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case_images: Clinical examinations in the sequential injection of B7-H3 CAR-T cell therapy. (A) Histological analysis of basal cell carcinoma sections by hematoxylin and eosin (H&E) and anti-B7-H3 staining for diagnosis. Slides were analyzed and scored by two individual pathologists independently. (B) Schematic diagram of B7-H3 CAR vector. VL, variable L chain; VH, variable H chain; TM, transmembrane domain. (C) The protocol of B7-H3 CAR-T injection. Intratumoral injection was performed at two tumor sites for a total of three times, on days 1, 14, and 62. (D) Detection of CAR-T (CD3+CAR+) cells in peripheral blood during immunotherapy. (E) Flow cytometry analysis of CD3+CAR+ cells in peripheral blood on days 67 and 69 after CAR-T cell injection. FITC-labeled recombinant B7-H3 protein was used to detect B7-H3-CAR transduced CD3+ cells.
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case_images: Immunohistochemical staining of the tumor tissue showed positive for B7-H3, with 80% positivity and ++/+++ intensity ( Figure 1A ); thus, the patient was accepted to enroll in the clinical trial of B7-H3-CAR-T immunotherapy. ZL 2020 1 0622482.X) was used to obtain the 4-1BB-based second-generation CAR-T ( Figure 1B ). In this course of CAR-T therapy, the patient received autologous anti-B7-H3 CAR-T cells through intratumoral injection three times with dose escalation on day 1, day 14, and day 62 ( Figure 1C ). Only 2.5% of total T cells in peripheral blood was CAR positive at day 5 after the third injection ( Figure 1D ), and the percentage of CAR-T cells was decreased to undetectable levels 2 days later ( Figures 1D, E ).
case_images: Clinical manifestation of multiple basal cell carcinoma after CAR-T cell therapy. (A) Abdominal tumor at site 1. After each injection, the tumor gradually shrank in size. (B) The large lesion of forehead tumor at site 2 became dry from original ulcer and bleeding. (C) Staining of B7-H3, CD3, and Ki67 of abdominal tumor at site 1 by immunohistochemistry (IHC). B7-H3+ cells showed sub-epithelial localization and were widely observed within the tumor tissue. CD3 staining showed an enrichment of T cells at the injection site within the tumor. (D) Expressions of TGF-beta and PDL1 within the tumor tissue examined by IHC. All IHC slides were analyzed and scored by two individual pathologists independently.
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case_images: The tumor-killing capacity of B7-H3-CAR-T cells was tested in vitro and in vivo; all results indicated the promising potential of CAR-T cells in killing B7-H3-positive tumor cells and controlling tumor growth in the tumor-bearing NSG mouse model ( Supplementary Figure 2 ). After three doses of injections, the volume of original lesion in the abdomen (site 1) was decreased by 40% (from 1.8 cm x 0.6 cm to 1.3 cm x 0.5 cm) in size, shrank from bulging to flat ( Figure 2A ). The large lesion in the forehead (site 2) became dry from original ulcer and bleeding ( Figure 2B ). IHC analysis indicated that the percentage of B7-H3-positive cells was decreased from 80% to about 40%, and the B7-H3 intensity was decreased from ++/+++ to + after the third injection of CAR-T cells ( Figure 2C ). Meanwhile, CD3 T cells were enriched in the injection site within the tumor; however, majority of these T cells failed to infiltrate into neighboring tumor area, which was stained positive for B7-H3 and Ki67 ( Figure 2C ). Subsequently, the expressions of two common inhibitory signaling molecules TGF-beta and PD-L1 were examined, and high expression level of TGF-beta was observed in the tumor area upon CAR-T treatment ( Figure 2D ).