF-RESCA-IL2 and gallium-68 (}68_Ga)-labeled 68_{Ga-Ga-NODAGA-IL2.}

Radiolabeling was optimized and stability was evaluated in human serum. In vitro receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs) and compared to 18_{F-FB-IL2. Ex vivo biodistribution studies were executed in immune competent}

mice. In vivo binding characteristics were studied in immunodeficient mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 minutes after hPBMCs inoculation and a 60-minute dynamic PET scan was acquired, followed by ex vivo biodistribution studies. Specific uptake was determined by co-injection of tracer with a blocking dose of unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only.

Chapter 1

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Several clinical imaging studies using immune checkpoint targeting molecules are ongoing.

Chapter 7 describes first-in-human PET imaging with the PD-L1 antibody 89_{Zr-atezolizumab.}

In this study the feasibility of imaging with 89_{Zr-atezolizumab in patients has been evaluated,}

as well as its potential to predict response to PD-L1 blockade. In this study, 22 patients with three different tumor types, received 89_{Zr-atezolizumab before the start of their atezolizumab}

therapy. Clinical response was correlated to this pre-treatment PET signal. Additionally, IHC analyses and RNA sequencing of the tumor biopsies were performed as well as in a few tumor tissues autoradiography. Tracer stability in vivo was evaluated in human blood samples. Internalization experiments were performed on both tumor cells lines, as well as human PBMCs and activated T-cells.

A summary of the results of this thesis, a general discussion and future perspectives are described in chapter 8.

General introduction

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REFERENCES

1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359-386.

2. Pilleron S, Sarfati D, Janssen-Heijnen M, Vignat J, Ferlay J, Bray F, et al. Global cancer incidence in older adults, 2012 and 2035: A population-based study. Int J Cancer. 2019;144:49-58.

3. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA

Cancer J Clin. 2018;68:394-424.

4. Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013;342:1432-1433.

5. Tang J, Shalabi A, Hubbard-Lucey V. Comprehensive analysis of the clinical immuno-oncology landscape. Ann Oncol. 2018;29:84-91.

6. Carbognin L, Pilotto S, Milella M, Vaccaro V, Brunelli M, Caliò A, et al. Differential activity of nivolumab, pembrolizumab and MPDL3280A according to the tumor expression of programmed death-ligand-1 (PD-L1): sensitivity analysis of trials in melanoma, lung and genitourinary cancers.