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1. Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

   https://doi.org/10.1016/j.bioactmat.2023.03.018  

   Chang, Yun; Jin, Gyuhyung; Luo, Weichuan; Luo, Qian; Jung, Juhyung; Hummel, Sydney N.; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Low, Philip S.; Lian, Xiaojun Lance; et al (September 2023, Bioactive Materials)

   Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy. 

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2. Generation of chimeric antigen receptor macrophages from human pluripotent stem cells to target glioblastoma

   https://doi.org/10.1016/j.iotech.2023.100409  

   Jin, G.; Chang, Y.; Bao, X. (December 2023, Immuno-Oncology and Technology)

   Background: Glioblastoma (GBM) is an aggressive brain tumor giving a poor prognosis with the current treatment options. The advent of chimeric antigen receptor (CAR) T-cell therapy revolutionized the field of immunotherapy and has provided a new set of therapeutic options for refractory blood cancers. In an effort to apply this therapeutic approach to solid tumors, various immune cell types and CAR constructs are being studied. Notably, macrophages have recently emerged as potential candidates for targeting solid tumors, attributed to their inherent tumorinfiltrating capacity and abundant presence in the tumor microenvironment. Materials and methods: In this study, we developed a chemically defined differentiation protocol to generate macrophages from human pluripotent stem cells (hPSCs). A GBM-specific CAR was genetically incorporated into hPSCs to generate CAR hPSC-derived macrophages. Results: The CAR hPSC-derived macrophages exhibited potent anticancer activity against GBM cells in vitro. Conclusion: Our findings demonstrate the feasibility of generating functional CAR-macrophages from hPSCs for adoptive immunotherapy, thereby opening new avenues for the treatment of solid tumors, particularly GBM. 

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3. Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

   https://doi.org/10.1136/jitc-2020-001877  

   Bandey, Irfan N; Adolacion, Jay R; Romain, Gabrielle; Paniagua, Melisa Martinez; An, Xingyue; Saeedi, Arash; Liadi, Ivan; You, Zheng; Rajanayake, Rasindu B; Hwu, Patrick; et al (March 2021, Journal for ImmunoTherapy of Cancer)

   null (Ed.)

   Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107a pos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit. 

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4. Chemotherapeutics and CAR‐T Cell‐Based Immunotherapeutics Screening on a 3D Bioprinted Vascularized Breast Tumor Model

   https://doi.org/10.1002/adfm.202203966  

   Dey, Madhuri; Kim, Myoung Hwan; Dogan, Mikail; Nagamine, Momoka; Kozhaya, Lina; Celik, Nazmiye; Unutmaz, Derya; Ozbolat, Ibrahim T. (October 2022, Advanced Functional Materials)

   Abstract Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically‐relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic‐flow based 3D bioprinted multi‐scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre‐defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose‐dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2‐targeting chimeric antigen receptor (CAR) modified CD8⁺T cells for 24 or 72 h. Extensive CAR‐T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically‐relevant tumor model for future translation of anti‐cancer therapies to personalized medicine. 

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5. T cell immune responses deciphered

   https://doi.org/10.1126/science.abq1679  

   Nourmohammad, Armita (May 2022, Science)

   An immune response involves a coordinated orchestra of antigen-recognizing cells ( e.g. , T cells) and signaling molecules to mount a specific response against a pathogen. Although systems immunology offers a growing list of molecular interactions that are involved in antigen-specific immune responses, an understanding of how a response is mediated by different antigen characteristics is still lacking. On page 880 of this issue, Achar et al. ( 1 ) address this question by using a robotic platform to survey a broad range of functional T cell responses to different antigen stimulations. Using machine learning, they construct a simplified map that separates six different stereotypical classes of antigen-dependent immune responses. Understanding this antigen-encoding could help guide immunotherapy, including engineering chimeric antigen receptor (CAR)–T cells and identifying vaccine antigens. 

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