Advances and challenges in CAR-NK cell immunotherapy for cancer

Abstract

Chimeric antigen receptor (CAR)-natural killer (NK) cell therapy has emerged as a promising strategy in the treatment of hematologic malignancies and solid cancers. Leveraging the innate immune properties of NK cells, CAR-NK cell therapies offer potential advantages for cell therapy, including safety of use in the allogeneic setting and reduced risk of toxicity. This Nutshell provides an overview of the latest advancements in CAR-NK cell therapy and the challenges that remain.

Graphical Abstract:

Introduction

CAR-T cell therapy has transformed the treatment landscape for hematologic malignancies, with six currently FDA-approved autologous products targeting CD19 or BCMA. Given the considerable time and cost constraints associated with generating autologous cell products, alongside the challenges inherent in engineering allogeneic CAR-T cells, and managing the related toxicity risks, such as cytokine release syndrome (CRS) and neurotoxicity, there is a growing interest in alternative “off-the-shelf” allogeneic therapies. Among these, NK cells represent a particularly promising allogeneic approach for CAR engineering. Leveraging their innate germline-encoded receptors and antibody-dependent cellular cytotoxicity (ADCC); NK cells can effectively target and kill cancer cells without requiring prior priming (reviewed in¹). This potentially enables them to overcome antigen escape mechanisms while minimizing the risk of graft-versus-host disease in allogeneic settings. Allogeneic CAR-NK cells have shown promise in clinical trials, demonstrating feasibility, safety, and efficacy (reviewed in²). Historically, NK cells have been classified based on surface expression of CD56 and CD16 into two primary subsets: CD56^brightCD16⁻, which are less cytotoxic but have a high capacity to produce cytokines and chemokines, and CD56^dimCD16⁺, which are highly cytotoxic with potent ADCC while also producing cytokines. However, recent advances in multi-omic profiling have deepened our understanding of NK cells by uncovering the complex states of their development, function, and heterogeneity in both healthy and disease states, highlighting the variations in their genomic, transcriptomic, proteomic, and epigenomic signatures.^3,4 This, along with advancements in genetic engineering, has provided new avenues to enhance CAR-NK cell therapy.

Sources of NK cells for CAR-NK cell therapy

Sources of NK cells for CAR-NK cell engineering include peripheral blood (PB), umbilical cord blood (UCB), NK cell lines, hematopoietic CD34+ stem cells and induced pluripotent stem cells (iPSCs). While PB from healthy donors offers a supply of mature, cytotoxic NK cells, the heterogeneity of PB-NK cells—affected by factors such as age and health—introduces significant inter-donor variability. Since NK cells comprise around 10% of peripheral lymphocytes, scalability for clinical applications is challenging. Progress has been made in ex vivo NK cell expansion using feeder cells. In addition, various studies have explored cytokine combinations to enhance NK cell potency and persistence, most notably the combination of IL-12, IL-15, and IL-18 to induce a memory-like phenotype.⁵ UCB is an attractive NK cell source, which makes up roughly 30% of UCB lymphocytes. These NK cells are relatively immature and highly proliferative. Our group has demonstrated the efficacy of UCB NK cells in CAR-redirected antitumor cytotoxicity.⁶ Nonetheless, inter-donor variability also significantly affects UCB-based CAR-NK therapies. We found that UCB units with lower nucleated red blood cell count and shorter collection-to-cryopreservation time (termed “optimal cords”) are critical indicators of clinical success.⁷ Alternative NK cell sources include CD34+ hematopoietic progenitor cells and iPSCs.^8,9 The NK cell line NK-92 is also used in clinical trials but requires irradiation prior to infusion. Indeed, the ideal NK cell source is still a topic of debate.

CAR-NK cell therapy in the clinic

The first clinical report on CAR-NK cell therapy was a 2018 phase I study¹⁰, which demonstrated the safety of CAR-NK cells using NK-92 cells engineered with a CD33-directed CAR for the treatment of acute myeloid leukemia. However, no clinical efficacy was achieved, likely attributed to the limited persistence of the CAR-NK-92 cells.

Our group explored cytokine armoring to improve CAR-NK cell persistence. In a phase I/II trial, we demonstrated the safety and efficacy of IL-15-armored UCB-derived CAR19 NK cells in patients with relapsed/refractory B-lymphoid malignancies. CAR19 NK cells were detectable for a year or longer post-infusion and were higher in responders, indicating improved persistence.⁶ The final report showed a treatment overall response rate (ORR) of 48.6%, one-year overall survival (OS) of 68%, and progression-free survival (PFS) of 32%. Notably, key predictors of improved outcomes included the use of optimal cord blood units, which resulted in a remarkable one-year OS of 94% and a one-year PFS of 69% among patients who received CAR-NK cells generated from these optimal cord blood units.⁷

Alternative sources of CAR-NK cells have also been explored in the clinic. Engineered iPSC-derived NK cells, such as FT596, which incorporates a high-affinity non-cleavable Fc receptor (hnCD16), a membrane-bound IL-15/IL-15R fusion protein, and an anti-CD19 CAR- have also shown promise in clinical settings. Interim results from a phase I trial presented at the 2021 American Society of Hematology meeting reported an ORR of 62%, with no dose-limiting toxicities.¹¹ Additionally, preliminary results from a phase I study of NKX019, an allogeneic CAR19-engineered PB-derived NK cell therapy, shared at the 2023 European Hematology Association conference, showed promising responses, especially in patients with non-Hodgkin lymphoma, without any rate-limiting toxicities.¹² As we eagerly await the final results from these clinical trials, these preliminary results underscore the potential of CAR-NK cell immunotherapies, with over 50 clinical trials currently registered exploring this innovative treatment approach.

Strategies to enhance CAR-NK cell immunotherapy

CAR-NK cell therapies face a number of challenges, including limited in vivo persistence and the immunosuppressive tumor microenvironment (TME), especially in solid tumors and myeloid malignancies. Next-generation CAR-NK cell therapies, leveraging cutting-edge genetic engineering, offer the potential to overcome these obstacles.

• An important challenge in NK cell therapy is the short lifespan of NK cells in the absence of cytokine support, resulting in limited in vivo persistence. Efforts to overcome this challenge include genetic engineering such as cytokine armoring (especially incorporation of the IL-15 and IL-21 transgenes) to improve NK cell survival, persistence and anti-tumor efficacy.^7,13 In particular, cytokine armoring with IL-15 has transformed the clinical application of CAR-NK cells in cancer therapy, effectively overcoming the short lifespan of non-engineered NK cells and resulting in improved persistence and in vivo proliferation. Cytokine-induced memory-like NK cells activated by IL-12/15/18 present a promising strategy to enhance NK cell effector function and persistence, resulting in diverse NK cell fates, including effector NK cells with a distinct memory-like population characterized by unique transcriptional and chromatin accessibility profiles.^5,14

• Another important consideration in CAR-NK cell therapy is optimization of the CAR design, crucial for enhancing their therapeutic potential and persistence of response. To leverage the ability of NK cells to target multiple tumor antigens, novel designs have integrated NK cell receptors like NKG2D into the CAR construct. Additional enhancements include identification of optimal transmembrane domains and inclusion of NK cell-specific adapter molecules like DNAM-1 and 2B4 to improve efficacy. Our group found that incorporating the T-cell centric CD28 costimulatory domain into CD70-directed CAR-NK cells resulted in superior cytotoxicity.¹⁵ CAR designs incorporating a PDZ binding motif to enhance synapse formation in NK and T cells have also been developed, resulting in improved effector function and prolonged survival in various tumor models.¹⁶

• Immunosuppression within the TME poses a significant challenge to the effectiveness of both T and NK cell adoptive therapies, resulting in upregulation of immune checkpoints and cell exhaustion. Factors such as hypoxia, nutrient deprivation, lactic acidosis, and immunosuppressive cytokines such as TGF-β together with suppressive cell types such as myeloid-derived suppressor cells and regulatory T cells, all contribute to a profoundly immunosuppressive TME. To counteract these effects, innovative strategies are being explored, including gene editing to disrupt immunosuppressive pathways. For instance, knocking out the CISH gene using CRISPR gene editing inhibits the intracellular cytokine checkpoint CIS, which enhances CAR-NK cell persistence and anti-tumor activity.¹⁷ Other promising approaches involve TGFBR2 knockout and checkpoint inhibition, such as targeting NKG2A with blocking antibodies line Monalizumab, currently in Phase 3 trials (PACIFIC-9 trial comparing durvalumab plus either oleclumab or monalizumab with durvalumab plus placebo in patients with unresectable, stage III NSCLC) or by gene knockout.^18,19 Early reports from CRISPR-based functional screens have identified key suppressors of CAR-NK cell function, like the calcium channel CALHM2.²⁰

• Although CAR-NK cells have shown an excellent safety profile in clinical settings, on-target off-tumor effects are important challenges for many targets, especially in solid tumors. Logic gating strategies have been developed to optimize CAR-NK cell function and improve safety. Our group demonstrated that CAR-NK cells can acquire target antigen expression through trogocytosis, which can lead to fratricide. This problem can be addressed by engineering CAR-NK cells with an inhibitory KIR-based CAR, as shown in both B-cell lymphoma and solid tumor models.²¹ Other efforts to enhance the safety profiles of CAR-NK cells include the integration of safety switches, such as the inducible caspase 9 system²², which can be pharmacologically activated to trigger apoptosis in transduced cells in response to potential adverse effects.

• Identifying the optimal tumor antigen for targeting continues to be a significant challenge in advancing cell therapies. Innovative multi-targeting approaches for CAR-NK cells are being explored to improve antigen recognition and overcome challenges like antigen loss during tumor progression. Strategies including dual targeting methods, natural receptor and ligand-based CARs, and switchable CAR designs aim to enhance tumor specificity (reviewed in²). Additionally, T-cell receptor (TCR) engineering of NK cells is being explored to enable NK cells to recognize and target tumors that express specific peptide-MHC complexes, thereby expanding their targeting capabilities to include intracellular antigens.²³ Our group has recently taken this approach to the clinic (NCT06083883).

Conclusion

CAR-NK cell therapy continues to show promise as a safe and readily available option for cell therapy. Advances in genetic engineering, cytokine armoring, and multi-targeting are significantly enhancing the efficacy of this approach. While challenges such as limited persistence and immunosuppressive tumor environments remain, ongoing research is rapidly evolving CAR-NK cell designs to improve outcomes in cancer treatment. Beyond oncology, CAR-NK cells hold promise for applications in autoimmune diseases and infections, further expanding the potential applications of this innovative strategy.