Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method.

Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using 'off-the-shelf' products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Unlocking the potential of allogeneic Vδ2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering.

Allogeneic Vγ9Vδ2 (Vδ2) T cells have emerged as attractive candidates for developing cancer therapy due to their established safety in allogeneic contexts and inherent tumor-fighting capabilities. Nonetheless, the limited clinical success of Vδ2 T cell-based treatments may be attributed to donor variability, short-lived persistence, and tumor immune evasion. To address these constraints, we engineer Vδ2 T cells with enhanced attributes. By employing CD16 as a donor selection biomarker, we harness Vδ2 T cells characterized by heightened cytotoxicity and potent antibody-dependent cell-mediated cytotoxicity (ADCC) functionality. RNA sequencing analysis supports the augmented effector potential of Vδ2 T cells derived from CD16 high (CD16Hi) donors. Substantial enhancements are further achieved through CAR and IL-15 engineering methodologies. Preclinical investigations in two ovarian cancer models substantiate the effectiveness and safety of engineered CD16Hi Vδ2 T cells. These cells target tumors through multiple mechanisms, exhibit sustained in vivo persistence, and do not elicit graft-versus-host disease. These findings underscore the promise of engineered CD16Hi Vδ2 T cells as a viable therapeutic option for cancer treatment.

Interleukin 15 in Cell-Based Cancer Immunotherapy.

Cell-based cancer immunotherapy, such as chimeric antigen receptor (CAR) engineered T and natural killer (NK) cell therapies, has become a revolutionary new pillar in cancer treatment. Interleukin 15 (IL-15), a potent immunostimulatory cytokine that potentiates T and NK cell immune responses, has demonstrated the reliability and potency to potentially improve the therapeutic efficacy of current cell therapy. Structurally similar to interleukin 2 (IL-2), IL-15 supports the persistence of CD8+ memory T cells while inhibiting IL-2-induced T cell death that better maintains long-term anti-tumor immunity. In this review, we describe the biology of IL-15, studies on administrating IL-15 and/or its derivatives as immunotherapeutic agents, and IL-15-armored immune cells in adoptive cell therapy. We also discuss the advantages and challenges of incorporating IL-15 in cell-based immunotherapy and provide directions for future investigation.

Engineering Induced Pluripotent Stem Cells for Cancer Immunotherapy.

Cell-based immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy, has revolutionized the treatment of hematological malignancies, especially in patients who are refractory to other therapies. However, there are critical obstacles that hinder the widespread clinical applications of current autologous therapies, such as high cost, challenging large-scale manufacturing, and inaccessibility to the therapy for lymphopenia patients. Therefore, it is in great demand to generate the universal off-the-shelf cell products with significant scalability. Human induced pluripotent stem cells (iPSCs) provide an "unlimited supply" for cell therapy because of their unique self-renewal properties and the capacity to be genetically engineered. iPSCs can be differentiated into different immune cells, such as T cells, natural killer (NK) cells, invariant natural killer T (iNKT) cells, gamma delta T (γδ T), mucosal-associated invariant T (MAIT) cells, and macrophages (Mφs). In this review, we describe iPSC-based allogeneic cell therapy, the different culture methods of generating iPSC-derived immune cells (e.g., iPSC-T, iPSC-NK, iPSC-iNKT, iPSC-γδT, iPSC-MAIT and iPSC-Mφ), as well as the recent advances in iPSC-T and iPSC-NK cell therapies, particularly in combinations with CAR-engineering. We also discuss the current challenges and the future perspectives in this field towards the foreseeable applications of iPSC-based immune therapy.

Minimally Invasive Preclinical Monitoring of the Peritoneal Cavity Tumor Microenvironment.

Intraperitoneal (i.p.) experimental models in mice can recapitulate the process of i.p. dissemination in abdominal cancers and may help uncover critical information about future successful clinical treatments. i.p. cellular composition is studied in preclinical models addressing a wide spectrum of other pathophysiological states such as liver cirrhosis, infectious disease, autoimmunity, and aging. The peritoneal cavity is a multifaceted microenvironment that contains various immune cell populations, including T, B, NK, and various myeloid cells, such as macrophages. Analysis of the peritoneal cavity is often obtained by euthanizing mice and performing terminal peritoneal lavage. This procedure inhibits continuous monitoring of the peritoneal cavity in a single mouse and necessitates the usage of more mice to assess the cavity at multiple timepoints, increasing the cost, time, and variability of i.p. studies. Here, we present a simple, novel method termed in vivo intraperitoneal lavage (IVIPL) for the minimally invasive monitoring of cells in the peritoneal cavity of mice. In this proof-of-concept, IVIPL provided real-time insights into the i.p. tumor microenvironment for the development and study of ovarian cancer therapies. Specifically, we studied CAR-T cell therapy in a human high-grade serous ovarian cancer (HGSOC) xenograft mouse model, and we studied the immune composition of the i.p. tumor microenvironment (TME) in a mouse HGSOC syngeneic model.