Navigating Regulatory Frameworks For Allogeneic CAR-NK Control Strategies

By Natalia Pripuzova, NPDK Bioconsulting

Allogeneic chimeric antigen receptor natural killer (CAR-NK) cell therapies are gaining rapid momentum as off-the-shelf alternatives to autologous CAR T cell therapies. These platforms combine the innate cytotoxicity of NK cells with engineered specificity, offering promise for scalable manufacturing and reduced risk of graft-versus-host disease.

However, the regulatory landscape for allogeneic CAR-NK products is complex, given the diverse mechanisms of action, source variability (e.g., iPSC-derived NKs), and genome editing components. To navigate these intricacies, regulatory professionals must strategically interpret existing FDA guidance documents on gene therapy, genome editing, potency, and overall CMC practices for IND and BLA.

This article summarizes key considerations across core guidance documents, offering a harmonized view of regulatory expectations for CAR-NK development at clinical stage.

The CMC Development Regulatory Landscape Is Complex And Changing

The expanding clinical demand for allogeneic cell therapies has prompted FDA to release several updated guidance documents addressing specific manufacturing and control strategies. Products, such as iPSC-derived CAR-NK cells, present additional complexity due to genome modifications using novel gene editing technologies, the necessity of multi-tier cell banking, and proprietary multi-component and stage-dependent bioprocesses.

FDA’s Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products guidance on development of CAR T cell products¹ and Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)² lay foundational expectations around manufacturing processes, control strategies, and overall IND requirements for both autologous and allogeneic cell therapies. FDA’s CAR T guidance¹ also touches on managing manufacturing changes and assessing comparability during the product life cycle and explains how the product CQAs will drive recommendations for the lot release testing. More recently developed guidance on Potency Assurance for Cellular and Gene Therapy Products³ focuses on several aspects, including:

• Risk of clinical hold in case of no adequate assurance of product potency at Phase 2 or 3 IND, which is intended to provide substantial evidence of effectiveness for a future BLA
• Establishment of a quality target product profile (QTPP), including potency-related characteristics of the product, based on understanding the product’s MOA
• Timely product characterization studies to drive the better understanding of the product’s MOA and progressive Implementation of a potency assurance strategy
• Matrixed strategies and multi-attribute methods
• Assay control and change management
• Importance of in-use studies and delivery device compatibility studies

Finally, the Human Gene Therapy Products Incorporating Human Genome Editing guidance⁴ discusses:

• release to include on-target editing efficiency and %/number of edited cells,
• assessing genomic integrity, off-target frequency, intra- and inter-chromosomal rearrangements,
• potential oncogenicity and insertional mutagenesis,
• editing modality and delivery choices must be justified,
• control for residual GE components,
• treating GE reagents as critical components of manufacture
• raw materials qualification across the supply chain, and
• looking for clonal expansion/unregulated proliferation of edited cells.

Considering the complexity of the manufacturing bioprocess of CAR-NK cells in particular, the new draft guidance on Considerations for the Use of Human- and Animal- Derived Materials and Components in the Manufacture of Cell and Gene Therapy and Tissue-Engineered Medical Products⁵ aligns the expectations on the control of human-derived feeder cells (or cell-derived particles) that are used for CAR-NK maturation and expansion stages.

The most recently released updated Recommendations for Determining Eligibility of Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products⁶ keeps the regulatory requirements and cross-references the disease-specific guidances for current screening/testing recommendations by agents (HIV/HBV/HCV/HTLV, CT/NG). That makes it easier to adopt updates per pathogen without reopening the umbrella.

FDA’s Federal Register notice announced the draft and the plan to issue the suite of disease-specific guidances, which is useful for understanding the transition timeline. However, the overall regulatory requirements in 21 CFR Part 1271 Subpart C are unchanged.

Together, these documents form the evolving framework within which developers must operate. Moreover, the CBER Guidance Agenda in 2025 includes:

• Recommendations for Validation and Implementation of Alternative Microbial Methods for Testing of Biologics, Draft Guidance for Industry
• Use of Platform Technologies in Human Gene Therapy Products Incorporating Human Genome Editing; Draft Guidance for Industry

Major Control Points And Methods For Implementation

To align with evolving FDA expectations, CAR-NK developers must adopt a proprietary control strategy that integrates product- and process-specific elements. Developers should clearly define their manufacturing platforms and genome editing approaches. Allogeneic CAR NKs, particularly iPSC-derived products, require stringent control over:

• donor cell characterization,⁶
• cell banking, safety testing, and genetic characterization,⁴
• use of fully controlled genome editing tools,⁴
• characterization of on- and off-target activity, integration profiles, and vector clearance,⁴
• control of raw materials used in bioprocess,⁵
• use of specific platforms and equipment, and
• potential bridging and comparability of all of the above when transitioning from early preclinical to clinical and then to commercial stage.⁷

Furthermore, iPSC-derived products must be accompanied by characterization of genome integrity, karyotyping, and evaluation of tumorigenic potential. FDA emphasizes the need to monitor for residual undifferentiated iPSCs to prevent teratoma formation. Integration site analysis and transgene copy number determination are also essential, especially for integrating vectors or knock-in strategies.

Potency remains a central focus of the control strategy. Potency assurance draft guidance³ encourages a matrixed approach using multiple analytical readouts that may include surrogate markers. For CAR-NKs, developers may implement assays measuring:

• cytotoxicity,
• target engagement,
• transgene expression, and
• downstream signaling.

Multiparameter flow cytometry and reporter assays can support potency matrices. In-process controls, lot release criteria, and orthogonal characterization methods must reflect the CQA defined during product characterization.

Conclusion

The regulatory landscape for allogeneic CAR-NK products continues to evolve, demanding proactive CMC strategy integration across guidance domains. Sponsors should anticipate regulatory expectations early by adopting modular, matrixed control strategies that incorporate genomic, functional, and safety testing. Leveraging FDA guidance documents collectively — rather than in isolation — will be key to achieving robust IND submissions and eventual licensure. As allogeneic therapies transition from innovation to commercialization, harmonized control frameworks will support both regulatory compliance and product consistency.

References:

U.S. Department of Health and Human Services (HHS), Food and Drug Administration (FDA) guidance documents

1. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products – 2024 (Final)
2. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy INDs - 2020 (Final)
3. Potency Assurance for Cellular and Gene Therapy Products - 2023 (Draft)
4. Human Gene Therapy Products Incorporating Human Genome Editing - January 2024
5. Use of Human- and Animal- Derived Materials and Components in the Manufacture (2024)
6. Recommendations for Determining Eligibility of Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps), 2025 (draft)
7. Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products, 2023

About The Author:

Natalia Pripuzova has a Ph.D. in virology. She completed postdoctoral training at the National Cancer Institute (NIH, Bethesda, Maryland) and at the U.S. FDA’s Office of Cellular, Tissue and Gene Therapies, CBER. She is the author of over 15 scientific publications. Since 2015, she had focused on regulatory CMC science, serving as product quality reviewer at the Office of Pharmaceutical Quality, FDA, CDER. In 2017, she transitioned to industry and held positions of director and head of regulatory CMC in U.S.-based and international biotech companies, promoting the development of mAbs, proteins, ADCs, and, more recently, allogeneic CAR-NK cell therapies. Natalia is currently the CEO of NPDK Bioconsulting, a regulatory CMC and patent consulting firm, aiming to promote the development and commercialization of various biotechnology and pharmaceutical products. Reach her at natalia.pripuzova@npdkbio.com or on LinkedIn. Learn more about NPDK Bioconsulting at www.npdkbio.com.