CAR NK-Cell Therapy Is Quickly Growing in Immunotherapy

Peter Riedell, MD

In hematologic oncology, advancements in chimeric antigen receptors (CARs) for T-cell therapy have led to new investigations and an emerging role for CAR-natural killer (NK) cell therapy. Here we review why CAR-NK cell therapy is an area of interest, how it differentiates from CAR T-cell therapy, its potential challenges, and the current stage of development of this form of treatment.

CAR NK-Cell Therapy: Background

The introduction of CAR engineering to adoptive cell therapy has led to immune effector cell treatments with improved cytotoxicity. This has been a major advancement in treatment for many patients with relapsed or refractory hematologic malignancies.¹

Autologous CAR T cells were used in pioneering therapies, and their efficacy has led to FDA approvals in hematologic malignancies.¹ For example, tisagenlecleucel (Kymriah), a CAR T-cell therapy, was approved for patients with relapsed/refractory acute lymphoblastic leukemia based on trial results showing an overall response rate of 81%, with 60% of patients achieving complete remission.^2,3

However, despite their clinical efficacy, CAR T cells have limitations.⁴ Not all patients are candidates for CAR T-cell therapy. For example, heavily pretreated patients may not have sufficient autologous T cells to achieve clinically relevant doses of CAR T cells.1,4 Also, generating individualized autologous CAR T-cell products for each patient can take weeks, which can lead to unacceptable treatment delays in patients with rapidly progressive disease. Furthermore, patients receiving CAR T cells are at risk of developing graft-vs-host disease (GVHD) even if human leukocyte antigen (HLA) matching between donor and recipient is performed.

This form of therapy has also yet to make significant headway in treating patients with solid tumors. Few patients with solid malignancies have achieved complete responses to date, potentially due to limited expansion or persistence of CAR T cells and the inability of these cells to penetrate solid tumors.⁵

Moreover, long-term persistence of CAR T cells may cause cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), which can be life threatening.^1,4,6 Lastly, target antigen loss after therapy can render CAR T immune cells ineffective due to their dependence on antigens for efficacy.

However, interest in CAR NK-cell therapy has resulted in ongoing research.^1,7

Peter Riedell, MD, assistant professor of medicine, Section of Hematology/Oncology, University of Chicago Medicine, provided his insights in an interview with Targeted Therapies in Oncology^TM.

“CAR T-cell therapies take time, and this can be problematic for patients [who] have more rapidly progressive disease and need therapy urgently,” said Riedell in an interview comparing CAR T-cell therapy to CAR-NK cell therapy. “Having a cellular therapy product which is ‘off-the-shelf’ is very attractive as it means we may be able to treat patients sooner rather than later with this therapy,” he added.”

CAR NK vs CAR T-cell Therapy

NK cells, which were discovered almost 50 years ago, can defend against tumors in most tissues without requiring detection of specific tumor antigens.⁷ Potent innate anti-tumor activity and favorable safety profile features have promoted interest in CAR-NK cell immunotherapy.

NK toxicity against tumor cells involves both innate and adaptive immunity.^7,8 For example, unlike T cells, NK cells can kill tumor cells without expression of major histocompatibility complex (MHC) molecules. As a result, CAR-expressing NK cells can eradicate heterogeneous malignancies that CAR T cells cannot, due to CAR T-cell dependence on MHC expression.⁸ Furthermore, NK cells are able to perform CD16-mediated anti- body-dependent cell-mediated cytotoxicity, giving them an added killing mechanism.^8,9

CAR-NK cells also differ from CAR T cells by having a shorter lifespan in the blood-stream. Less potential for long-term off-tumor toxicities in CAR-NK cells is theorized as a result.⁹ “Healthy cells express CD19 as well as malignant cells. While having CAR T cells remain in the body for longer periods may be associated with continued clinical benefit in maintaining remissions, when CAR T cells remain in the body for prolonged periods, this may also lead to B-cell aplasia and hypogammaglobulinemia,” explained Riedell.

Additionally, preclinical and phase 1/2 trials have shown that allogeneic CAR-NK- cell infusions decrease the risk of GVHD.^1,9,10 This allows the expansion of NK-cell production beyond autologous cells or only 1 cell line source. Persistence of allogeneic CAR NK cells has been observed in patients for at least 1 year despite HLA mismatching.^11,12

Furthermore, NK cells can be administered without a requirement for full HLA matching.¹⁰ “This allows for the use of allogeneic sources for CAR NK cells, including healthy donors, umbilical cord blood units, or induced pluripotent stem cells,” Riedell noted. Importantly, manufacturing failures and out-of-specification products can also be avoided with off-the-shelf therapy.¹⁰

“This allows for the use of allogeneic sources for CAR NK cells, including healthy donors, umbilical cord blood units, or induced pluripotent stem cells. Products are able to come off the shelf without the need to navigate collection of patients’ T cells and await their engineering and manufacture, which can take weeks,” Riedell noted. Importantly, manufacturing failures and out-of-specification products can also be avoided with off-the-shelf therapy.¹⁰

“CAR-NK cell therapy may be associated with a lower incidence and severity of CRS and neurologic toxicity, which is another reason this therapy is being explored,” explained Riedell. These less severe adverse events may be due to the release of milder cytokines such as granulocyte-macrophage colony-stimulating factor and interferon-γ.⁹ CAR T cells induce the release of more cytotoxic cytokines, such as interleukin-1 (IL-1) and IL-6, that are associated with CRS.

Roadblocks for CAR-NK Therapy

Despite the safety and promising clinical efficacy of unmodified allogeneic NK cells, several challenges to using CAR-NK cells have emerged from clinical trials.

“While CAR-NK cell therapy has been shown to be technically feasible, there is overall limited data in regard to the efficacy and safety of this treatment approach. Given that these cellular therapy products are allogeneic, there is a concern for emergence of graft-vs-host disease,” Riedell said. “There are many current clinical trials being conducted that evaluate CAR-NK cell therapy and we eagerly await the results of these trials to better understand the impact of this treatment approach,” he added.

Furthermore, NK cells have a short lifespan of only 1 to 2 weeks, and without cytokine support, infused cells do not persist in the donor, which restricts efficacy.¹³

“It is unknown if responses seen with this treatment may be durable and associated with continued remissions or if this therapy may be better utilized to induce responses and remissions in patients and then consolidate those remissions with an allogeneic stem cell transplant,” explained Riedell.

Techniques to enhance the stability of CAR-NK cells include incorporation of transgenes encoding exogenous cytokines, such as IL-15.11 However, exogenous cytokines have undesirable adverse effects and can promote the activation of other immune sub- sets, such as regulatory T cells, which may suppress the effector functions of NK cells.¹⁴

Another challenge with CAR-NK cells is that NK cells are limited in number and often require ex vivo expansion and actiation. NK cells represent a minor fraction of peripheral blood leukocytes, and thus the generation of sufficient numbers of NK cells remains a major challenge for adoptive immunotherapy.

NK-92 is an established NK cell line that can be used as a source of cells for CAR- NK therapies, representing an alternative to patient- or donor-derived NK cells. An advantage of this process is easier manufacture of off-the-shelf CAR-NK products; however, a drawback is that NK-92 cells are from a tumor cell line and have a potential tumorigenicity risk.¹⁵

Lastly, CAR NK approaches are limited by approaches to gene transfer in NK cells. Gene transduction may lead to random intergration of DNA into the target cell genome, and can encourage off-target effects, including the silencing of essential genes or expression of tumor suppressor genes.⁹

Viral transduction results in low levels of transgene expression in NK cells and adversely impacts their survival. Nonviral vectors have been explored and are considered safe alternatives, but their relative overall benefits remain unclear.¹¹

Trial Results and Ongoing Trials of Interest

Several phase 1 and 2 trials for CAR-NK therapy are ongoing, with some published results.

In a phase 1/2 study (NCT03056339), patients with B-cell lymphoid malignancies were administered cord blood–derived, HLA-mismatched, anti-CD19 CAR-NK cells.¹² The cells were transduced with a retroviral vector that expressed genes encoding anti- CD19 CAR, IL-15, and inducible caspase 9 (safety switch).

Of 11 heavily pretreated patients with CD19-positive lymphoma or chronic lymphocytic leukemia (CLL), 8 had an objective response (73%) and 7 had complete remission (64%) without major toxic effects. There were no recorded events of CRS, neurotoxicity, hemophagocytic lymphohistiocy- tosis, or GVHD.

Myelotoxicity was observed, which the investigators attributed to the lymphodeplet-ing chemotherapy prior to infusion. Many responses were seen within 30 days of infusion. Also, the CAR-NK cells expanded and persisted for at least 12 months.

A second study, a phase 1 trial (NCT04245722), evaluated the safety and efficacy of FT596, a multi-antigen–targeted, pluripotent stem cell–derived, off-the-shelf, anti-CD19 CAR-NK cell therapy. In the study, 20 heavily pretreated patients with relapsed/ refractory B-cell lymphoma or CLL were treated with FT596, either alone or in combination with rituximab (Rituxan).

Responses were seen in 8 of 11 efficacy-evaluable patients, 7 of which were complete respons- es. No GVHD or ICANS was observed in any of the 20 treated patients, and only 2 cases of CRS were reported.¹⁶

Several other clinical trials of interest are ongoing. A phase 1 study (NCT05247957) evaluating NKG2D, a cord blood–derived CAR-NK therapy, in patients with relapsed/refractory acute myeloid leukemia is expected to conclude at the end of 2022.

Another phase 1 study (NCT04887012) of HLA haploidentical anti-CD19 CAR-NK cells in relapsed/refractory B-cell non-Hodgkin lymphoma is ongoing. Finally, an early phase 1 study (NCT05215015) of CAR-NK cells targeting CD33 in patients with acute myeloid leukemia is ongoing.

“CAR-NK cell therapy will likely become much more common and an area of increasing research focus should we be able to gain a better understanding that this treatment approach is safe and efficacious,” Riedell noted. “Additional studies are needed in order to understand optimal CAR-NK cell constructs, the best antigens to target, and strategies to bolster CAR-NK cell manufacturing, storage, and delivery,” he added.”

_REFERENCES:

_{1. Basar R, Daher M, Rezvani K. Next-generation cell therapies: the emerging role of CAR-NK cells. Blood Adv. 2020;4(22):5868-5876. doi: 10.1182/bloodadvances.2020002547}

_{2. FDA approves tisagenlecleucel for B-cell ALL and tocilizumab for cytokine release syndrome. FDA. September 7, 2017. Accessed March 23, 2022. https://bit.ly/38mmisI}

_{3. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5):439-448. doi:10.1056/NEJMoa1709866}

_{4. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021;11(4):69. doi:10.1038/s41408-021-00459-7}

_{5. Wagner J, Wickman E, DeRenzo C, Gottschalk S. CAR T cell therapy for solid tumors: bright future or dark reality? Mol Ther. 2020;28(11):2320-2339. doi:10.1016/j.ymthe.2020.09.015}

_{6. Morris EC, Neelapu SS, Giavridis T, Sadelain M. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy. Nat Rev Immunol. 2022;22(2):85-96. doi:10.1038/s41577-021-00547-6}

_{7. Marofi F, Saleh MM, Rahman HS. CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies. Stem Cell Res Ther. 2021;12(1):374. doi:10.1186/s13287-021-02462-y}

_{8. Farag SS, Caligiuri MA. Human natural killer cell development and biology. BloodRev. 2006;20(3):123-137.doi:10.1016/j.blre.2005.10.001}

_{9. Xie G, Dong H, Liang Y, Ham JD, Rizwan R, Chen J. CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine. 2020;59:102975. doi:10.1016/j.ebiom.2020.102975}

_{10. Lupo KB, MatosevicS. Natural killer cells as allogeneic effectors in adoptive cancer immunotherapy. Cancers. 2019;11(6):769.doi:10.3390/cancers11060769}

_{11. Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, GermeraadWTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021;14(1):73. doi:10.1186/s13045-021-01083}

_{12. Liu E, Marin D, Banerjee P. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med. 2020;382(6):545-553. doi:10.1056/NEJMoa1910607}

_{13. Malmberg KJ, Carlsten M, Björklund A, Sohlberg E, Bryceson YT, Ljunggren HG. Natural killer cell-mediated immunosurveillance of human cancer. Semin Immunol. 2017;31:20-29. doi:10.1016/j.smim.2017.08.002}

_{14. Pedroza-Pacheco I, Madrigal A, Saudemont A,et al. Interaction between natural killer cells and regulatory T cells: perspectives for immunotherapy. Cell Mol Immunol. 2013;10(3):222-229.doi:10.1038/cmi.2013.2}

_{15. Zhang C, Oberoi P, Oelsner S, et al. Chimeric antigen receptor-engineered NK-92 cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol. 2017;8:533.doi:10.3389/fimmu.2017.00533}

_{16. Bachanova V, Ghobadi A, Patel K, et al. Safety and efficacy of FT596, a first-in-class, multi-antigen targeted, off-the-shelf, iPSC-derived CD19 CAR NK cell therapy in relapsed/refractory b-cell lymphoma. Blood. 2021;138(suppl 1):823. doi:10.1182/blood-2021-151185}