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Volume 21, Issue 4, Pages 904-912 (April 2013) Treatment of Metastatic Renal Cell Carcinoma With CAIX CAR-engineered T cells: Clinical Evaluation and Management of On-target Toxicity Cor HJ Lamers, Stefan Sleijfer, Sabine van Steenbergen, Pascal van Elzakker, Brigitte van Krimpen, Corrien Groot, Arnold Vulto, Michael den Bakker, Egbert Oosterwijk, Reno Debets, Jan W Gratama Molecular Therapy Volume 21, Issue 4, Pages 904-912 (April 2013) DOI: 10.1038/mt.2013.17 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Consort diagram showing compliance to eligibility criteria and protocol treatment. CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; mAb, monoclonal antibody; MTD, maximum tolerated dose. Molecular Therapy 2013 21, 904-912DOI: (10.1038/mt.2013.17) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 T cell infiltration in liver. Immunohistochemistry of biopsy of liver taken from patient 8 at day 4, i.e., 2 days after last CAR T cell infusion (original magnification ×200, all sections), showing liver parenchyma (L), portal triangle (P) with bile duct (B) and stained for expression of CAIX antigen and for CD3, CD4, and CD8 T cells. CD8 T cells were lining at the basal side (arrowheads) or infiltrating (arrow) the epithelium of the bile duct. CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor. Molecular Therapy 2013 21, 904-912DOI: (10.1038/mt.2013.17) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 CAR-specific IFN-γ production by post-treatment PBMC correlates with pre-infusion CAR T cell IFN-γ production potency. Maximum IFN-γ production by CAR T cells in 1 × 106 PBMC per 24 hours is calculated from the IFN-γ production of pre-infusion CAR T cells and the proportion of CAR T cells in PBMC and is expressed as pg IFN-γ/106 PBMC/24 hours, see Supplementary Table S3b. The measured IFN-γ production in post-treatment PBMC correlates with the calculated maximum IFN-γ production by CAR T cells in PBMC; this correlation is significant (Pearson's correlation analysis: r = 0.452; P = 0.04; regression line: y = 23 + 0.32x). CAR, chimeric antibody receptor; IFN, interferon; PBMC, peripheral blood mononuclear cell. Molecular Therapy 2013 21, 904-912DOI: (10.1038/mt.2013.17) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Blood cytokine levels. Blood cytokine levels were assessed in plasma samples obtained before, during, and following CAIX CAR T cell therapy by cytokine bead arrays. For both treatment cycles, pre-treatment (day 1) and peak levels are shown for individual patients. Peak day is presented as median and range. CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. Molecular Therapy 2013 21, 904-912DOI: (10.1038/mt.2013.17) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions