What are the different types of drugs available for CAR-NK?

Introduction to CAR-NK Therapy

Definition and Basic Concepts
Chimeric Antigen Receptor‐Natural Killer (CAR‐NK) therapy is a form of cellular immunotherapy that relies on genetically engineered natural killer (NK) cells to recognize and kill cancer cells. In this approach, NK cells—innate lymphocytes that are capable of rapid cytotoxic responses—are modified to express synthetic receptors called chimeric antigen receptors (CARs). These receptors typically comprise an extracellular antigen‐binding domain (usually a single‐chain variable fragment, or scFv) that recognizes specific tumor‐associated antigens, a hinge region, a transmembrane domain, and intracellular signaling domains that drive cell activation and subsequent cytotoxic functions. Unlike CAR‐T cell therapies, CAR‐NK products offer the benefit of intrinsic safety properties, such as minimal risk of graft‐versus‐host disease (GVHD), because NK cells do not require strict HLA matching. In addition, their innate ability to sense stress ligands through receptors like NKG2D combined with the added specificity of a CAR construct makes them an attractive alternative targeting tool in cancer treatment.

Overview of CAR-NK in Cancer Treatment
CAR-NK therapies are emerging as promising candidates to treat both hematological malignancies, including B-cell leukemias and lymphomas, as well as several solid tumors like glioblastoma, breast, ovarian, and pancreatic cancers. Their mechanism of action integrates both CAR‐mediated targeting and natural cytotoxicity pathways. Upon engagement with specific tumor antigens, CAR‐NK cells release cytotoxic granules containing perforin and granzymes, induce apoptosis through death receptor ligands (e.g., Fas ligand, TRAIL), and can also mediate antibody‐dependent cellular cytotoxicity (ADCC) via receptors such as CD16. With ongoing early-phase clinical trials demonstrating promising safety profiles—including reduced incidences of cytokine release syndrome (CRS) and neurotoxicity—CAR-NK therapies are gradually gaining acceptance in the broader oncology community. Moreover, these cell therapies can be engineered to secrete supportive cytokines such as IL-15 to improve persistence and function after infusion.

Types of Drugs in CAR-NK Therapy

Classification of Drugs
The “drugs” in CAR-NK therapy are primarily living cell products developed using advanced bioengineering techniques. They can be classified from multiple perspectives, including the origin of the NK cells, the design of their CAR constructs, and additional molecular modifications made to improve efficacy and safety. Several key classifications include:

Based on Cell Source
NK Cell Lines:
The NK-92 cell line is a commonly used model for CAR-NK studies due to its ease of expansion and genetic manipulation. However, to ensure safety, especially regarding their proliferation in patients, these cells are often irradiated prior to infusion to prevent long-term engraftment.
Peripheral Blood NK (PB-NK) Cells:
These are mature NK cells isolated from donor blood. They provide a ready source of highly cytotoxic cells, yet their expansion and transduction can be challenging due to heterogeneity and limited life-span post-thaw.
Umbilical Cord Blood (UCB)-Derived NK Cells:
UCB-derived NK cells offer a more “off-the-shelf” approach, with advantages such as an immature phenotype that gives them a higher tolerance to genetic manipulation and potential for large-scale expansion while exhibiting sufficient cytotoxicity once activated.
Induced Pluripotent Stem Cell (iPSC)-Derived NK Cells:
iPSC-derived NK cells provide an unlimited and uniform source for therapy. Their derivation and expansion processes are more controlled, which can contribute to improved consistency in the final product, though the manufacturing process is relatively lengthy and requires specialized expertise.

Based on CAR Construct Generations and Formats
First-Generation CAR Constructs:
These usually incorporate only the basic CD3ζ activation domain. While they provide some level of activation, their overall efficacy may be lower compared to later-generation constructs.
Second-Generation CAR Constructs:
These include an additional co-stimulatory domain such as CD28 or 4-1BB (CD137) alongside CD3ζ. These modifications can promote enhanced activation, persistence, cytokine production, and proliferation of the NK cells.
Third-Generation CAR Constructs:
They combine multiple co-stimulatory domains in tandem (e.g., CD28 and 4-1BB) alongside the CD3ζ activation domain. This design further augments cytotoxicity and in vivo persistence, which is particularly beneficial for tackling solid tumors.
"Armored" CAR-NK Cells:
These are advanced constructs engineered to secrete additional cytokines (commonly IL-15) or to express other molecules such as safety switches (e.g., inducible caspase-9) to mitigate toxicity. The armored design also includes modifications to overcome the inhibitory effects imposed by the tumor microenvironment.
Dual-Targeting or Multi-specific CAR Constructs:
Some designs incorporate dual antigen recognition domains or use logic gate configurations (AND/OR gates) to improve specificity and reduce the risk of antigen escape, enhancing tumor targeting without harming normal tissues.

Based on Additional Genetic Modifications and Safety Features
Safety Switches:
Incorporation of suicide genes such as inducible caspase-9 (iC9) allows clinicians to rapidly ablate CAR-NK cells in the event of severe adverse effects.
Cytokine Gene Inclusion:
Some CAR-NK therapies are further modified to secrete supportive cytokines (e.g., IL-15) to augment persistence, proliferation, and overall anti-tumor activity after infusion.
Engineering for Enhanced ADCC:
Certain products are engineered to express high-affinity, non-cleavable forms of CD16 to potentiate ADCC in combination with therapeutic monoclonal antibodies.

Based on Manufacturing Platforms and Delivery Strategies
Viral Vector-Based Gene Transfer:
Lentiviral and retroviral vectors are traditionally used to introduce CAR constructs into NK cells. Such approaches, while efficient, pose concerns related to insertional mutagenesis and high production costs.
Non-Viral Gene Transfer Methods:
Recent innovations include mRNA electroporation and lipid nanoparticle (LNP)-mediated delivery systems that offer safer and more transient expression profiles, reducing the long-term risks and potential for genotoxicity.
Ex Vivo Expansion and Priming:
Some CAR-NK therapies are co-cultured with feeder cell lines or cytokine cocktails to achieve massive expansion (thousands-fold) while also priming the NK cells for enhanced cytotoxicity. This process, although not a “drug” per se, critically defines the quality and efficacy of the final therapeutic product.

Mechanisms of Action
The various drug types in the CAR-NK landscape function through a combination of direct and indirect immunologic mechanisms:

CAR-Mediated Specific Cytotoxicity:
The engineered CAR on NK cells enables direct recognition of target antigens on tumor cells. This binding triggers intracellular signaling cascades (via CD3ζ and co-stimulatory domains such as CD28 and 4-1BB) that stimulate the NK cells to release cytotoxic granules containing perforin and granzymes, which induce apoptosis in the tumor target.

Natural Cytotoxicity Push:
Even when tumor cells downregulate the specific target antigen (an issue known as antigen escape), NK cells retain their native activating receptors (e.g., NKG2D, NKp30, NKp44, NKp46, and DNAM-1) that can recognize stress ligands on tumor cells. This dual mechanism enhances overall tumor cell killing.

ADCC (Antibody-Dependent Cellular Cytotoxicity):
Some CAR-NK products are engineered to express high-affinity forms of the CD16 receptor. This receptor engages the Fc region of therapeutic antibodies, thereby bridging innate immune mechanisms with antibody-mediated therapies. When these NK cells interact with antibody-coated tumor cells, they execute ADCC, further enhancing anti-tumor effects.

Cytokine Support and Modulation:
Armored CAR-NK cells engineered to secrete cytokines like IL-15 not only support their own survival and proliferation but also modulate the tumor microenvironment (TME) to be less immunosuppressive, thus potentiating the overall anti-tumor response.

Safety Mechanisms:
The incorporation of inducible safety switches (such as inducible caspase-9) allows for the rapid elimination of CAR-NK cells when severe adverse events occur. This mechanism adds an extra layer of control and safety to the therapy, ensuring that cells can be ablated if necessary.

Current Development and Availability

Approved Drugs and Clinical Trials
Although CAR-T therapies have already received FDA approval, the field of CAR-NK therapy is still evolving with several early-phase clinical trials underway. Multiple clinical trials provide evidence of the safety and efficacy of CAR-NK therapies, particularly in hematological malignancies and increasingly in solid tumors. For example:

Anti-CD19 CAR-NK Cells:
Clinical studies, such as the multi-center trial referenced with clinical trial identifier NCT03056339, have demonstrated promising response rates and safety profiles when using anti-CD19 CAR-NK cells for relapsed/refractory B-cell malignancies. Notably, these trials report significant clinical responses without the severe toxicities (e.g., CRS, neurotoxicity, or GVHD) often associated with CAR-T therapies.

Other Targeted CAR-NK Products:
Additional candidates under clinical investigation include “armored” CAR-NK products that are engineered to secrete IL-15, dual-targeting CAR constructs that combine CD19 and additional synergistic signaling domains (such as NKG2D–2B4–CD3ζ), and products incorporating high-affinity CD16 for enhanced ADCC.

Advanced Design Pioneers:
Early-phase trials have highlighted candidates designed using both viral vector systems and more novel non-viral transfection methods like mRNA-LNP platforms. These strategies aim to provide an “off-the-shelf” product that can be rapidly administered and offer better therapeutic control.

While no CAR-NK drug has yet reached full regulatory approval at the scale of CAR-T therapies, the extensive pipeline of clinical trials and preclinical studies suggests that these products are approaching maturity in their development. The collective efforts in refining CAR structure, enhancing cell persistence, and ensuring safety indicate significant potential for future approval and widespread use.

Leading Pharmaceutical Companies
A variety of organizations, including biotechnology companies, academic institutions, and research consortia, are actively involved in the development of CAR-NK cellular products. Some of the key players include:

ImmunityBio, Inc. and Artiva Biotherapeutics, Inc.:
These companies have made notable advancements in designing NK cell–based therapies, with several collaborations aiming to combine CAR-NK cells with other treatment modalities such as monoclonal antibodies.

NKGen Biotech, Inc.:
NKGen is engaged in developing CAR-NK cell platforms, including combination therapies that integrate CAR modifications with enhanced ADCC properties through high-affinity Fc receptor engineering.

Affimed GmbH and Related Entities:
Affimed and its collaborations have contributed to the development of CAR-NK strategies, particularly in partnering with other biopharmaceutical companies to enhance combination therapies that target solid tumors and hematological malignancies.

Academic Institutions:
Major cancer centers such as The University of Texas MD Anderson Cancer Center are prominent in conducting early-phase clinical trials that underline the feasibility and safety of CAR-NK therapies. These institutions often collaborate with biotechnology companies to develop and test next-generation CAR-NK designs.

Furthermore, numerous patents filed by organizations highlight the intellectual property and innovative efforts in optimizing CAR-NK production and therapeutic applications. These collaborative research and development efforts underscore the industry’s commitment to bringing CAR-NK therapies into clinical practice.

Challenges and Future Directions

Current Challenges in Drug Development
While the promise of CAR-NK therapy is substantial, several challenges must be addressed to optimize their clinical performance and ensure their safe, widespread use:

Manufacturing and Expansion:
NK cell products are inherently sensitive to ex vivo manipulations. Their expansion, genetic modification, and effective transduction remain challenging. For instance, primary NK cells (either from PB or UCB) require sophisticated culture conditions and feeder-cell systems to achieve sufficient yields, and NK cells are more sensitive to freezing and thawing processes compared with T cells. Furthermore, variability in donor cell quality and the heterogeneity of NK cell populations can affect the consistency of the final product.

Gene Transfer Efficiency:
Efficient methods for transducing NK cells with CAR constructs are paramount, but NK cells are notoriously difficult to transfect, which has led to the exploration of both viral (lentiviral, retroviral) and non-viral (mRNA electroporation, lipid nanoparticle-mediated) gene delivery platforms. While viral vector methods offer higher transduction efficiencies, concerns regarding insertional mutagenesis and cost persist. Non-viral methods, though safer, currently suffer from lower gene transfer efficiencies.

In Vivo Persistence and Expansion:
Another considerable challenge is the relatively short persistence of CAR-NK cells once infused into the patient. Unlike CAR-T cells, NK cells have a limited in vivo lifespan, which might necessitate multiple infusions to maintain an effective anti-tumor response. Although engineering strategies such as cytokine gene incorporation (IL-15) and “armoring” techniques can extend their persistence, optimizing these modifications while preserving safety remains complex.

Tumor Microenvironment (TME) and Antigen Escape:
The immunosuppressive nature of the TME in solid tumors poses a significant hurdle. Inhibitory cytokines like TGF-β and suppressive cells (e.g., MDSCs, Tregs) can impair NK cell function. Additionally, tumor cells may decrease or change the expression of the target antigen (antigen escape), making them less susceptible to CAR-mediated recognition. Dual-targeting designs and combination therapies might mitigate these challenges, but they add layers of complexity to the drug development process.

Safety Concerns:
Although CAR-NK cells are associated with a lower incidence of severe toxicities such as CRS and neurotoxicity compared with CAR-T cells, ensuring absolute safety remains crucial. The incorporation of suicide genes (e.g., inducible caspase-9) is a promising approach, yet fine-tuning these systems to function reliably under diverse patient conditions is an ongoing challenge.

Future Prospects and Innovations
Looking forward, numerous innovations and future directions aim to address the current challenges and further optimize CAR-NK therapies:

Next-Generation CAR Designs:
Future CAR constructs may incorporate multiple co-stimulatory domains, dual-targeting capacities, and additional signaling motifs that are uniquely tailored for NK cell signaling. Innovations such as logic-gated CARs (AND/OR gates) offer enhanced specificity and reduce off-target toxicities by ensuring that activation occurs only when multiple tumor antigens are present. Moreover, “armored” CAR-NK cells that secrete cytokines such as IL-15 and incorporate inhibitory receptor deletions (e.g., targeting PD-1, TIGIT) offer promise for overcoming an immunosuppressive TME.

Enhanced Gene Transfer Methods:
Refining non-viral techniques—such as the utilization of mRNA-LNP technologies that have proven effective for COVID-19 vaccines—may revolutionize the efficiency and safety of CAR gene delivery into NK cells. These methods have the potential to provide faster, more cost-effective production without the risks associated with viral vectors.

Optimization of Expansion and Manufacturing Protocols:
Advances in NK cell expansion protocols, including optimized cytokine cocktails and feeder cell systems, can significantly increase the yield and functionality of CAR-NK products. Standardizing these protocols will be key to reducing inter-donor variability and ensuring reproducibility at clinical scale.

Combination Therapies:
Future treatments may combine CAR-NK cells with other immunomodulatory agents such as immune checkpoint inhibitors or therapeutic antibodies to enhance their efficacy. For example, CAR-NK cells engineered with high-affinity CD16 variants can effectively mediate ADCC in synergy with monoclonal antibodies, offering a multi-faceted attack on tumors. Additionally, multi-component strategies that target both the immunosuppressive TME and the tumor cells directly are under active development.

Personalized and “Off-the-Shelf” Approaches:
Thanks to the stem cell–derived NK cells and standardized cell lines (e.g., NK-92), it is becoming increasingly feasible to generate allogeneic, off-the-shelf CAR-NK products. This scalability could reduce treatment costs and make therapies more accessible, particularly if robust manufacturing protocols and regulatory pathways are established.

Advanced Safety Mechanisms:
The inclusion of built-in safety switches, such as inducible caspase-9, and suicide gene systems allows precise control over the infused cells in the event of adverse reactions. Future innovations may further refine these systems to ensure rapid, controllable ablation of the cells if necessary, paving the way for broader clinical acceptance.

Emerging Biomarkers and Real-Time Monitoring:
To ensure optimal therapeutic outcomes, the development of biomarkers to monitor CAR-NK cell expansion, persistence, and functional status in patients is of paramount importance. Innovations in imaging and molecular monitoring can provide real-time feedback on cellular responses, allowing for timely interventions and personalized dosing strategies.

Conclusion
In summary, the different types of drugs available for CAR-NK therapy are best understood as a diverse class of engineered cellular products characterized by variations in cell source, CAR construct design, and additional genetic modifications that enhance their function and safety. Starting from NK cell lines, peripheral blood, or umbilical cord blood to iPSC-derived NK cells, each source offers distinctive advantages and challenges in terms of expansion, transduction efficiency, and clinical efficacy. CAR constructs used in these therapies have evolved from first-generation formats, featuring a basic CD3ζ domain, to more advanced second- and third-generation designs that include co-stimulatory domains such as CD28, 4-1BB, and even innovative “armored” designs that secrete cytokines like IL-15 or incorporate safety switches.

Mechanistically, these drugs enable NK cells to target tumor cells through both CAR-mediated recognition—which triggers cytotoxic granule release and apoptosis—and natural innate killing via receptors like NKG2D and CD16. They also employ additional mechanisms such as ADCC and are engineered to overcome the immunosuppressive effects of the tumor microenvironment. Current clinical development is robust, with numerous early-phase trials (e.g., anti-CD19 CAR-NK cell trials) demonstrating safety and promising efficacy profiles in both hematological cancers and solid tumors. Leading pharmaceutical companies and academic institutions, including ImmunityBio, Artiva Biotherapeutics, NKGen Biotech, and The University of Texas MD Anderson Cancer Center, are at the forefront of advancing these innovative therapies.

Nonetheless, challenges remain related to manufacturing, efficient gene transfer, in vivo persistence, and overcoming the tumor microenvironment. Future directions focus on next-generation CAR designs, improved non-viral transfection methods, sophisticated expansion protocols, combination therapies with checkpoint inhibitors or monoclonal antibodies, and enhanced safety features to tailor an effective and robust off-the-shelf product.

Overall, CAR-NK cell therapy represents a paradigm shift in cancer immunotherapy—a move toward safer, off-the-shelf, and multifaceted anti-cancer drugs that leverage both engineered specificity and innate immune functionality. The integration of advanced genetic engineering techniques and manufacturing innovations is poised to broaden the therapeutic horizon for patients with hard-to-treat cancers, offering a promising complement or alternative to current CAR-T therapies. As clinical trials progress and translational research continues to innovate, CAR-NK drugs are likely to become a cornerstone in the next generation of immuno-oncology treatments, combining general immune activation with specific and potent anti-tumor activity while maintaining an excellent safety profile.