Feasibility of IV Tc-99m-tilmanocept for Imaging of M2-like TAMs in Metastatic Melanoma (Tilmanocept)

Pilot Study to Test the Feasibility of IV Injected Tc-99m-tilmanocept for Imaging of M2-like Tumour Associated Macrophages in Metastatic Melanoma

This study in 20 patients is designed as a monocentric, open-label and uncontrolled, exploratory pilot study. Patients diagnosed with advanced melanoma (stage III-IV) and scheduled for anti-PD-1 immunotherapy will be recruited for this project. Patients will receive IV 250 µg Tilmanocept, labelled with 370 MBq of Tc-99m (bolus injection) according to the Navidea's protocol in our GMP certified radiopharmaceutical unit, before the first cycle of clinically scheduled immunotherapy. Scintigraphy images will be acquired dynamically from time of injection to +30 minutes. Quantitative SPECT/CT (xSPECT/CT, Siemens Symbia Intevo, Erlangen, Germany) imaging will be performed up to 1 hour p.i. to evaluate hyperaemia, and up to 3 hours p.i. to image and measure the CD206 receptor uptake. The data of the scans will be compared to immunohistochemistry results from biopsy staining for TAMs and M2-like TAMs and retrospectively with response to the immunotherapy to determine any correlation between M2-like TAMs and treatment response. For the planned retrospective comparison we will use the FDG - PET/CT data that is done after the immunotherapy as standard of care. We will analyse the lesion size and FDG - uptake in standard of care PET/CT of CD206+ and CD206 negative lesions in Tilmanocept SPECT/CT before and after immunotherapy to determine any correlation between CD206 related uptake and treatment response.

According to the Swiss Federal Statistical Office, cancer has been the leading cause of death in Switzerland for men aged between 45 and 84 and women aged between 25 and 84. Melanoma, represents the deadliest and most aggressive form of skin cancer. The incidence of melanoma is rising and in Switzerland it is the fourth most occurring malignancy. In the recent years many advances have been achieved in the treatment of disseminated disease, namely targeted and immunomodulatory treatments (checkpoint inhibitors), either as monotherapy or in combination. Immune checkpoint inhibitors, such as anti-PD-1 are standard of care for advanced melanoma. However, despite all the success by checkpoint inhibitors, still many patients do not benefit from treatment. Therefore, several attempts have been made to investigate possible prediction of outcome to PD1/PD-L1 treatment. Scientific studies reported mutational load, neoantigens or PD1/PD-L1 expression and/or CD8 infiltrates in melanoma to be predictive for response. However, it has further been shown that also PD-L1 negative tumours respond to PD1/PD-L1 blockade. One possible explanation could be the heterogeneity of intra-tumoural PD-L1 expression or the heterogeneity among different tumour sites in melanoma patients. Overall, the PD-L1 expression is highly dependent on extrinsic factors, as for example hypoxia and therefore highly versatile at different sites and over time. Another important mechanism of resistance lies in the tumour microenvironment, mainly in inhibitory immune cells of the host. Among others, such as regulatory t-cells (Tregs) or myeloid derived stem cells (MDSC), tumour associated macrophages (TAMs) seem of particular importance in the immune-modulation of the tumour microenvironment. TAMs in the tumour microenvironment play an essential role in cancer progression. Particularly, the alternatively activated M2 polarized phenotype (M2-like) is confirmed as indicator of poor patients' outcome. During melanoma progression the anti-tumoural M1 polarized phenotype (M1-like) shifts towards the M2 phenotype, which can directly suppress immunity for example via production of cytokines as TGFb or IL-10, cooperating in the reduction of specific CD8 derived immune response. The evolution in understanding the macrophage compartment led therefore to the development of further treatment strategies as CSFR-1 directed antibodies, showing promising synergizing effects with PD-1 directed treatment and reversed resistance to checkpoint inhibitor treatment. CD206, also termed as MRC1 (C-type mannose receptor 1), is an M2-like macrophage marker in both mouse and human. Its upregulation in macrophages renders them to produce IL-10 and TGFb, identifying this TAM population as anti-inflammatory subtype. Tc-99m-tilmanocept is a FDA/EMA approved drug (Lymphoseek™, NAVIDEA Biopharmaceuticals, OH, USA). The radio-peptide targets CD206 with nano-molar affinities and is licensed for mapping and localization of lymph nodes draining breast cancer or melanoma using subcutaneous, intradermal, subareolar, or peritumoral injection. Studies have shown superiority to conventional sentinel node procedures. Technetium Tc-99m binds to the diethylenetriaminepentaacetic acid (DTPA) moieties of the Tilmanocept, making it a tracer, suitable for lymph node scintigraphy/SPECT/CT. Furthermore, recent evidence establishes its role to detect systemic inflammation. Due to its high specificity, even small targets as atherosclerotic plaques in mice can be visualized. Most recent trials have investigated Tc-99m Tilmanocept as intravenous injection (IV) in patients with Rheumatoid Arthritis. A dosage of 150 µg Tilmanocept IV, labelled with 370 MBq of Tc-99m-has demonstrated to be safe and was specific in affected joints of all subjects who had undergone multiple RA flares, which demonstrates CD206+ macrophage infiltration of these joints. Several trials currently investigate the role of Tc-99m-tilmanocept IV and show promising results, for example in patients with Kaposi Sarcoma (NCT03157167) or in patients with liver metastases (NCT03029988). This study aims to investigate the feasibility and efficacy of Tc-99m-tilmanocept as a marker for CD206+ M2-like TAMs in lesions in melanoma patients. The identification of a correlation of M2-like TAMs presence with the outcome of patients receiving anti-PD1 immunotherapy, would open up new ways to predict ICH treatment response. Therefore, we want to take the first step with this pilot study to evaluate the feasibility of Tc-99m-tilmanocept as a marker for imaging of M2-like TAMs in melanoma metastasis. This Project falls under risk category C. Intravenous Injection had been tested in a Phase I dose escalation trial in subjects with active rheumatoid arthritis. Subjects received an IV injection of 50, 200 or 400µg Tilmanocept labelled with 37, 185 or 370 MBq of Tc-99m. This study reported that IV injection of Tc-99m-tilmanocept was well tolerated and no adverse drug reactions were observed in any group. Nevertheless, the approval for Tilmanocept does not include IV as route of administration. Therefore, the risk category is C. There are no data available determining the risk for pregnant woman or the foetus. But, as for every nuclear medicine exam, pregnancy will be tested before any intervention and is part of the standard of care precaution. Pregnancy is an exclusion criterion. Although no serious hypersensitivity reactions were reported in clinical trials with Tc-99m-tilmanocept, there might be a risk of such reaction due to its chemical similarity to dextran. Patients will be ask before administration about previous hypersensitivity reactions to dextran and modified forms of dextran. In case of emergency, standard emergency procedures will be employed. There is no immediate benefit to the study participants. The results of the study might provide a better understanding of the diversity of responses to immunotherapy and might allow in future to predict the response of a melanoma patient to immunotherapy. This prediction could allow a more personalized treatment plan for future melanoma patients and could therefore also prevent unnecessary toxicity reactions caused by immunotherapy. General study design This study in 20 patients is designed as a monocentric, open-label and uncontrolled, exploratory pilot study. Patients diagnosed with advanced melanoma (stage III-IV) and scheduled for anti-PD-1 immunotherapy will be recruited for this project. Patients will receive IV 250 µg Tilmanocept, labelled with 370 MBq of Tc-99m (bolus injection) according to the Navidea's protocol in our GMP certified radiopharmaceutical unit, before the first cycle of clinically scheduled immunotherapy. Scintigraphy images will be acquired dynamically from time of injection to +30 minutes. Quantitative SPECT/CT (xSPECT/CT, Siemens Symbia Intevo, Erlangen, Germany) imaging will be performed up to 1 hour p.i. to evaluate hyperaemia, and up to 3 hours p.i. to image and measure the CD206 receptor uptake. The data of the scans will be compared to immunohistochemistry results from biopsy staining for TAMs and M2-like TAMs and retrospectively with response to the immunotherapy to determine any correlation between M2-like TAMs and treatment response. For the planned retrospective comparison we will use the FDG - PET/CT data that is done after the immunotherapy as standard of care. We will analyse the lesion size and FDG - uptake in standard of care PET/CT of CD206+ and CD206 negative lesions in Tilmanocept SPECT/CT before and after immunotherapy to determine any correlation between CD206 related uptake and treatment response.

Patients diagnosed with advanced melanoma (stage III-IV) will receive IV 250 µg Tilmanocept, labelled with 370 MBq of Tc-99m before the first cycle of clinically scheduled anti-PD-1 immunotherapy.

The primary endpoint is the imaging result obtained by scintigraphy/SPECT at day 0 as lesion number and site, compared to standard of care clinical imaging (FDG - PET/CT) per lesion and per patient.

Secondary endpoint will be the proportion of macrophages and the ratio of macrophage subtype (CD206 positive (M2-like) versus CD206 negative (M1-like)), obtained by immunohistochemistry in biopsies in correlation with Tc-99m-tilmanocept uptake.

For retrospective analysis of correlation of M2-like TAM proportion and response to anti-PD1 immunotherapy we will compare the three most active lesions (uptake of Tc-99m-tilmanocept) in scintigraphy/SPECT images with SOC follow up imaging PET/CT (or if needed other anatomical imaging as MRI or contrast enhanced CT) in each patient.

Inclusion Criteria: Female and male Age ≥ 18 Diagnosis of having advanced melanoma , stage III-IV Scheduled for clinically indicated anti-PD-1 immunotherapy Informed Consent as documented by signature FDG PET/CT within 4 weeks before screening Biopsy available Minimum 3 lesions detected in FDG PET/CT Exclusion Criteria: Age under 18 Ocular melanoma Women who are pregnant or breast feeding, Intention to become pregnant during the course of the study, Previous enrolment into the current study, Enrolment of the investigator, his/her family members, employees and other dependent persons, Previous immunotherapy, History of any disease or relevant physical or psychiatric condition or abnormal physical finding which may interfere with the study objectives at the investigator judgment The subject has a known allergy to or has had an adverse reaction to dextran exposure Insufficient knowledge of project language, inability to give consent or to follow procedures The patient makes use of his/her "right not to know" and refuses to be informed about incidental findings

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