Single-Cell Analysis of Target Antigens of CAR-T Reveals a Potential Landscape of "On-Target, Off-Tumor Toxicity"

Abstract

Cellular immunotherapy represented by CD19-directed chimeric antigen receptor T (CAR-T) cells has achieved great success in recent years. An increasing number of CAR-T therapies are being developed for cancer treatment, but the frequent and varied adverse events, such as "on-target, off-tumor toxicity", limit CAR-T application. Here, we identify the target antigen expression patterns of CAR therapies in 18 tissues and organs (peripheral blood mononuclear cells, bone marrow, lymph nodes, spleen, heart, ascending aortic tissue, trachea, lung, skin, kidney, bladder, esophagus, stomach, small intestine, rectum, liver, common bile duct, and pancreas) from healthy human samples. The atlas determines target antigens expressed on some normal cell types, which facilitates elucidating the cause of "on-target, off-tumor toxicity" in special tissues and organs by targeting some antigens, but not others. Moreover, we describe the target antigen expression patterns of B-lineage-derived malignant cells, acute myeloid leukemia (AML), and solid tumors. Overall, the present study indicates the pathogenesis of "on-target, off-tumor toxicity" during CAR therapies and provides guidance on taking preventive measures during CAR treatment.

Keywords: chimeric antigen receptor T cells (CAR T cells); malignant cells; off-tumor toxicity; on-target; single-cell RNA sequencing; target antigen.

Figure 1

Identification of the expression pattern of CAR target antigens in normal tissues and organs at single-cell transcriptome level. (A) Schematic representation of selected tissues and organs for analyzing target antigens. (B) Dot plot shows the expression levels of CAR target antigens in PBMCs. (C) Dot plot shows the expression levels of CAR target antigens in BM. UMAP projections of heart-derived cells (D), lung-derived cells (E), kidney-derived cells (F), and liver-derived cells (G), colored by clusters, and dot plots showing the expression level of CAR target antigens in different clusters.

Figure 2

Expression patterns of B-lineage-specific antigens (CD19, MS4A1, CD22, TNFRSF17, CD38, SDC1, SLAMF7, GPRC5D, and TNFRSF8) in human normal tissues and organs. (A) Dot plots show the expression levels of B-lineage-specific antigens in PBMCs, BM, LN, and SP at mRNA level or protein level. (B) Flow cytometric analysis of abandoned human PBMCs after medical examination. Representative FACS dot plots for CD30 in normal CD8⁺ T cells (DAPI⁻CD4⁻CD8⁺), CD4⁺ T cells (DAPI⁻CD4⁺CD8⁻ but excluding Treg), and Treg cells (DAPI⁻CD4⁺CD8⁻CD127^low/−CD25⁺). Frequency histogram of CD30+ cells in CD8⁺ T cells, CD4⁺ T cells, and Treg cells. Violin plots show the expression level of B-lineage-specific antigens in heart-derived clusters (C), lung-derived clusters (D), liver-derived clusters (E), and pancreas-derived clusters (F). (G) Schematic diagram of high B-lineage-specific antigen-expressing cell types in different tissues and organs.

Figure 3

Expression pattern of AML antigens (CD33, IL3RA, and CLEC12A) in human normal tissue and organs. CD33, IL3RA, and CLEC12A-expressing proportions (expression value >0) of PBMC/BM-derived cells (A), heart-derived cells, ascending aortic tissue-derived cells (B), lung-derived cells (C), skin-derived cells (D), liver-derived cells, common bile duct-derived cells, pancreas-derived cells (E), are illustrated in UMAP plots. (F) Schematic diagram of high AML antigen-expressing cell types in different tissues and organs.

Figure 4

Expression patterns of solid tumor antigens (GPC3, B4GALNT1, and ERBB2) in human normal tissues and organs. (A) GPC3 and ERBB2-expressing proportions (expression value >0) of PBMC-derived cells. (B) Violin plots show the expression levels of GPC3, B4GALNT1, and ERBB2 in heart-derived clusters and ascending aortic tissue-derived cells. GPC3, B4GALNT1, and ERBB2-expressing proportions (expression value >0) of lung-derived cells (C), kidney-derived cells (D), and pancreas-derived cells (E). (F) UMAP plots show the expression level of ERBB2 in small intestine-derived cells and rectum-derived cells. (G) UMAP plots show the expression levels of GPC3, B4GALNT1, and ERBB2, in skin-derived clusters. (H) Schematic diagram of high GPC3/B4GALNT1/ERBB2-expressing cell types in different tissues and organs.

Figure 5

The difference of expression patterns of solid tumor antigens (GPC3, B4GALNT1, and ERBB2) in malignant cells and nonmalignant cells. (A) Dot plot shows the expression level of target antigens (CD19, MS4A1, CD22, TNFRSF17, CD38, SDC1, SLAMF7, and TNFRSF8) in B lineage-related cells derived from lymphomas (DLBCL, FL, and tFL) and rLN. (B) Dot plot shows the expression level of target antigens (CD33, IL3RA, and CLEC12A) in hematopoietic stem/progenitor-like cells obtained from BM samples of AML patients and healthy donors, and in normal liver/heart EC clusters. “AML314.D31” represents the sample of patient AML314 after 31 days of treatment. BM5.34p means the sample of BM CD34-positive cells derived from healthy donor BM5, and BM5.34p38n means the sample of BM CD34-positive and CD38-negative cells derived from healthy donor BM5. (C) The expression level of IL3RA in liver sinusoidal endothelial cells and macrovascular endothelial cells. (D) GPC3 expression levels in adjacent liver and hepatocellular carcinoma at scRNA-seq level. (E) GPC3 and ERBB2 expression levels of the epithelial lineages in normal primary gastric tissues and gastric cancers.