Quality standards for NK cell immunotherapies

Valentin von WerzOliver Spadiut^*Aleksander SzarzynskiSara Zigon-Branc

• Technische Universitat Wien, Vienna, Austria

Natural killer (NK) cells are innate lymphocytes capable of directly targeting and killing tumor cells without prior sensitization 1 . Due to their inherent cytotoxicity, NK cells have emerged as a very promising modality for cancer immunotherapy, offering outstanding advantages over chimeric antigen receptor-T (CAR-T) cell therapies, such as reduced risks of the cytokine release syndrome and neurotoxicity [1][2][3] . CAR-engineered NK cells further enhance therapeutic potential by improving target specificity and cytotoxic efficacy, thereby complementing and extending the clinical success of current CAR-T cell therapies 1,4 . However, the lack of formally defined critical quality attributes (CQAs) currently limits cross-trial and cross-study comparability, compromises safety evaluation, and impedes clinical efficacy optimization for NK cell-based therapies.CQAs play a pivotal role in biopharmaceutical development as they describe the required product quality, safety and efficacy 5 . According to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline Q8(R2), a CQA is a "physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality" 6 . Despite their central importance, CQAs have not yet been formally defined for NK cell therapies yet. The current FDA guidance only roughly defines parameters for quality testing, recommending that CQAs should minimally consist of identity, quality, purity, and potency 7,8 . Regulatory agencies currently refrain from defining generalized acceptance criteria or fixed limits, as these must be adapted to each manufacturing platform, therapeutic indication, and target patient group. While clearly defined CQAs with specified ranges would bring obvious benefits, overly rigid or premature standardization could stifle innovation, particularly for next-generation therapies tailored to specific indications or specific patient populations. In contrast, the current high variance in trial release criteria is complicating product comparison and poses challenges for a consistent regulatory evaluation. We are convinced that a harmonized effort for defining CQAs -anchored in a Quality by Design (QbD) framework and aligned with ICH and regulatory guidelines -is required and essential for ensuring the consistency, safety, and efficacy of NK cell therapies.To potentially initiate this harmonization effort, we systematically analyzed the reported release criteria from all completed Phase 1 and 2 clinical trials of NK cell therapies targeting cancer, which were conducted in the US and Europe with reported release criteria 9-14 (Table 1).In all the clinical trials the accepted range for NK cells was uniformly set at 46 highlighting its importance as a benchmark linked to clinical performance. Purity was defined 47 by the of other cell types in the final NK cell cell were 48 identified by surface NK cells (CD56+) , T cells (CD3+), B cells (CD19+, 49 CD20+), (CD14+), and NK-like T cells (CD16+CD3+). Activation (NKG2D, 50 NKp44, NKp46) and inhibitory receptors (NKG2A) were reported a variable extent, 51 and NK cell purity criteria of the final product ranged from ≥30% to ≥90% 9-12, 14 . T and B cell 52 contaminants were strictly limited between ≤0.2% to <3%