Jun 20, 2024

Summary

CAR-T cell therapies have enabled unparalleled success in treating patients with liquid tumors. Yet, access to these therapies remains limited by various manufacturing hurdles, including their high cost and autologous sourcing. Therefore, a priority in the field is to develop ready-made-cellular therapies that can be expedited to the clinic at a lower price and serve a broader patient population. Now, a group at the Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, led by Dr. Lili Yang, has developed a reliable platform for producing CAR-NKT cells at scale. Their preclinical findings support that the resulting universal CAR-NKT cells have high anti-tumor efficacy and can be engineered to persist and prevent adverse reactions following allogeneic infusion.

Background

CAR T cells have proven ideal therapies for blood malignancies, such as B cell lymphomas, acute lymphoblastic leukemia, and multiple myeloma. Long-term outcomes of CAR-T cell therapy are being evaluated and support the efficacy and limited toxicity for some modalities, such as those targeting the B-lymphocyte antigen CD19.^1,2 These amazing cellular drugs continue to evolve as scientists make considerable strides to improve CAR-T cell designs further to achieve greater efficacy against solid tumors. However, patients’ access to these game-changing and extraordinarily costly cell therapies remains a critical limitation. Off-the-shelf cellular therapies can address this barrier to treatment. Yet, a primary hurdle to overcome in designing universal cell therapies is the potential for adverse reactions and allorejection following infusion.

Currently, CAR-T cells are engineered from patient-derived T cells, which imposes several limitations beyond cost, including the availability of sufficient and healthy autologous T cells and timely access to the final product. Now, work by a team at the University of California, Los Angeles, led by Dr. Lili Yang, addresses these critical limitations, providing a path toward the next generation of universal CAR-T cells.³

Leveraging the benefits of unconventional T cells

Dr. Yang and her team have focused on unconventional T cells and specifically invariant natural killer T cells (NKT) to engineer new CAR-T cell therapies applicable to a broader patient population. Invariant NKT cells, or iNKT cells, constitute a unique subset of T cells that share features of conventional T cells and NK cells. iNKT cells express an invariant T cell receptor (i.e., TCR α chain (Vα24-Jα18) and TCR β chains that use limited Vβ segments (Vβ11) in humans) and recognize CD1d-presented lipid targets inducing innate and adaptive immune responses.^4,5 Activation of iNKT cells promotes secretion of various cytokines (e.g., interferon-α and interleukin-4) and other factors contributing to dendritic cell maturation and recruitment of cellular immunity, such as cytotoxic T cells. Unlike conventional circulating T cells, iNKT cells are abundant in tissues, functioning in homeostatic regulation and in protection from infection and cancer.^4,5

Various clinical studies have evaluated iNKT cells as immunotherapy in cancer. Several properties of iNKT cells make them attractive for this application, including their capacity to infiltrate tumors and engage in tumor cell killing, induce the activation of dendritic cells, and promote tumor antigen-specific T-cell clonal expansion and B cell activation.^5,6 Lastly, allogeneic iNKT cells do not induce graft-versus-host disease (GvHD), presenting a huge opportunity to engineer safer cellular therapies for the masses.

Engineering off-the-shelf CAR-NKT cells

In conventional CAR-T cell engineering, sourcing cells in sufficient numbers is generally achievable, whether these are obtained from the patient or a donor. However, despite all the advantages of iNKT cells for engineering therapies, they are present in very limited numbers.⁵ Therefore, Yang’s team had first to resolve this critical limitation. In their newly published work, the team has leveraged hematopoietic stem cells (HSCs) for genetic engineering and differentiation to secure the production of universal CAR-NK cells that do not induce GvHD and are resistant to immune rejection. Additionally, Yang’s team implemented a feeder-free culture system to support CAR-NKT cell production at scale.³

Universal CAR-NKT cell manufacturing workflow. Retrieved from Li et al. 2024.³ CC BY 4.0 Deed | Attribution 4.0 International | Creative Commons

Their cell engineering strategy had two main goals: first, to eliminate the potential for immune rejection of the cell product, and second, to establish target specificity. To this end, CRISPR-Cas9 genome editing enabled the team to knockout the expression of HLA molecules, making these cells undetectable by conventional T cells. A lentiviral vector was used to deliver a construct consisting of iNKT TCR alpha/beta sequences and a B cell targeting CAR (i.e., anti-BCMA). Additionally, an IL-15 sequence was included in the lentivector to promote anti-tumor activity of the final cell product.

“The synthetic gene fragments were obtained from GenScript”³

Once modified, HSCs were differentiated and expanded through a 6-week culture process yielding universal BCAR-NKT cells in high numbers ideal for clinical use. Li and colleagues found that these cells expressed the expected NKT cell identity markers and significantly produced high levels of cytokines and other cytotoxic molecules to support anti-tumor functionality.

Expansion and differentiation of engineered HSCs under feeder-free conditions. For the last expansion, the team tested two feeder conditions, including α-galactosylceramide (αGC)-loaded PBMCs and artificial antigen-presenting cells (aAPCs). Ultimately, while feeder-free and both feeder conditions enabled the desired differentiation of HSCs, Yang’s team achieved greater cellular expansion with feeder aAPCs. Retrieved from Li et al. 2024.³ CC BY 4.0 Deed | Attribution 4.0 International | Creative Commons

In vitro, universal BCAR-NKT cells did not induce significant responses from allogeneic T cells. Moreover, when infused in a mouse model to evaluate allorejection, BCAR-NKT cells expanded and persisted without inducing GvHD.

In a significant discovery, Yang’s team found that universal BCAR-NKT cells had superior activity in targeting and killing tumor cells in vitro compared to BCAR-T cells. Whether against multiple myeloma cell lines or primary tumor tissue, universal BCAR-NKT cells were more effective than their conventional counterparts due partly to their capacity to recognize various tumor targets (i.e., BCMA, CD1d, and NK ligands).

Universal BCAR-NKT cells targeting mechanisms. “Diagram showing the CAR/TCR/NKR triple tumor-targeting mechanisms of U15BCAR-NKT cells.” Retrieved from Figure 4, Li et al. 2024, only panel G is shown.³ CC BY 4.0 Deed | Attribution 4.0 International | Creative Commons

Lastly, in vivo experiments leveraging tumor-bearing mice tested the efficacy of universal BCAR-NKT against that of conventional BCAR-T cells. Yang’s team found that under different conditions of tumor burden and heterogeneity tested, BCAR-NKT cells were more efficacious, even in tumors without BCMA and CD1d expression. Moreover, universal BCAR-NKT cells showed prolonged survival without adverse outcomes, such as GvHD.

Conclusion

Overall, Dr. Lili Yang’s team has developed a scalable platform for manufacturing highly efficacious universal CAR-NKT cells. These cells show robust tumor-killing activity and can reach multiple targets safely in an allogeneic setting. The established platform relies on donor HSCs for the engineering, differentiation, and expansion of CAR-NKT cells under conditions compatible with clinical use.

Reference

1. Cappell, K.M., Kochenderfer, J.N. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20, 359–371 (2023). https://doi.org/10.1038/s41571-023-00754-1

2. Mitra A, Barua A, Huang L, Ganguly S, Feng Q, He B. From bench to bedside: the history and progress of CAR T cell therapy. Front Immunol. 2023 May 15;14:1188049. https://doi.org/10.3389/fimmu.2023.1188049

3. Li YR, Zhou Y, Yu J, Zhu Y, Lee D, Zhu E, Li Z, Kim YJ, Zhou K, Fang Y, Lyu Z, Chen Y, Tian Y, Huang J, Cen X, Husman T, Cho JM, Hsiai T, Zhou JJ, Wang P, Puliafito BR, Larson SM, Yang L. Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy. Mol Ther. 2024 Apr 6:S1525-0016(24)00221-1. https://doi.org/10.1016/j.ymthe.2024.04.005

4. Crosby, C.M., Kronenberg, M. Tissue-specific functions of invariant natural killer T cells. Nat Rev Immunol 18, 559–574 (2018). https://doi.org/10.1038/s41577-018-0034-2

5. Kitayama S, Zhang R, Liu TY, Ueda N, Iriguchi S, Yasui Y, Kawai Y, Tatsumi M, Hirai N, Mizoro Y, Iwama T, Watanabe A, Nakanishi M, Kuzushima K, Uemura Y, Kaneko S. Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells. Stem Cell Reports. 2016 Feb 9;6(2):213-27. https://doi.org/10.1016/j.stemcr.2016.01.005

6. McEwen-Smith RM, Salio M, Cerundolo V. The regulatory role of invariant NKT cells in tumor immunity. Cancer Immunol Res. 2015 May;3(5):425-35. https://doi.org/10.1158/2326-6066.cir-15-0062