This study investigates whether a single intradermal (i.d.) injection of low-dose anti-CTLA-4 (ipilimumab), given at the tumour site, can enhance immune activation when combined with standard intravenous (i.v.) anti-PD-1 therapy in patients with advanced melanoma. While combined checkpoint inhibition is effective, it is associated with high toxicity, creating a need for strategies that maintain efficacy with fewer side effects.

Preclinical and early clinical data suggest that local (intradermal) CTLA-4 blockade can stimulate systemic anti-tumour immune responses with reduced toxicity, potentially by reactivating suppressed T cells in tumour-draining lymph nodes. This study compares systemic immune effects of intradermal versus standard intravenous CTLA-4 administration, both combined with nivolumab.

The primary objective is to assess systemic immune activation by measuring changes in CD4+ and CD8+ T-cell frequencies and ICOS expression in peripheral blood. Additional immune monitoring includes blood sampling, tumour biopsies, and advanced imaging using FDG-PET/CT and a novel CD8-targeted PET tracer. The study is a prospective, open-label pilot trial in patients with metastatic melanoma, with follow-up for clinical outcomes and immune response over approximately 13 weeks.

Rationale Immune checkpoint inhibitors (ICI) represent a drastic breakthrough in the treatment of meta static melanoma. Inhibitors of the PD-1 immune checkpoint (e.g., nivolumab and pembroli zumab), when given as monotherapy, are more effective than monotherapy with CTLA-4 checkpoint inhibitors (e.g., ipilimumab), with objective response rates of 30 - 40 %. The com bination of anti-PD-1/anti-CTLA-4 has shown to increase the response rate up to 58 % with a median survival of 72 months (1). However, this also leads to significant increased rates of toxicity (59 % grade 3 or higher adverse events versus 22 % for nivolumab monotherapy) (2). Thus, there is a need to identify effective treatments with less side effects. In clinical stage II melanoma, intradermal (i.d.) administration of low, 20 mg dose anti-CTLA-4 tremelimumab at the primary tumour excision site has shown promising immune-modulatory effects in a previ ous trial (3). Effects were seen both in the draining sentinel lymph node and systemically with out any serious toxicity as seen with systemic therapy. We assume that the increased systemic rates of tumour antigen-specific T cells observed is due to "re-awakening" of previously primed, but immune suppressed T cells in the primary tumour-draining lymph nodes. Based on these interesting results it is hypothesized that the clinical response to monotherapy anti-PD-1 could be improved, even in more advanced stages, by combining with a low dose of i.d. anti-CTLA 4 delivered to the primary tumour-draining lymph basin.

Systemic effects of blockage of CTLA4 can be effectively measured by increase of expression of ICOS on different immune subsets (4). Indeed, the systemic changes after i.d. injection of anti-CTLA-4 in the previous study by van de Pul et al. resulted in dose-dependent increases in ICOS expression on CD4+/CD8+ cells in all patients (> 200% change in the highest dose cohort of 20 mg tremelimumab at 3 weeks), and prolonged decrease in activated Tregs and myeloid derived suppressor cells (MDSCs) in all dose cohorts starting already after one week(3). As patients in the IpiD study will have more advanced disease, we hypothesize that systemic CTLA4 blockage effects will lead to at least 50% increase in ICOS expression CD4+/CD8+ cells from baseline to on treatment. Changes in the immune response upon treat ment can be monitored locally in tumour biopsies, which are cumbersome and thus less at tractive for repeat assessments. Systemic effects can also be evaluated in peripheral blood mononuclear cells (PBMCs) as described above, or with molecular imaging. The latter has the advantage to provide whole body, localised information of biological processes related to the mechanism of action as well as tumour responses. For example, in melanoma patients treated with ipilimumab monotherapy, an early Fluorine-18 Fluorodeoxyglucose ([18F]FDG) PET/CT scan can predict outcome after two cycles for treatment with melanoma (5), and baseline spleen-to-liver ratio of [18F]FDG uptake was associated with poor outcome(6, 7). However, [18F]FDG is a generic tracer to visualise metabolic activity and as such will not discriminate between tumour cells and immune cells. Novel tracers focussing on immune effector cells, like the CD8+ T cell tracers (among others GEH200520/GEH200521 (18F)), can be of added value here to study the systemic effect of i.d. anti CTLA-4. This study protocol therefore will combined with the accompanying CD8 T cell imaging protocol (NCT05629689; EU Trial number: 2024 515218-42-00).

Primary objective To determine the systemic immunological effects of a single dose of 20 mg ipilimumab locally administered i.d. in combination with i.v. standard of care nivolumab or systemic standard of care i.v. administration of ipilimumab and nivolumab as determined with the frequency and functionality of CD4+/CD8+ T cells in peripheral blood cells.

Main trial endpoints T cell subset frequencies (as percentage of parent) and , and systemic activation (determined as a 50 % or greater increase in expression of ICOS/CD278 (inducible T-cell costimulatory) in PBMCs CD4+ and CD8+ T cells from baseline to on-treatment in both groups. We hypothesize a ≥ 50% increase in ICOS expression in at least 8 out of 12 patients.

Trial design Prospective monocentre open-label, pilot clinical trial. Patients included in this pilot study will also be included in an imaging study with a novel [18F]CD8 tracer (A Phase 1a/1b, Multi-Centre, Open-Label, Dose-Escalation and Dose-Expansion Study in Patients with Solid Tumour Ma lignancies to Evaluate GEH200520 Injection / GEH200521 (18F) Injection Safety and Tolera bility, Positron Emission Tomography Imaging, Pharmacokinetics, and Changes in Imaging after Treatment; NCT05629689, EU Trial number: 2024-515218-42-00). Trial duration for safety is expected to be 13 weeks (92 days), follow up for survival will be collected during standard of care.

Trial population Group 1: Adult patients with advanced/metastatic melanoma with (a) tumour lesion(s) that can be biop sied, who are eligible according to standard of care for first-line systemic treatment with i.v. nivolumab. 2.7.2 Group 2 Adult patients with advanced/metastatic melanoma with (a) tumour lesion(s) that can be biop sied, who are eligible according to standard of care for first-line systemic treatment with i.v. nivolumab.

Group 2: Adult patients with advanced/metastatic melanoma with (a) tumour lesion(s) that can be biop sied, who are eligible according to standard of care for first-line systemic treatment with i.v. ipilimumab and nivolumab.

Interventions Group 1: Patients will be treated once by local i.d. injection of 20 mg ipilimumab around (the excision site of the) primary tumour or around a skin metastasis, in combination with i.v. nivolumab according to standard of care (continuous)

Group 2: Patients will be treated according to standard of care with i.v. ipilimumab (4 cycles) combined with i.v. nivolumab (continuous).

For all patients, nivolumab will be continued until complete response (iCR), confirmed progres sive disease (iCPD) or intolerance of treatment according to standard clinical practice. After 24 weeks, it is allowed to stop anti-PD1 treatment in case of a confirmed partial remission (iPR), according to standard practice or the Safe-Stop study protocol (NCT05652673), which evaluates the safety and efficacy of early discontinuation after 24 weeks in patients with a favourable response. This approach is consistent with emerging clinical practice (8-10). Pa tients will have regular follow up according to standard clinical practice, including radiological evaluation (RECIST v1.1 and iRECIST with CT and/or MRI imaging: 1st evaluation at 3 months, follow-up evaluations every 3 months in case of SD and every 4 months in case of PR/CR until disease progression/death or stop treatment due to progression of disease/toxicity).

If not already performed as part of standard of care staging, all patients will undergo a [18F]FDG PET/CT according to EARL criteria before start of treatment, and a tumour biopsy at baseline and on treatment. Blood samples (total volume 150 mL) will be collected at in total 5 timepoints (at baseline, and 1 week, 3 weeks, 32 days, and 12 weeks after start of treatment).

All patients will participate in: A Phase 1a/1b, Multi-Centre, Open-Label, Dose-Escalation and Dose-Expansion Study in Patients with Solid Tumor Malignancies to Evaluate GEH200520 Injection / GEH200521 (18F) Injection Safety and Tolerability, Positron Emission Tomography Imaging, Pharmacokinetics, and Changes in Imaging after Treatment; NCT05629689, EU Trial number: 2024-515218-42-00.

REFERENCES

1. Wolchok JD, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, et al. Long-Term Outcomes With Nivolumab Plus Ipilimumab or Nivolumab Alone Versus Ipilimumab in Patients With Advanced Melanoma. J Clin Oncol. 2022;40(2):127-37. 2. Hodi FS, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Cowey CL, et al. Nivolumab plus ipilimumab or nivolumab alone versus ipilimumab alone in advanced melanoma (CheckMate 067): 4-year outcomes of a multicentre, randomised, phase 3 trial.

Lancet Oncol. 2018;19(11):1480-92. 3. van Pul KM, Notohardjo JCL, Fransen MF, Koster BD, Stam AGM, Chondronasiou D, et al. Local delivery of low-dose anti-CTLA-4 to the melanoma lymphatic basin leads to systemic T(reg) reduction and effector T cell activation. Sci Immunol. 2022;7(73):eabn8097.

4. Fu T, He Q, Sharma P. The ICOS/ICOSL pathway is required for optimal antitumor responses mediated by anti-CTLA-4 therapy. Cancer Res. 2011;71(16):5445-54. 5. Kudura K, Dimitriou F, Basler L, Forster R, Mihic-Probst D, Kutzker T, et al. Prediction of Early Response to Immune Checkpoint Inhibition Using FDG-PET/CT in Melanoma Patients. Cancers (Basel). 2021;13(15). 6. Sachpekidis C, Anwar H, Winkler J, Kopp-Schneider A, Larribere L, Haberkorn U, et al. The role of interim (18)F-FDG PET/CT in prediction of response to ipilimumab treatment in metastatic melanoma. Eur J Nucl Med Mol Imaging. 2018;45(8):1289-96. 7. Wong A, Callahan J, Keyaerts M, Neyns B, Mangana J, Aberle S, et al. (18)F-FDG PET/CT based spleen to liver ratio associates with clinical outcome to ipilimumab in patients with metastatic melanoma. Cancer Imaging. 2020;20(1):36.

Inclusion Criteria:

• Patient must be of age ≥ 18 years, and have a histologically confirmed diagnosis of locally advanced, surgically incurable, or metastatic cutaneous melanoma.

• European Cooperative Oncology Group (ECOG)/World Health Organisation (WHO) performance status of 0 or 1.

• Patient must be eligible for anti-PD-1 treatment with nivolumab (group 1) or with ipilimumab + nivolumab (group 2) according to the treating physician.

• Patient must have one or more tumour lesions of which a biopsy can safely be obtained according to standard clinical practice.

• Patients must have a life expectancy of 3 months or greater.

• Patients must have measurable disease (according to RECIST v1.1) with at least one cutaneous metastasis. Note: measurable disease defined as: at least 1 visceral or nodal/soft tissue melanoma lesion that can be accurately and serially measured in at least 1 dimension and for which the longest diameter is ≥ 10 mm as measured by CT scan or MRI. Lymph nodes must measure ≥ 15 mm in their short axis to be considered measurable by CT-scan or MRI.

• Adequate bone marrow, hepatic, renal and coagulation function (to be conducted within 7 days prior to start therapy, during the baseline period):

  • Leukocyte count ≥ 3,5 × 109 / L
  • Platelets ≥ 100 × 109 / L.
  • Total bilirubin ≤ 3 × the upper limit of normal (ULN).
  • ASAT and ALAT ≤ 3.0 × ULN; except patients with documented liver metastases ASAT and/or ALAT ≤ 5.0 × ULN.
  • (Estimated) creatinine clearance ≥ 45 mL/min/1,73 m2.
  • Albumin ≥ 30g / L
  • LDH ≤ 2 x ULN

• Women of childbearing potential (WOCBP) must use contraception during the study and for 23 weeks after the last dose of nivolumab.

• Men who are sexually active with WOCBP must use contraception during the study plus 7 months after the last dose of nivolumab.

• Written and signed informed consent.

Exclusion Criteria:

• Primary uveal or mucosal melanoma.

• Prior treatment with CTLA-4 inhibitor or agonist, or anti-PD1, except for adjuvant nivolumab > 6 months ago.

• Prior radiotherapy is permitted except on RECIST v1.1 target lesions within 2 weeks of start of trial treatment. Irradiated lesions without progression before start of treatment cannot be target lesions. Note: Patients must have recovered from all radiation-related toxicities, and not require corticosteroids.

• Patient has 12-lead ECG significant findings during screening, per Investigator's as sessment.

• History of another malignancy (that is progressing or requires active treatment) within the previous 5 years. Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or in situ cancer that has undergone potentially curative therapy.

• Patient has a confirmed active SARS-CoV-2 infection.

• Patient has serious non-malignant disease or conditions that, in the opinion of the Investigator, could compromise patient safety or protocol objectives.

• Patient has brain or bone-marrow metastasis that, in the opinion of the Investigator, could compromise patient safety or protocol objectives.

• Active systemic infections requiring therapy, or signs or symptoms of a systemic infection within two weeks prior to baseline.

• Patient has used systemic corticosteroids to treat inflammatory or autoimmune symptoms within 15 days or other immunosuppressive drugs within 30 days prior to screening. Exceptions:

  • Patients that require intermittent use of inhalation or topical corticosteroids are eligible for the study.
  • Patient has received acute, low-dose, systemic immunosuppressant medications (e.g., a one-time dose of dexamethasone for nausea) that, in the opinion of the Investigator, will not compromise protocol objectives.

• Patient has had treatment with systemic immunosuppressive medications (including but not limited to prednisone, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumour necrosis factor agents) within 2 weeks prior to baseline.

• Patient has had treatment with systemic immunostimulatory agents (including but not limited to interferons [IFNs] or interleukin-2 [IL-2]) within 6 weeks or 5 half-lives of the drug, whichever is shorter, prior to baseline.

• Known history or evidence of immunodeficiency states (e.g., organ transplant, leukaemia, human immunodeficiency virus (HIV), hepatitis B, hepatitis C or known acquired immunodeficiency syndrome (AIDS)). NOTE: Testing for HIV must be performed at sites were mandated locally.

• Patient has had any major surgery within 4 weeks prior to enrolment or major surgery is scheduled during the study, with the exception of procedures that are part of the study site IIS.

• Patient has any safety laboratory test results (clinical chemistry, haematology, and urinalysis) that, in the opinion of the Investigator, could compromise patient safety or protocol objectives.

• Patient has any other diseases, metabolic dysfunction, physical examination finding, or clinical laboratory finding giving reasonable suspicion of a disease or condition that contraindicates the use of the ICI treatment, or that may affect the interpretation of the results or render the patient at high risk from complications.

• Patient has history of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanised antibodies or fusion proteins or known allergy to the study IMP ingredients and/or the proposed ICI therapy.

• Pregnancy or breastfeeding.

• Patient has a history of alcohol or drug abuse within the last year.

• Currently participating in or has participated in a study of an investigational agent within 30 days of baseline or has not recovered from adverse events due to agents administered more than 4 weeks earlier, except A Phase 1a/1b, Multi-Centre, Open-Label, Dose-Escalation and Dose-Expansion Study in Patients with Solid Tumor Malignancies to Evaluate GEH200520 Injection / GEH200521 (18F) Injection Safety and Tolerability, Positron Emission Tomography Imaging, Pharmacokinetics, and Changes in Imaging after Treatment; NCT05629689, EU Trial number: 2024-515218-42-00.

• For any reason, patient is considered by the local investigator to be an unsuitable candidate to participate in this study.