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NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity

ABSTRACT: CD93 CAR T cells eliminate AML in preclinical models without targeting hematopoietic progenitor cells, and a NOT-gated CAR engineering strategy mitigates on-target, off-tumor toxicity to endothelial cells. Chimeric antigen receptor (CAR) T cells hold promise for the treatment of acute myeloid leukemia (AML), but optimal targets remain to be defined. We demonstrate that CD93 CAR T cells engineered from a novel humanized CD93-specific binder potently kill AML in vitro and in vivo but spare hematopoietic stem and progenitor cells (HSPC). No toxicity is seen in murine models, but CD93 is expressed on human endothelial cells, and CD93 CAR T cells recognize and kill endothelial cell lines. We identify other AML CAR T-cell targets with overlapping expression on endothelial cells, especially in the context of proinflammatory cytokines. To address the challenge of endothelial-specific cross-reactivity, we provide proof of concept for NOT-gated CD93 CAR T cells that circumvent endothelial cell toxicity in a relevant model system. We also identify candidates for combinatorial targeting by profiling the transcriptome of AML and endothelial cells at baseline and after exposure to proinflammatory cytokines.

Significance:

CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies. See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549

SUBMITTER: Richards R

PROVIDER: S-EPMC8580619 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Project description:BackgroundLiver hepatocellular carcinoma (LIHC) is one of the malignant tumors with high incidence as well as high death, which is ranked as the sixth most common tumor and the third highest mortality worldwide. CD93, a transmembrane protein, has been widely reported to play an important role in different types of diseases, including many types of cancer by mainly functioning in extracellular matrix formation and vascular maturation. However, there are few researches focusing on the role and potential function of CD93 in LIHC.MethodsIn this study, we comprehensively analyzed the relationship between CD93 and LIHC. We not only discovered transcriptional expression of CD93 in LIHC by using the TIMER, GEPIA and UALCAN database, but also performed WB and IHC to verify the protein expression of CD93 in LIHC. Meantime, Kaplan-Meier Plotter Database Analysis were used to assess the prognosis of CD93 in LIHC. After knowing close correlation between CD93 expression and LIHC, there were STRING, GeneMania and GO and KEGG enrichment analyses to find how CD93 functions in LIHC. We further applied CIBERSORT Algorithm to explore the correlation between CD93 and immune cells and evaluate prognostic value of CD93 based on them in LIHC patients.ResultsThe transcriptional and protein expression of CD93 were both obviously increased in LIHC by above methods. There was also a significant and close correlation between the expression of CD93 and the prognosis of LIHC patients by using Kaplan-Meier Analysis, which showed that LIHC patients with elevated expression of CD93 were associated with a predicted poor prognosis. We found that the functions of CD93 in different cancers are mainly related to Insulin like growth factor binding protein 7 Gene (IGFBP7)/CD93 pathway via STRING, GeneMania and functional enrichment analyses. Further, our data obtained from CIBERSORT Algorithm suggested CD93 was also associated with the immune response. There is a close positive correlation between CD93 expression and the infiltration levels of all six types of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). Importantly, CD93 can affect the prognosis of patients with LIHC partially due to immune infiltration.ConclusionOur results demonstrated CD93 may be a candidate predictor of clinical prognosis and immunotherapy response in LIHC.

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NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity

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