Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The top LNP was subsequently optimized using an orthogonal design of experiments and the physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding an LNP formulation that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor cell killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform demonstrates potential for engineering a range of human CAR-M immunotherapies to treat various types of solid tumors.

This phase 1 study evaluated the safety and feasibility of fully human anti-mesothelin chimeric antigen receptor-T cells (huCART-meso) in patients with lung adenocarcinoma, ovarian cancer, and mesothelioma. huCART-meso cells were administered intravenously, intrapleurally, and/or intraperitoneally with or without lymphodepletion. The huCART-meso cells are autologous T cells engineered to express a fully human extracellular single-chain antibody with mesothelin specificity and 4-1BB/TCRζ intracellular signaling domains. All patients (n = 20) received a dose of 1-3 × 107 CAR+ cells/m2 (1 patient with lung adenocarcinoma, 5 patients with mesothelioma, and 14 patients with ovarian cancer). Peak expansion was observed within the first 14 days, and huCART-meso cells were detectable in 16/20 patients at day 21 and in 5 patients at 12 months, with 1 patient showing detectable cells for over 2 years. The most common serious adverse event was cytokine release syndrome (7/20 patients, 35%). The best overall response was stable disease (12/20 patients, 60%), with a maximum reduction in target tumor volume of 41%. The median overall survival was 26.1 weeks, and the median progression-free survival was 12.3 weeks. These results establish the feasibility, safety, and preliminary efficacy of huCART-meso therapy, providing a rationale for future trials, ideally in combination with other therapies.

We show continuous tumor exposure results in a loss of chimeric antigen receptor (CAR) T cell (CART) endocytic activity due to downregulation of Rab5. Loss of endocytic activity exacerbates the effects of trogocytosis, the bidirectional transfer of tumor target antigens and CARs between malignant cells and CARTs, resulting in CART dysfunction and fratricide. Constitutive expression of Rab5 within the CARTs reduced fratricide by reducing the amount of trogocytosed antigens on the cell surface, while simultaneously enhancing CAR availability through dissociation of CAR from target, recycling unbound CAR back to the plasma membrane, and limiting CAR capture by tumor cells. Rab5-expressing CARTs exhibited superior antitumor activity in both BCMA-CARTs isolated from the bone marrow of treated patients and mesothelin-specific CARTs in a solid tumor model. These studies uncover an unexpected relationship between endocytosis and CART function and suggest that pairing Rab5 with CAR expression could improve the clinical efficacy of CART therapy.

Abstract Allogeneic chimeric antigen receptor (CAR) T-cell therapies hold promise for certain malignant disorders, as they allow for scalability, on-demand availability, and enhanced product quality. Currently, allogeneic CAR T-cell approaches focus on deleting the αβ T-cell receptor complex and major histocompatibility complex (MHC)-I/II from CAR T cells to prevent graft-versus-host disease and rejection of allogeneic cells by the recipient’s immune system. However, genetic ablation of MHC-I leads to natural killer (NK) cell activation in recipients because of the missing-self response. Here, we demonstrate the successful generation of universal allogeneic CAR T (UCART) cells that evade the NK cell’s missing-self response and display similar performance to autologous CAR T cells both in vitro and in vivo. An HLA single-chain trimer platform enabled the demonstration that HLA-E presentation of a defined signal peptide sequence, VMAPRTLIL (designated as HLA-ESP-1C), confers resistance to NKG2A+ cell populations with negligible activation of NKG2C+ cell populations. Examination of additional receptor-ligand interactions that may impact NK cell activation confirmed that CD54 and CD58 ablation on UCART cells, in combination with HLA-ESP-1C expression, led to optimal resistance to heterogeneous NKG2A+/C+ NK cell populations. Finally, in a humanized mouse model reconstituted with allogeneic NK cells, we demonstrated that UCART19 cells promote stringent tumor control. Our work suggests an updated approach for allogeneic CAR T-cell therapy in clinical applications.

Chimeric antigen receptor T cells (CART) have transformed cancer therapy by inducing durable remissions of B cell cancers. Dogs spontaneously develop B cell cancers and autoimmune diseases with similar pathophysiology as humans. In a first-in-canine trial, anti-CD20 CART with canine 41BB-CD3ζ (cBBζ) cytoplasmic domains exerted selective pressure against CD20+ B cell lymphoma outgrowth but did not persist or induce remission. Here we show that canine CARTs incorporating human (h)BBζ demonstrate superior therapeutic function, mediated by FceRgI. HBBζ-CART showed greater cytolysis and CD8 T cell outgrowth than cBBζ-CART in repetitive killing assays and a canine B cell leukemia xenograft model. Transcriptional profiling revealed significant upregulation of FCER1G, encoding the Fc epsilon receptor I gamma chain (FceRgI), in CD8 hBBζ- versus cBBζ-CARTs. CRISPR-mediated FCER1G deletion and pharmacologic inhibition of Syk indicated that FceRgI enhances hBBζ-CART cytolytic potency via a Syk-NFkB axis, associated with increased granzyme B and IFNg expression. Correspondingly, bulk and sc-RNAseq analysis from human B cell lymphoma subjects receiving anti-CD19/20 hBBζ-CART revealed that FCER1G was significantly upregulated in CD8 CARTs from responders versus nonresponders. Collectively, these data indicate that FceRgI enhances CD8 CART therapeutic function and establishes a mechanistic understanding for strategies to enable durable remissions of B cell-mediated diseases across species. Veterinary and Comparative Immunology (VET)

Figure S6. Isoplexis-tSNE plots and polyfunctionality heatmap of single-cell secretome

Supplementary Figures and Figure Legends

Figure S5. Statistical analysis of the cytotoxicity of expanded-NK cells against different tumor targets and their degranulation.

Figure S3. Expressions of activating receptors and cell death ligands on freshly isolated-NK cells and 2 expanded-NK cells.

Figure S2. Representative flow plots of SSA against FSA and the expression of CD56 and CD3 of NK 2 cells as well as the gating strategy for counting NK cells

BackgroundMultiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.MethodsAs a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.ResultsSilenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).ConclusionsTitratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.

Comparative oncology has advanced cancer immunotherapy, although cellular mechanisms governing chimeric antigen receptor T cell (CART) therapy in canines are poorly understood. In a first-in-canine trial, anti-CD20 CART with canine 4-1BB-CD3ζ (cBBζ) domains induced CD20-negative lymphoma outgrowth but did not persist or deplete B cells. Here we show that canine CARTs incorporating human BBζ (hBBζ) demonstrate superior therapeutic function, mediated by FcεRγI. hBBζ-CART showed greater cytolysis and CD8 T cell outgrowth than cBBζ-CART in repetitive killing assays and a canine B cell leukemia xenograft model. Transcriptional profiling revealed upregulation of FCER1G and innate-like genes in CD8 hBBζ versus cBBζ-CARTs. CRISPR-mediated FCER1G deletion and pharmacologic Syk/NF-κB inhibition indicated that Syk-NF-κB signaling regulates FcεRγI-mediated enhancement of hBBζ-CART cytotoxicity, associated with increased granzyme B and IFN-γ/TNF-α production. Syk-NF-κB signaling promotes FcεRγI expression in hBBζ CARTs, and CAR-TCR interactions potentiate NF-κB signaling to upregulate FcεRγI and enhance CART function. These studies identify a potent therapeutic subset of innate-like canine CARTs induced by hBBζ signaling, which holds potential to improve both canine and human CART therapy.

Figure S1. mbIL-21 structure and the effect of K562 expressing CD40 on NK cell expansion

Antibody list for 28-color Symphony A5 NK pane

The efficacy of chimeric antigen receptor (CAR) T cells against solid tumors is limited. The molecular mechanisms underlying CAR T cell resistance are yet to be elucidated and new strategies need to be developed to improve treatment outcomes. Here we report that solid tumors respond to CAR T cells by upregulating the secretion of small extracellular vesicles carrying tumor antigens, which are horizontally transferred to CAR T cells, leading to antigen recognition and CAR T cell fratricide. Engineered CAR T cells armored with Serpin B9, a major granzyme B inhibitor, show decreased fratricide and increased vitality, tumor infiltration, and antitumor activity in female mice. Moreover, Serpin B9-armored CAR T cells show higher efficacy than parental CAR T cells in treating solid tumors when combined with the anti-programmed death 1 antibody. Our study demonstrates a mechanism that limits CAR T cell function and suggests an improved strategy in tumor treatment. Zhong et al. show that, in response to chimeric antigen receptor (CAR) T cell targeting, solid tumors increase the secretion of small extracellular vesicles carrying tumor antigens that are transferred to CAR T cells, resulting in fratricide and reduced antitumor activity.

Figure S7. Pearson correlation, heatmap, and GSEA analysis of Bulk RNA-Seq data

Figure S4. Expressions of the immune checkpoints on freshly isolated-NK cells and expanded-NK cells

AbstractHuman natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.