Volume 25, Issue 5, Pages 1248-1258 (May 2017) Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers  Chengcheng Zhang, Zhe Wang, Zhi Yang, Meiling Wang, Shiqi Li, Yunyan Li, Rui Zhang, Zhouxing Xiong, Zhihao Wei, Junjie Shen, Yongli Luo, Qianzhen Zhang, Limei Liu, Hong Qin, Wei Liu, Feng Wu, Wei Chen, Feng Pan, Xianquan Zhang, Ping Bie, Houjie Liang, Gabriele Pecher, Cheng Qian  Molecular Therapy  Volume 25, Issue 5, Pages 1248-1258 (May 2017) DOI: 10.1016/j.ymthe.2017.03.010 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Procedure of Clinical Trial and Characteristics of CAR-T Cells (A) Trial schedule. Basic imagological examinations and blood tests were needed for enrollment screening. Between the completion of lymphodepletion and initiation of CAR-T infusion, blood tests were administered on day 0 to reconfirm whether the patient could tolerate cell infusion. CAR-T infusion was executed on days 1, 2, and 3, and 10%, 30%, and 60% of the total cell dose were administrated, respectively. (B–E) Characteristics of CAR-T cells were analyzed by FCM results for quality control of the CAR-T cell products. For each product, the ratios of T cells, NK cells, NKT cells, and CAR-T cells were measured before infusion. There was no difference in CD4/8 between CAR+ cells and CD3+ cells. Tm cells and checkpoint inhibitors were also detected. E, enrollment. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 CRP and Cytokines Detection for Immune Monitoring CRP levels were divided into two groups according to the DLs. (A and B) The DL1/2/3 (A) and DL4/5 (B) levels are shown. (C) All cytokines and CRP of Ps 7 (left) and 10 (right) are shown. Most factors exhibited a similar trend as an immune response. (D) Spearman’s correlation of IFN-γ and CRP with other cytokines. n = 93. Each correlation coefficient and significance is shown. IFN-γ correlated with all factors except IP-10 and MIG. (E and F) The IFN-γ and CRP levels of Ps 7 (E) and 10 (F) receiving DL 5 were compared. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Clinical Evaluation of CAR-T Therapy (A) Most patients showed the decreased CEA levels in response to CAR-T therapy. (B) PET/CT assessment of P9-2 receiving DL5 before (left) and 4 weeks after (right) CAR-T infusion. (C) MRI assessment of P10 receiving DL4 before (left) and 4 weeks after (right) CAR-T infusion. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Detection of DNA Copies of CAR-T Cells in Peripheral Blood Detection of DNA copies of CAR-T cells in peripheral blood. The panels (A)–(E) are represented as an individual patient. DNA copies were detectable after a high dose of CAR-T infusion and proliferation of CAR-T cells were also observed in some patients a few weeks after CAR-T infusion. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 CAR-T Trafficking in Tumor Tissues (A) CD3+ cells were detected in metastatic tumor tissues obtained at the fourth week after CAR-T infusion by IHC staining. Ps 5 (left), 6 (middle), and 7-2 (right) receiving DL2, DL3, and DL5 are shown, respectively. (B) DNA copies of CAR-T cells in tumor tissue of Ps 6 and 7-2 are presented. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 Immune-Suppressive Factor Levels after CAR-T Infusion and CAR-T Distribution in Tumor Site (A and B) Follow-up detection of MDSCs, Tregs, the PD-1 proportion of PBMCs, and T cells in peripheral blood. n = 10. The CD8− gate was regarded as CD4+. (C) CD3 staining of puncture tissue of P7-2. T cells showed much greater accumulation in the mesenchyma than the parenchyma. Molecular Therapy 2017 25, 1248-1258DOI: (10.1016/j.ymthe.2017.03.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions