Abstract 1443: Sensitive detection and quantification of CAR-T cells in the treated patients
Authors: An Xiaoyu , Tu Xiaolong , Feng Xinhe , Zhang Jianjian , Song Dandan , Yu Haijuan , Li Henry 
Published: August 2019

Abstract
Background. Cell therapy for cancers has gained great momentum as a promising new cancer treatment approach. In particular, autologous T cells modified with a chimeric antigen receptor (CAR-T) has recently been regulatory-approved for the treatment of the patients of CD19+ acute B-cell leukemia [1][2]. However effective monitoring and characterizing the CAR-T cells after administration, vital to understand the treatment effect and its underneath mechanism, remain to be an urgent unmet need for CAR-T therapy in the clinics. There have been two major methods to monitor the presence of CAR-T in patients at present: a) flow cytometry (a single cell based assay recognizing the tag engineered in CAR molecule), but with extremely poor sensitivity ~1%); and b) Real-Time PCR (amplifying the engineered tag sequences), being more sensitive, but without the ability to identify and characterizing the individual CAR-T cells [3][4]. Thus, a method with high sensitivity and also the ability to characterize individual CAR-T is in high demand.
Methods. We are attempting to establish a novel platform, based on rare cell detection in pathology format, to detect and character rare CAR-T cells in patients treated by CAR-T using Rarecyte technology. First, 1x106 white blood cells (PBMC from C57BL/6 mice), spiked with the serial dilution of 0-250 human breast cancer MCF-7 cells, were smeared on a pathology slide and subjected to the staining by antibodies against human Cytokeratin 18 (Abcam, ab181597) and mouse CD45 (R&D, AF114), followed by either flow cytometry or Rarecyte system, in order to test the detection sensitivity and quantification dynamic ranges. Second, PBMC obtained from the DLBCL patient treated with CD19 CAR-T cells were (100 μL) were subjected to the same two analysis as above with the CAR-T specific staining (CD3-FITC, CD19-CAR-PE, DAPI (nucleus)), as controlled by the PBMC from healthy donors.
Results. Our preliminary data from the spiked study indicated that our method has high sensitivity down to 2 cells (&gt;90% recovery), and good linear range of quantification. The sensitivity (&lt; 1/1-million) is at least 3-orders of magnitude above that of flow cytometry (~1%). Our CAR-T treated clinical data demonstrated the detection and quantification of CAR-T in patients that flow cytometry cannot. In addition, the single CAR-T can be picked up for further characterization, e.g. single cell genomics.
Conclusions. Our method detection, monitoring and characterization of CAR-T offers an great opportunity in the clinical setting to monitor patient treatment and to understand the treatment mechanisms, that otherwise is unavailable. This could become an useful diagnostics to provide guidance on the treatment and prognosis of patients.
Citation Format: Xiaoyu An, Xiaolong Tu, Xinhe Feng, Jianjian Zhang, Dandan Song, Haijuan Yu, Henry Li. Sensitive detection and quantification of CAR-T cells in the treated patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1443.