CD20-specific nanoCAR T-cells incubated with CD70+ JD033T organoid line

CD70-specific nanoCAR T-cells incorporating 4-1BB as costimulatory domain show the highest efficacy

CD70-specific nanoCAR T-cells incubated with CD70+ JD033T organoid line

Differentially expressed genes in nanoCAR T-cells from cluster CD8WT vs CD4WT

CD70KO improves the functionality of CD70-specific nanoCAR T-cells.

Differentially expressed genes in bulk RNAseq data from CD70-4_1BB:ζ KO vs CD70-4_1BB:ζ WT

CD70KO has no effect on CD20-specific nanoCAR T-cells.

Differentially expressed genes per cluster

CD70 WT and KO CD70-specific nanoCAR T-cells have a distinct transcriptional profile after production.

Differentially expressed genes in nanoCAR T-cells from cluster CD8KO vs CD4KO

CD70 expression on nanoCAR T-cells.

Differentially expressed genes in nanoCAR T-cells from cluster CD4KO vs CD4WT

Differentially expressed genes in nanoCAR T-cells from cluster CD8KO vs CD8WT

Surface expression of CD70-BFP and nanoCAR-eGFP fusion proteins.

Single cell analysis of nanoCAR T-cell treated mice result in 12 defined clusters.

CD70-specific nanoCAR T-cells incubated with CD70- JD041T organoid line

Differential gene expression reveals distinct differences between the clusters.

Abstract CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months nation. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted states. Three of six vaccinated patients experience disease recurrence during the follow-up period Collectively, these data support the feasibility, safety, and immunogenicity of this treatment NSCLC.