Evaluation of Intratumoral Influenza Vaccine and Systemic Pembrolizumab in Patients with Early pMMR Colorectal Cancer

The primary objective of this study is to evaluate the efficacy of combining an **intratumoral flu vaccine** with systemic **pembrolizumab** in inducing tumor regression in patients with early pMMR colorectal cancer. This investigation is clinically relevant as it explores a novel therapeutic approach that could enhance the immune response against colorectal cancer, potentially leading to improved patient outcomes.

Secondary objectives include the assessment of specific safety events during the trial. Additionally, the study aims to characterize the cancer-killing immune cells using various methods and to analyze the functional changes within the tumor microenvironment. These secondary objectives are crucial for understanding the broader implications of the treatment on the immune system and its safety profile.

The clinical trial involves participants diagnosed with **colorectal cancer**, specifically those with histologically confirmed localized pMMR stage cT1N0M0 to cT4N2M0 (stage I to III) colorectal carcinoma. The study population includes both male and female subjects aged 18 years and older. Participants are required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, indicating they are fully active or restricted in physically strenuous activity but ambulatory. The trial does not include a vulnerable population. Participants must have adequate bone marrow, kidney, and liver function, as defined by specific laboratory criteria. Lifestyle considerations include the requirement for participants and their partners to use acceptable methods of contraception during the study and for a specified period after receiving pembrolizumab. The sponsor has not provided information regarding the total number of participants in the trial.

The clinical trial is designed to evaluate the efficacy and safety of combining a **flu vaccine** with **pembrolizumab** in patients with early-stage pMMR colorectal cancer. This is a Phase 4, randomized, double-blind, controlled trial. The trial is expected to last approximately five years, with an estimated end date in May 2028. Participants will be involved in the study from the screening visit until the end-of-study visit, with the total duration of individual participation varying based on the treatment period and follow-up requirements.

The sequence of study visits begins with an inclusion (screening) visit, where eligibility criteria are assessed, including histological confirmation of localized pMMR colorectal carcinoma and adequate organ function. Following successful screening, participants will be randomized to receive either the investigational combination treatment or a control. The treatment phase involves the administration of the flu vaccine via intralesional use and pembrolizumab via intravenous administration. Follow-up visits will be scheduled to monitor safety outcomes, tumor response, and any adverse events. The primary endpoint is the achievement of a major pathological response in 50% or more of the patients, defined as less than 10% viable tumor cells. Secondary endpoints include safety assessments and detailed immunological analyses of tumor and blood samples.

The expected length of participant involvement is determined by the treatment period, which is up to three months, followed by a post-treatment follow-up. Conditions that may lead to early termination from the study include the occurrence of significant adverse events, withdrawal of consent, or non-compliance with study procedures. Participants are required to adhere to specific conditions regarding fertility, pregnancy, and lactation, and must use acceptable contraception methods during and after the study as specified in the protocol.

The clinical trial involves the administration of **Influvactetra**, a suspension for injection, which is utilized as an experimental treatment. This vaccine contains a combination of influenza virus strains, including B/Phuket/3073/2013-like virus, A/Victoria/2570/2019 (H1N1)pdm09-like strain, Influenza virus A/Darwin/9/2021 (H3N2), and Influenza virus B/Austria/1359417/2021-like strain. The vaccine is administered via **intralesional use** at a dosage of 0.5 mL per administration. The maximum treatment period is one day, with a single dose being administered. The vaccine is produced by Viatris APS and is authorized for use in Denmark under the marketing authorization number 61929.

Another experimental treatment in the trial is **KEYTRUDA** (pembrolizumab), a concentrate for solution for infusion. Pembrolizumab is a monoclonal antibody used in immunotherapy and is administered through **intravenous administration**. The dosage is set at 200 mg per infusion, with a maximum treatment period of three weeks. This treatment is produced by Merck Sharp & Dohme BV and is authorized for use in the European Union under the marketing authorization number EU/1/15/1024/002.

The trial also includes the use of **Influvac Tetra Saison 2024/2025**, another suspension for injection, which serves as a comparator treatment. This vaccine contains strains such as B/Phuket/3073/2013-like virus, Influenza virus B/Austria/1359417/2021-like strain, Influenza A/Victoria/4897/2022 (H1N1), and Influenza A virus/Thailand/8/2022 (H3N2)-like strain. It is administered via **intralesional use** at a dosage of 0.5 mg/L per administration, with a maximum treatment period of one day. This vaccine is produced by Viatris Healthcare GmbH and is authorized for use in Germany under the marketing authorization number PEI.H.11881.01.1.

Efficacy in the clinical trial titled "Combining intratumoral flu vaccine and systemic pembrolizumab in patients with early pMMR colorectal cancer – the FLU-IMMUNE trial" will be assessed using both primary and secondary endpoints. The primary endpoint is defined as achieving a major pathological response (MPR) in 50% or more of the patients. MPR is characterized by less than 10% viable cells, corresponding to Mandard tumor regression grade 1-2 (MTRG).

Secondary endpoints include specific safety outcomes such as tumor site perforation and delay of surgery, as well as changes in recovery assessed by the QoR-15 questionnaire. Additionally, the trial will involve comprehensive analyses of tumor and blood samples. Tumor samples will undergo immunohistochemical analysis of CD3+ and CD8+ T cells, spatial protein expression analyses of immune-infiltrated regions at different time points, flow cytometry, and full transcriptomic analyses including single-cell analysis. Blood samples will also be analyzed using flow cytometry and full transcriptomic analyses, including single-cell analysis.