Generation of Clinically Applicable CAR-NKT Cells

Research Background

Chimeric Antigen Receptor (CAR) T cell therapy has been approved by the Food and Drug Administration (FDA) for the treatment of B-cell malignancies and multiple myeloma. However, the use of autologous CAR-T cell products presents challenges such as high costs, long manufacturing times, and difficulty in obtaining cells from patients. This is particularly problematic for patients with disease progression or those who have undergone prior treatments, potentially lacking sufficient or functional T cells for CAR-T cell production. In order to develop “off-the-shelf” cell therapy products, two approaches are being explored: one involves using conventional αβT cells with the deletion of endogenous TCR expression to reduce the risk of graft-versus-host disease (GVHD), and the other involves using cell types with an inherently low risk of GVHD, such as macrophages, NK cells, and invariant natural killer T (iNKT or NKT) cells.

NKT cells are a unique type of αβT cell characterized by an invariant TCRα chain and NK markers. Due to their recognition of non-polymorphic CD1d molecules, these cells do not trigger GVHD like conventional T cells. NKT cells demonstrate potential for the development of “off-the-shelf” cell therapies, possessing strong tumor-killing activity, tumor infiltration ability, and the capacity to connect innate and adaptive immune responses. However, the development of NKT cell therapies is limited by the scarcity of human blood NKT cells and the difficulty in expanding them from peripheral blood mononuclear cells (PBMCs). Therefore, identifying clinically feasible methods to generate allogeneic NKT cells from other sources is meaningful.

Research Origin

This research was collaboratively authored by Yan-Ruide Li, Yang Zhou, Jiaji Yu, and others from multiple institutions, including the University of California, Los Angeles. The study was submitted on August 23, 2023, accepted on March 28, 2024, and will be published online in the journal “Nature Biotechnology”.

Research Process

Detailed Process

1. Research Design:

   • Cryopreserved human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) were obtained from commercial suppliers and used to generate NKT cells.
   • Phase 0: CD34+ HSPCs were thawed and transduced with viral vectors to insert CARs and other genes of interest.
   • The entire culture process was conducted in a feeder-free and serum-free environment to meet clinical requirements.

2. Cell Generation Process:

   • Phase 1: HSPC expansion (2 weeks).
   • Phase 2: NKT cell differentiation (1 week).
   • Phase 3: Further NKT cell differentiation (1 week).
   • Phase 4: NKT cell expansion (2 weeks).

3. Experimental Details and Sample Handling:

   • During the NKT cell expansion phase, two alternative strategies were tested using human feeder cells: α-galactosylceramide (αGC)-loaded healthy donor PBMCs or K562-based artificial antigen-presenting cells (AAPCs) to evaluate clinical development feasibility.
   • Differentiated NKT cells underwent flow cytometry analysis to determine cell phenotype and function.

4. Main Experiments:

   • The anti-tumor efficacy, expansion capability, and persistence of the generated allogeneic CAR-NKT (alloCAR-NKT) cells were tested in a multiple myeloma (MM) model.
   • Flow cytometry and single-cell TCR sequencing were performed in spleen, bone marrow, and peripheral blood to verify uniform TCR expression and tumor antigen combinations.

5. Data Analysis:

   • Single-cell RNA sequencing (scRNA-seq) was used for gene analysis to assess gene expression profiles of different stages and treated cells.
   • Gene expression, phenotype analysis, and function testing data were integrated to reveal the performance and mechanisms of alloCAR-NKT cells in the anti-tumor environment.

Experimental Results

1. Cell Generation and Expansion:

   • The generated alloCAR-NKT cells showed high consistency in yield and purity, achieving over one million-fold expansion from input CD34+ HSPCs to mature alloCAR-NKT cells.

2. Phenotype and Function:

   • alloCAR-NKT cells expressed typical NKT cell phenotypes in flow cytometry analysis.
   • In in vitro experiments, allo/15bCAR-NKT (containing IL-15 enhancement) cells demonstrated strong immune function, including high levels of effector cytokines and cytotoxic molecules.
   • ELISA and immunofluorescence analyses showed the anti-tumor effects of these cells, including multi-target tumor cell killing via CAR, TCR, and NK receptors.

3. Anti-Tumor Efficacy:

   • In multiple primary multiple myeloma samples, allo/15bCAR-NKT cells exhibited significant tumor cell killing ability.
   • In human MM xenograft mouse models, a single dose of allo/15bCAR-NKT cells achieved tumor elimination and long-term animal survival.

4. Tumor Environment and Immune Evasion:

   • allo/15bCAR-NKT cells showed a selective clearance effect on immunosuppressive cells within the tumor microenvironment (TME).
   • Effective tumor killing was achieved without significant GVHD or cytokine release syndrome (CRS).

Research Conclusions

The generated alloCAR-NKT cells demonstrated excellent characteristics in terms of process, performance, and safety, including: - High purity and consistency in cell production. - Multi-target anti-tumor capability and superior cell survival and effector functions. - No significant GVHD or CRS under certain conditions, showing good clinical application potential.

Research Significance

This study demonstrates that by optimizing clinically guided cell culture methods, highly effective allogeneic CAR-NKT cells can be successfully produced, providing a wide range of application potential without relying on three-dimensional culture and xenogeneic feeder conditions. This achievement opens new opportunities for cell therapy for various hematological cancers and solid tumors, and may extend to the treatment of other diseases.