Manufacturing and clinical applications of non‐ CAR ‐T immune effector cells

The field of Transfusion Medicine (TM) has evolved over the years to include much more than traditional blood banking. In many institutions TM includes apheresis services and other labs, such as coagulation, HLA, and cell therapy. Cell Therapy (CT), along with Regenerative Medicine (RM), is a complex, highly regulated field, and thus having the lab housed in TM within Laboratory Medicine & Pathology makes sense. While CT is much more complex than traditional blood banking, the “blueprint” and lessons learned from blood banking serve as a framework for operations, allowing labs to meet both standards set by professional organizations (e.g., AABB, FACT) and regulations from FDA and other relevant authorities. Technologists trained in blood banking are often recruited to CT labs, as they are accustomed to using aseptic technique while working with bags/tubing sets to manipulate blood components. Finally, physicians trained in TM and CT lab processing are best prepared to be laboratory and medical directors of CT labs, as their training is directly geared toward directorship of a clinical lab. In addition to focusing on hematopoietic progenitor cells (HPCs; i.e., marrow, apheresis products, and umbilical cord blood) in support of blood and marrow transplant programs, many CT labs house a translational research and development section. Often the translational lab will play a role in the development of protocol-specific immune monitoring assays, the preparation of protocol-specific sample and data collection, review and reporting plans. However, the main responsibility of the lab is the development of novel CT and RM products. Most translational labs will follow this general roadmap: (1) evaluation of research and pre-clinical studies, (2) scale-up and optimization, and (3) methods validation. Evaluation of research and pre-clinical studies. At this stage, the research and pre-clinical data is evaluated for feasibility to move forward into the clinical arena. This evaluation includes an assessment from several perspectives, including medical, scientific, technical and regulatory. The results of toxicology and ‘proof of concept’ studies, including studies of cell survival kinetics and distribution, are reviewed. The overall goal is to define the desired characteristics of the cellular product, establish possible methods of cell manufacturing and product testing, and identify appropriate instrumentation and optimal reagents. Often, the reagents and instrumentation used in early studies are typically not suitable for human use and are of smaller scale, respectively. Project management commences with timelines and milestones established, and a pre-IND meeting/call may occur at this stage as well. Scale-up and optimization. Prior to the initiation of the clinical trial, development of a reproducible, large-scale and clinically relevant methodology is required. A technology transfer from the research-based lab to the translational development lab occurs. Frequently, research-based methods are not refined, lacking standard operating procedures (SOPs) and details that would allow smooth transition into current good manufacturing practices (cGMP) manufacturing. One obvious example required for this stage is the determination of culture requirements (e.g., media type, components, vessel selection, media changes and other requirements). Confirmation of seeding density and media depth requirements will guide culture vessel selection. Once the details for culture are determined, SOPs and a batch production record (BPR) can be developed. Methods validation. The validation phase begins when an optimized and clinically appropriate method has been defined. The SOPs and BPR are established, and a validation plan is written and then executed. Typically, a validation consists of three successive runs which must pass pre-established specifications; often most or all of these specifications become the product lot release criteria. The Chemistry, Manufacturing and Controls (CMC) section [i.e., the product manufacturing methods which accompanies the investigational new drug (IND) application] can now be finalized by the translational lab. The CMC and validation summary are provided to FDA in support of the IND application and, in some cases, the Institutional Review Board (IRB) submission. Following acceptance by the FDA, or other relevant authority if outside the USA, this process leads to the cGMP production of the novel therapy in support of a phase I clinical trial. Figure 1 provides an overview of the general manufacturing schema for cellular therapies. Currently in the USA there are 46 licensed cell and gene therapies (https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products; accessed June 10, 2025).1 A growing number of these products are immune effector cells (IECs), now primarily chimeric antigen receptor (CAR)-T cells. While dendritic cells [Provenge (Dendreon) approved in 2010 for prostate cancer] and T cell receptor (TCR) T cell therapies [Tecelra (Adaptimmune) approved in 2024 for synovial sarcoma] are also licensed, here we will focus on a few of the other types of IECs, some of which are represented on the list of licensed products (e.g., Amtagvi, Iovance Biotherapeutics, Inc.), and others that are at various stages of development on the pipeline toward biologics license application (BLA). Natural killer (NK) cells were independently described by Rolf Kiessling and Ronald B. Herberman in the 1970s and subsequently named for their ability to kill malignant and infected cells without prior sensitization.2-4 This marrow-derived special subset of lymphocytes plays a critical role in the innate immune system. Since the discovery and early understanding of NK cells, their biology and function have been shown to be quite complex with their functional fate (i.e., cytotoxicity) being determined by cumulative inputs (i.e., inhibitory and activating) from interaction with their potential target and other cells, as well as the environment. NK cells lack markers for B and T cells, and they are often characterized at a basic level as being CD3 (pan T cell marker)-negative and CD56 (isoform of neural cell adhesion molecule, NCAM)-positive. They account for 5%–15% of circulating lymphocytes and have the morphology of the “large granular lymphocyte”. NK cells are non-phagocytic and are cytotoxic to IgG-coated targets, specializing in antibody-dependent cellular cytotoxicity, or ADCC. They have two approaches to cell killing within the ADCC paradigm—the granule exocytosis pathway (i.e., perforin/granzyme) and the death receptor pathway (i.e., Fas/Fas ligand).5 NK cells are a logical selection for both anti-cancer6 and anti-viral purposes.7 Current approaches to NK cell manufacturing utilize a variety of sources as starting material. The apheresis peripheral blood mononuclear cell (PBMC) product is likely most common.8 However, umbilical cord blood (UCB)9 has been used, and a growing number of alternatives (e.g., induced pluripotent stem cells (iPSCs) and NK 92, a lymphoma cell line) offer some exciting promise.10, 11 Although autologous starting material has been used, most starting material is obtained from allogeneic sources, often haploidentical.6 As interest in establishing off-the-shelf therapies increases, more sources and different approaches to manufacturing are being considered. The earliest experience with NK cells in clinical trials actually involved so-called lymphokine activated killer (LAK) cells.12 Most such approaches involved collection of PBMCs, no further manipulation, and a simple incubation with IL-2. With the availability of immunomagnetic selection devices, the next generation of manufacturing involved depletions and/or enrichments to better purify the NK cells before incubation and activation with IL-2 or Il-15.8 Some groups have focused on depletions (e.g., CD3 and CD19 depletions) to avoid potential negative consequences of accessory cells (i.e., T cell-mediated graft-vs-host disease (GvHD) and B cell-mediated passenger lymphocyte syndrome (PLS) or EBV reactivation/post-transplant lymphoproliferative disease (PTLD)).6, 13 Others have found success employing enrichment strategies by including CD56 selection to highly purify the starting material and then move forward into culture.14 Some protocols include other cytokines or combinations of cytokines (e.g., IL-12, IL-18, and IL-21). 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