CAR-NK Cell Therapy: Analysis From Six Dimensions

Natural killer cells (NK cells) are a type of innate lymphocytes that have the ability to recognize and eliminate virus-infected cells and tumor cells. With the disruptive success of CAR-T cell therapy, interest in the potential of NK cells in anti-tumor immunotherapy has doubled.

Since the launch of CAR-T therapy, CAR-NK cell therapy has mushroomed and ushered in an investment frenzy, and major cell companies or related companies have made plans. However, clinical advancement does not seem to be as smooth as CAR-T cell therapy. The experience of CAR-T cell therapy is still worth learning from CAR-NK cell therapy, but the differences caused by the mechanism also need to be resolved.

Viral vectors and transduction selection

Currently, the CAR-T products approved for commercial use are all produced using lentiviral or retroviral vector transduction and are only for autologous use due to the risk of GVHD . Among them, vesicular stomatitis virus G protein (VSV-G) lentivirus is the most commonly used pseudotyping receptor for the production of CAR-T cells.

Compared to retroviral vectors, lentivirus-based transduction is safer due to lower genotoxicity and insertional mutagenesis. Typically, lentiviral transduction in primary NK cells is less efficient and often requires multiple rounds of transduction.

At the same time, the risk of GVHD with CAR-NK cell therapy is very small. However, it also has low expression levels of the low-density lipoprotein receptor, which is the primary transduction receptor for VSV-G. Therefore, it is necessary to select vectors specific for NK cell transduction.

It is currently believed that baboon enveloped pseudotype lentiviral vector (BaEV-LV) can significantly improve the transduction efficiency of NK cells . In addition, delivery of CRISPR genome editing components via virus-like particles is becoming a popular method. Transduction of CAR and gene knockout can be performed together.

Mol Ther Methods Clin Dev.

Transfection methods such as electroporation and lipofection are also used to deliver exogenous genes into NK cells. Currently, famous transposon systems include PiggyBac and Sleeping Beauty. Compared with viral transduction, NK cell transfection is associated with faster transgene expression, lower levels of apoptosis, less inter-individual variability, and higher gene transfer efficiency.

However, the applicability of the transposon system in transducing primary NK cells still requires further modification to overcome problems such as low transduction efficiency and the cytopathic effect of plasmid DNA electroporation on NK cells.

CAR-NK cell persistence

CAR-NK cells are not as durable as CAR-T cells, which is one of the reasons for the limited clinical efficacy of NK cell therapy. It is currently believed that short-term exposure to IL-12, IL-15, and IL-18 produces longer-lasting memory-like NK cells .

Preclinical pretreatment is also one of the factors influencing the durability of clinical application of CAR-NK cells . Typically, the fludarabine/cyclophosphamide combination is used before receiving CAR-NK cell therapy to reduce host rejection of NK cells, reprogram the immunosuppressive tumor microenvironment, and reduce tumor burden.

In terms of cell dosage, before adoptive transfer of unmodified NK cell therapy, the high-dose regimen can better enable the expansion and persistence of NK cells in the body to meet the needs, while the low-dose regimen cannot .

But what is the appropriate level of persistence? Is the longer the better? Not really.

It is currently believed that enhancing NK cell persistence only needs to be emphasized in specific tumors. Experience with CAR-T cell therapy shows that in types such as lymphoma, patients can develop durable responses without prolonging the duration of CAR-T therapy. This may be something that CAR-NK cells can learn from.

So far, most goals in optimizing NK cell CAR structures have focused on improving cytotoxicity rather than persistence . The low immunogenicity of CAR-NK also enables it to be administered multiple times to compensate for persistence issues.

Migration ability of CAR-NK cells

Ways for CAR-T cells to improve their migration ability ( trafficking ) include: expressing chemokine receptors through CAR design , combined administration , changing administration routes , etc. All three strategies are useful for CAR-NK cells.

NK cells themselves use chemokine signals in normal immunity. NK cells modified to express CXCR2, CXCR4, CCR5 or CCR7 have been proven to enhance the migration function of mice and enhance their anti-tumor ability. However, it does not affect the degree of cytotoxic cytokine production and the direct tumor killing activity of NK cells.

Nutritional metabolism of CAR-NK cells

Nutritional metabolism is mainly for indications of solid tumors . In the tumor microenvironment, considerable nutritional and metabolic barriers prevent immune cells from functioning, including: hypoxia, lack of nutrients, low pH, and elevated levels of various metabolites.

CAR-T cell products with optimized metabolic functions, such as higher oxidative phosphorylation, have been shown to be more effective clinically. For example, in CLL patients, CD8+ T cells show impaired activation and reduced glucose uptake after stimulation, and long-term exposure affects metabolic homeostasis, leading to abnormal metabolic reprogramming upon stimulation. CLL-derived CD8+ T cells display impaired mitochondrial biogenesis when stimulated.

Strategies to improve metabolism include:

• By directly manipulating cell metabolism during in vitro expansion;
• Genetically engineer T cells. This also applies to NK cells.

For example, studies using mIL-21 to expand NK cells and CAR-NK cells in vitro have shown that the expanded NK cells related to enriched metabolic pathways are less differentiated, have non-depleted phenotypes, and have memory-like phenotypes. The ratio is increased and can inhibit the "cannibalism" of CAR-NK cells.

NK cells are more susceptible to the effects of hydrogen peroxide in the tumor microenvironment than T/B cells . Some studies have genetically modified NK cells to overexpress peroxiredoxin-1(PRDX1). The cells have shown effective anti-tumor activity against breast cancer cells under oxidative stress.

Impact of tumor antigen loss

Current exploration of CAR-T cells includes:

• Design dual-target CAR-T cells;
• Sequential administration of CAR-T cells with different CARs;
• Combined oncolytic virus administration.

Different from CAR-T cells, NK cells can retain CAR-independent killing ability through innate receptors ( NCR, CD16, DNAM-1 or NKG2D/DAP10, etc. ) in a tumor escape environment where CAR antigen is lost or downregulated.

In addition, dual-target CAR-NK cells are currently in preclinical development, such as CD3-targeted CAR expression containing NKG19D transmembrane and 2B2/CD4ζ signaling domains.

Effects of Cytosis

Trogocytosis : effects on signaling, loss of antigens, and cannibalism.

Positive  effects of cell gnawing:

Through cytosis, NK cells can obtain chemokine receptors ( such as CCR5, CXCR4, CCR7 or TYRO3, etc. ) . The acquisition of CCR7 enhances the ability of NK cells to home to lymph nodes, while TYRO3 improves effector function and proliferation levels.

Inhibitory  effects of cytophagia:

Through cytosis, CAR-NK cells can obtain immunosuppressive proteins to inhibit their cytotoxic effects. For example: in the context of leukemia, NK cells can obtain PD-1 from leukemia cells; in ovarian cancer, NK cells can obtain CD9 from ovarian cancer cells to inhibit cytotoxicity. Overcoming cytosis-mediated signaling is the use of blocking antibodies directed against the target antigen. For example, in ovarian cancer, there are studies using CD9-blocking antibodies to restore NK cell cytotoxicity in vitro in the presence of CD9.

Cell Rep.

Cytosis and loss of target cell antigens

Another consequence of cytosis is the loss of target cell antigens. While this has been shown with CAR-NK cells, it has been studied even more with CAR-T cells. Since antigen density affects CAR function, downregulation or internalization of the target antigen can lead to tumor escape. Therefore, overcoming antigen loss caused by phagocytosis may improve tumor clearance.

The first method is by adjusting the affinity of the CAR to the target antigen . The low-affinity structure is thought to reduce cytosis while maintaining efficacy. In CAR-T cell therapy, preclinical studies have proven that low-affinity CD19 CAR-T cells can significantly limit cytology while maintaining anti-tumor activity and clinical efficacy.

Another approach is to adjust the signal domain of CAR . Because cytosis has been shown to affect CARs with CD28 or 4-1BB signaling domains to varying degrees. However, the effects of this mechanism vary widely and depend mainly on antigen density.

The final result of the cytosis is to transfer the antigen to NK cells, which mediates the killing of other NK cells by NK cells. Knocking out target antigens in effector cells can overcome sibling killing in the context of endogenous T cell or NK cell antigens such as CD7 or CD38 , but this approach is not applicable to antigens transferred to effector cells via cleavage.

Summary and future outlook

Let’s just say it’s not popular, CAR-NK has many corporate deployments in both hematological tumors and solid tumors. On May 6, 2023, the National Center for Drug Evaluation (CDE) accepted the IND application for Qihan Biotech’s allogeneic CD19 CAR-NK ;

Speaking of hot topics, many CAR-NK cell products that have attracted much attention have been delayed in clinical advancement . Fate, an industry pioneer, also announced the termination of its cooperation with Johnson & Johnson on January 5, 2023. At the same time, it chose to focus on the development of second-generation CD19 CAR-NK therapy and terminated the development of the FT596 project.

This just shows that the iterative needs and technologies in CAR-NK cell structure and design have caught up with the progress of actual clinical trials. This also gives more opportunities to subsequent companies, and overtaking through technical corners may not be impossible.