The project focuses on the development, validation, and clinical implementation of aHyC immunohybrid therapy for the treatment of triple-negative breast cancer (TNBC). It brings together Slovenian and Italian partners to establish a GMP-compliant production process, prepare the required regulatory and ethical documentation, and enable cross-border patient inclusion in a clinical study. The project officially began with a virtual kick-off meeting on 7 September 2023, where partners reviewed the project objectives, work package activities, administrative obligations, implementation timeline, and expected results. A Steering Committee was also established, with representatives of all project partners, to support effective coordination between the project partners and the lead partner.

Development of the aHyC Preparation Process
A central part of the project has been the adaptation of aHyC preparation protocols for the TNBC indication. During the first phase of implementation, partners ZTM, VBC, and Celica aligned the protocol for dendritic cell production using the NANT-XL bioreactor and defined the specifications of the leukapheresis product required for monocyte isolation. These activities were reviewed during the first periodic meeting, held in person on 26 January 2024 at Celica’s premises in Ljubljana. At this stage, the aHyC preparation protocols were adapted for TNBC, and a draft clinical trial protocol for treating TNBC patients with aHyC was prepared. The protocol later entered the initial implementation phase and is being further adjusted according to tumor cell characteristics. Immunohybridomas are already being prepared in accordance with Good Manufacturing Practice (GMP) requirements, allowing production for patients to begin after clinical and regulatory conditions are fulfilled.

GMP-Compliant Production and NANT Technology Validation
The implementation of NANT technology in a GMP-compliant environment has been one of the main technical pillars of the project. Following the second regular project meeting on 7 August 2024, partners continued work on harmonizing the quality standards and preparation of leukapheresis products. In cooperation with VBC, the implementation of NANT technology in a GMP-compliant environment began. VBC later installed the NANT-XLP bioreactor at Celica’s facilities, upgraded the software, and optimized the dendritic cell isolation protocol. ZTM provided five leukapheresis samples, which were processed both manually and using the NANT-XLP bioreactor for validation purposes. Validation of the NANT-XLP bioreactor was completed through two additional validation cycles. The results confirmed that the quality of cells obtained through the bioreactor is comparable to manually prepared cells. This represents an important step toward standardizing the production process and increasing future production capacity.
The next technical step is a detailed evaluation of differentiation outcomes between the manual and bioreactor-based preparation methods.

Leukapheresis Optimization and Quality Indicators
ZTM optimized the apheresis procedure, including component separation, and carried out a collection from a healthy donor. The leukapheresis products were prepared according to the optimized ZTM protocol and passed the required biological tests for further processing. Several biological parameters were measured, including platelet activation and the proportion of immunosuppressive monocytes - CD14+DR lo/neg. These parameters may serve as quality indicators for future procedures and help support process control in the clinical preparation of the therapeutic product.

Regulatory and Ethical Preparation
Regulatory and ethical preparation has been a major component of the project from the early stages. By the end of the second implementation phase, documentation was prepared for submission to the Public Agency of the Republic of Slovenia for Medicinal Products and Medical Devices - JAZMP and the Commission of the Republic of Slovenia for Medical Ethics. In November 2024, a workshop was organized in cooperation with JAZMP to address the legal and ethical requirements for including patients in a cross-border clinical study. This was especially important because the project involves both Slovenian and Italian partners and aims to support cross-border patient recruitment. Following review by JAZMP and the National Ethics Commission, questions were addressed in the revised clinical study protocol. In the fifth project period, after the periodic meeting on 17 October 2025, the partners finalized the timeline for the remaining activities and submitted a renewed application for clinical study approval to JAZMP and the Commission of the Republic of Slovenia for Medical Ethics. An interim assessment was received, and the identified ambiguities were addressed. The clinical study was approved in February 2026.

Clinical Trial Plan
The approved clinical trial will begin with a Phase 1 study involving 10 patients with TNBC. The main purpose of this phase is to assess the safety profile of the therapy. After Phase 1 is completed, the project team will analyze the collected data. If the safety profile of the therapy is favorable, the study will continue into Phase 2, with an estimated enrollment of 72 patients. A patient information and consent document has already been prepared for patients who meet the inclusion criteria and wish to participate in the study. Following approval in February 2026, the project can move from regulatory preparation toward patient enrollment and clinical implementation.

Cross-Border Patient Inclusion
The project is designed as a cross-border clinical initiative involving partners from Slovenia and Italy. In Italy, PP5-ASUFC and PP4-UNIUD identified suitable hospitals and began their involvement in the study preparation. They also submitted a draft clinical protocol to the Italian regional ethics committee. Italian patients are continuously informed about the possibility of enrolling in the study. ASUFC monitors an annual cohort of more than 50 TNBC patients, providing an important basis for future patient recruitment. During the third project period, LP1-Celica organized a stakeholder meeting at JAZMP headquarters with participation from all project partners. The purpose of the meeting was to define criteria for cross-border patient inclusion. Partners PP5-ASUFC, PP4-UNIUD, and VBC are also developing a proposal for a cross-regional patient recruitment platform. The platform will include information on requirements and obligations for both patients and hospitals and will support coordinated recruitment across regions.

Project Timeline and Key Milestones
he project started on 1 September 2023, while the virtual kick-off meeting took place on 7 September 2023. Partners reviewed the project content, planned activities, administrative obligations, work package structure, and timeline for achieving results.
The first periodic meeting was held on 26 January 2024 at Celica in Ljubljana. Partners aligned the dendritic cell production protocol, defined leukapheresis product specifications, and adapted aHyC preparation protocols for TNBC.
A related interview was also published in local media:
https://sis-egiz.eu/2024/01/30/preboji-so-na-pragu/

The second regular meeting took place on 7 August 2024 and focused on key project milestones. Partners worked on harmonizing leukapheresis quality standards, implementing NANT technology in a GMP-compliant environment, optimizing apheresis procedures, and measuring biological quality indicators. A workshop was held in cooperation with JAZMP in November 2024 to address legal and ethical requirements for including patients in a cross-border clinical study.

The third periodic meeting was held remotely in February 2025. Partners reviewed the status of the aHyC preparation protocol, NANT-XLP implementation, leukapheresis sample processing, and validation of the bioreactor-based method. Selected project content was presented to the broader public in the RTV SLO 1 science programme Ugriznimo znanost, in the episode titled “Human Cell”, aired on 8 May 2025:
https://365.rtvslo.si//arhiv/ugriznimo-znanost/175130194

The fourth periodic partner meeting was held virtually in July 2025. Partners reviewed progress and agreed on next steps. The project duration was extended until the end of 2025.

The fifth periodic meeting was held on 17 October 2025. Partners finalized the timeline for remaining activities and submitted a renewed clinical study application to JAZMP and the Commission of the Republic of Slovenia for Medical Ethics.

A final project meeting was held on 5 December 2025 at the Institute of Oncology in Ljubljana. Project partners presented the activities carried out and the achievements reached during the project period.
In December 2025, the project team also organized an online press conference to present the project and its results to journalists and the general public.
The clinical study was approved in February 2026, enabling the project to proceed toward patient enrollment and implementation of the Phase 1 TNBC clinical trial.