For most people today, cancer is still perceived as an incurable disease. Whether it is the experience of the disease itself or the conventional treatment process, both are extremely painful, with a high probability of recurrence and a rapidly increasing incidence year by year—hence the widespread fear of cancer. However, with the continuous development of biomedical technology, scientists have invented a cellular therapy called "CAR-T," which has greatly enhanced the effectiveness of cancer treatment. Its principle is to equip our immune guardians—"T cells"—with a high-tech weapon called "CAR" (chimeric antigen receptor), thereby enhancing their ability to recognize and kill tumor cells.

CAR-T cell therapy has matured with the discovery of effective targets and continuous improvements in CAR design, achieving remarkable success in a wide variety of cancers, particularly in hematological malignancies such as lymphoma and leukemia. Yet this did not satisfy scientists' aspirations and exploration for a complete cure for cancer, and thus a "newer" type of anticancer warrior was born—CAR-NK. The underlying principle also employs genetic engineering to equip NK cells with the high-tech "CAR" weapon.

Process of developing CAR-NK cell immunotherapy

Since the addition of "CAR-NK," our "war" against tumors has advanced with unstoppable momentum! Although both warriors are proficient in fighting cancer, CAR-NK possesses unique advantages over CAR-T in the treatment of solid tumors. Let us take a look at the advantages of CAR-NK! (Shenzhen Cell Valley boasts a highlevel domestic CAR-NK preparation capability; for details, please refer to our previous post: "Research Progress | Shenzhen Cell Valley Achieves Major Breakthrough in CAR-NK Manufacturing Process.")

Now that we have mentioned the advantages of CAR-NK, we must also discuss the "birthplace" of this hero. Currently, there are four main sources of NK cells for CAR-NK therapy: human peripheral blood (PB), umbilical cord blood (CB), stem cell-derived NK cells, and the NK-92 cell line.

Although NK cells may come from different origins, their deep-seated aversion to cancer cells never changes, and their ultimate goal is always to "exterminate" cancer cells. So let us learn more about the origin stories of these heroes.

Sources of NK Cells

Peripheral Blood (PB) NK Cells:

Advantages: NK cells can be relatively easily isolated from the patient (autologous PB-NK) or from healthy donors (allogeneic PB-NK) and expanded. Therefore, most preclinical CAR-NK studies use PB-NK. Limited by disease and treatment, patient autologous NK cells may have impaired function, and allogeneic NK cells are preferred clinically, but T cells must be carefully removed to reduce the risk of GVHD. PB-NK cells are already mature, require no induction of differentiation, and have strong killing activity.

Disadvantages: 1. Allogeneic PB-NK cells show significantly reduced viability and cytotoxicity after cryopreservation; 2. Gene transduction efficiency is relatively low, and extended in vitro expansion time affects killing function; 3. Typically, 10⁵-10⁸ cells/kg body weight are needed, but PB-NK cell proportions are low, making large-scale culture difficult.

With its strong R&D capabilities, Shenzhen Cell Valley has successfully overcome the above bottlenecks and can provide PB-NK cells with high quantity, high purity, high gene transduction efficiency, and potent killing activity.

Umbilical Cord Blood (CB) NK Cells:

Advantages: "Off-the-shelf," ready for use, with good cryopreservation tolerance. CB-NK cells have high proliferative efficiency; a universal NK cell bank can be established, allowing HLA-mismatched products to be selected on demand. They exhibit stronger bone marrow homing capacity. Cord blood cells have strong proliferative potential—only 10% of a single CB unit can generate a pure cell pool of nearly 10⁹ NK cells within two weeks, typically sufficient for one treatment cycle.

Disadvantages: CB-NK cells are not fully differentiated; natural killer receptor expression is relatively low, and cytotoxic capacity is limited. In addition, the higher proportion of hemoglobin and red blood cells in cord blood can affect PBM separation and culture, requiring careful handling during experiments.

Stem Cell-Derived NK Cells:

Advantages: NK cells are typically induced from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), with an expansion cycle of 3-5 weeks, which can avoid the heterogeneity between donor and recipient NK cells.

Disadvantages: iPSC-derived NK cells carry potential malignant transformation capacity, posing a tumorigenic risk in vivo. They also have potential immunogenicity, which may trigger unintended immune responses.

NK-92 Cell Line:

Advantages: "Off-the-shelf," ready for use; cell line expansion and activity maintenance are convenient. NK-92 lacks CD16-mediated ADCC, but this can be obtained through modification. NK-92 cells are amenable to genetic manipulation without the need for viral vectors; electroporation can effectively introduce exogenous genes.

Disadvantages: Because NK-92 is a tumor-derived aneuploid immortalized cell line, it requires irradiation before use to inhibit its proliferative capacity in vivo, which limits its lifespan and reduces in vivo residence and efficacy.

Peripheral Blood NK Cells vs. Umbilical Cord Blood NK Cells

Among the four sources, the peripheral blood NK cell and umbilical cord blood NK cell "brothers" appear most frequently, with the former being most widely used clinically. These two brothers each have their own characteristics in fighting solid tumors, and there are also differences in efficacy in CAR-NK therapy (for details, see our previous post "Comparison of CAR-NK Cells Derived from Adult Peripheral Blood and Umbilical Cord Blood"). These differences depend on multiple factors, including the quality of the source cells and preparation techniques. (Whether CAR-NK is prepared from PB-NK or CB-NK, Shenzhen Cell Valley achieves cell viability and purity both above 90%, CAR transduction positivity up to 60-80%, and expansion fold up to tens of thousands.)

"Four sources of CAR-NK cells (from Clin Transl Immunology. 2021 Apr 28;10(4):e1274.)"

Both brothers are "born" in the bone marrow and serve as the first line of defense against cancer in our body. Moreover, their response speed is second to none. They are particularly "sensitive" to various pathogenic agents and harmful cells, remaining vigilant at all times—as soon as these "villains" appear in our body, they can react immediately and eliminate them. Of course, all this credit goes to their ability to release a "trump card" that terrifies these villains—cytotoxic granules—which can induce apoptosis or programmed cell death in target cells.

As the "elder brother," peripheral blood NK cells, being mature and experienced, possess more diverse populations and greater functional variability, which better enables them to respond to various threats and navigate the complex landscape of anticancer defense with ease. In contrast, umbilical cord blood NK cells, as the "younger brother," have a lower degree of differentiation and relatively limited diversity, resulting in less experience in handling cancer cells and a slightly weaker killing capacity.

In summary, although the two brothers come from different origins, they are both "stalwart warriors" on our anticancer journey, bringing the dawn of hope to mankind in the formidable challenge of cancer. On the persistent and arduous "battlefield" of fighting solid tumors, the elder brother, with his maturity, stability, and solid skills, demonstrates stronger killing effects and has achieved much. The younger brother, although his direct killing ability is somewhat weaker compared to the elder, has important applications in hematopoietic stem cell transplantation and regenerative medicine. It can be said that both are "shining" and "contributing" in their respective fields for the benefit of mankind!

References

[1] Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif Kerbauy L, Overman B, Thall P, Kaplan M, et al. CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med. 2020;382(6):545-53.

[2] Gong Y, et al. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. Journal of Hematology & Oncology, 2021. 14(1): p. 73.

[3] Xie GZ, Dong H, Liang Y, et al. CARNK cells: A promising cellular immunotherapy for cancer. EBioMedicine, 2020, 59: 102975.