Funding:

Project Acronym: NKBOOST

Project Name: NKBOOST - Quality by design-centered platform for the manufacturing of CAR-engineered NK cells and extracellular vesicles for immunotherapy

Activity years: 2022 - 2024

Funding: Fundação para a Ciência e a Tecnologia, I.P

Topic: Chemical Engineering

Budget: 249.999,99 €

Reference: PTDC/EQU-EQU/3708/2021

Host Institution: Associação do Instituto Superior Técnico para a Investigação e o Desenvolvimento (IST-ID)

Partners: Instituto Superior de Engenharia de Lisboa (ISEL) e STEMLAB, S.A.

Researchers (ISEL):
Professor  Cecília Ribeiro da Cruz Calado - Ciência ID: 9418-E320-3177

Description:

This past decade marked the beginning of an incomparable success story for cancer therapy when chimeric antigen receptors (CAR) were first used to redirect the specificity of Tcells, culminating in two cellular-based Advanced Therapy Medicinal Products (ATMPs) receiving market authorization to treat B-cell malignancies. Contrarily to T cells, natural killer(NK) cells can lyse infected or malignant cells without prior activation or human leukocyte antigen (HLA) restriction. Moreover, NK cells can be isolated from umbilical cord blood(UCB), which represents a very attractive, allogeneic, and off-the-shelf source, capable of providing more than 100 doses/unit of NK cells for immunotherapy. Recently, a Phase I/IIclinical trial using allogeneic UCB-derived CAR-NK (cNK) cells reported rapid responses for most patients, without the development of major side effects, confirming UCB cNK as apowerful alternative to CAR-T cells. In the end of 2019, the observation that nanosized extracellular vesicles (EVs) secreted by CAR-modified cells carry CAR on their surface andexpress a high level of cytotoxic molecules capable of tumor growth inhibition, opened the door to a potential cell-free CAR-based immunotherapy. EVs have a low risk of toxicity andtherefore have the potential to be an off‑the‑shelf therapeutic. Moreover, due to their nanoscale size they can be used for solid tumor therapy. With these promising results it isexpected that the combined and/or alternate use of cNK cells and derived EVs (cNK cells/EVs) will undoubtedly strengthen the use of cNK-based cancer therapy and will rapidlyprogress towards clinical studies, stressing the need for a clearly standardized manufacturing process for these products to meet commercial and clinical demands. In this context,the main goal of NKBoost is to design and develop a scalable quality-by-design (QbD)-centered manufacturing platform for human cNK cells/EVs. Several challenges are expected:(i) identification of the critical quality attributes (CQAs) of cNK cells/EVs and definition of the quality target product profile (QTPP), with the establishment of a standardizedmethodology to measure them; (ii) development of a protocol to efficiently transduce NK cells in large scale as this represents a limiting step for clinical applications; (iii) removal ofnon-transduced NK-cells from the bioreactor in a closed, and automated way, using an innovative continuous flow-sorting technique; (iv) optimization of cNK cell expansionconcomitantly with the production of cNK-EVs in bioreactors (e.g. stirred tank and wave bioreactors); (v) integration of a novel scalable continuous purification process for theparallel isolation and purification of cNK cells and EVs comprising unit operations that are robust, selective and cost-effective; and (vi) implementation of QbD principles throughout the manufacturing process. Process analytical technologies (PAT) along upstream and downstream processes will be used to identified critical process parameters (CPPs) and to studytheir impact in CQAs of cNK cell/EVs and on QTPP. NKBoost combines the expertise of researchers from different groups, that will work in a synergistic way to overcome those sixchallenges. The PI (from IST-ID) has vast experience in the establishment of culture systems for the expansion of different cell types, including hematopoietic stem/progenitor cells.More recently, in collaboration with the co-PI (from ISEL), specialist on bioprocess monitoring, modelling and control, through the joint supervision of master students initiated thestudies of how different culture conditions affected mesenchymal stromal cells (MSC) and derived EVs infrared (IR) spectra profiles (Section 3.4, reference 5). The IST-ID team alsoincludes experts on the purification of biological products using scalable and cost-effective methodologies, that collaborate with the PI on the development of a purification processfor MSC-derived EV (Appendix A). NKBoost will also benefit from the expertise in immunology/hematology of Dr. Rita Azevedo and MD Isabel Bogalho as consultants. The innovativemanufacturing platform established herein, integrated with a novel flow-sorting method, is expected to pave a new way for the development of new cell/EV engineering tools,eliminating time- and labour-consuming procedures. Moreover, to ensure product quality and consistency throughout development-to-commercialization, the whole process shouldfollow Good Manufacturing Practices (GMP) guidelines. In that context, NKBoost will benefit from having in the team, the R&D director of Crioestaminal (Stemlab SA, Portugal), withexpertise in the development and quality assurance of ATMP. Overall, it is expected that this platform will support the development of more clinical trials aimed to corroborate thetherapeutic benefits of cNK cells observed in pre-clinical and early trials, paving the way toward its commercialization.