Volume 143, Issue 5, Pages 1375-1384.e5 (November 2012) Redirected T Cells That Target Pancreatic Adenocarcinoma Antigens Eliminate Tumors and Metastases in Mice  Amit Maliar, Charlotte Servais, Tova Waks, Markus Chmielewski, Ron Lavy, Peter Altevogt, Hinrich Abken, Zelig Eshhar  Gastroenterology  Volume 143, Issue 5, Pages 1375-1384.e5 (November 2012) DOI: 10.1053/j.gastro.2012.07.017 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 The experimental system: T-bodies engineered with a TAA-specific CAR. (A) CAR design. Schematic representation of the CAR used to redirect the T-bodies. α714 was built in a similar manner to α743. (B) FACS analysis of cultured Capan-1 cells using (I) anti-CD24 antibodies or (II) anti-Her2/neu and isotype controls (either murine monoclonal antibody [for CD24] or human monoclonal antibody [for Her2/neu]). (C) Cytotoxic activity of T-bodies using luciferase-labeled Capan-1 cells in a 19-hour assay at an effector-to-target ratio of 0.3:1. αTNP (Sp6) T-bodies were used as irrelevant effector controls. All of the T-bodies displayed significant cytotoxicity compared with the control (P < .05). There was no significant difference between the HER2-743 and CD24 T-bodies, both displaying a significant difference from HER2-714 (P < .05). (D) Interferon gamma production by various T-bodies following 48-hour coculture with Capan-1 cells was determined by enzyme-linked immunosorbent assay. All T-bodies secreted significantly higher levels of interferon gamma compared with the Sp6 (P < .05). Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Antitumor activity of intratumorally injected T-bodies. Established subcutaneous Capan-1 tumors were treated with 4 intratumoral injections of T-bodies (1 × 107 T-bodies per injection) on alternate days. Numbers in parentheses represent the number of tumor-free mice in each group (n = 7) 70 days after onset of T-body treatment. A–C represent the tumor volume of individual mice, and D shows the average tumor diameter of each group, as represented by the cubic root of the volume. Transduction efficiencies for each T-body in this experiment were as follows: irrelevant (anti-TNP), 37%; anti-CD24, 30%; anti-HER2-743, 46%. This experiment represents one of 3 experiments that gave similar results. Two-way analysis of variance between the treated groups showed a statistically significant difference (P < .001) versus the group treated with T-bodies of irrelevant specificity. Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Antitumor activity of systemically administered T-bodies. Total bioluminescence counts of established (3 weeks) orthotopically transplanted Luciferase-Capan-1 tumors in SCID mice treated with 3 consecutive intravenous injections of 1 × 107 T-bodies of CD24, HER2-743, or irrelevant TNP specificity as control. The figures in parentheses represent the number of tumor-free mice in each treatment group (n = 7) 63 days after the onset of T-body treatment. A–C show the bioluminescence of individual mice, and D shows the cubic root of average bioluminescence of each group. The area shaded in gray at the bottom of each graph denotes the region below the detection threshold. From day 21, significant differences were observed between the anti-CD24–treated group and the group receiving irrelevant T-body (P < .001) and between the anti-CD24 group and the anti–HER2-743 T-body group (P < .001). The response to anti-HER2-743 was significantly different from the irrelevant group only between days 14 and 21 (P < .05). Transduction efficiencies in this experiment were as follows: irrelevant, 39%; anti-CD24, 33%; anti-HER2, 38%. Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Tumor elimination by T-bodies: effect of repeated administration. Luciferase-labeled Capan-1 cells were orthotopically grafted into the pancreas. After 4 weeks, mice were irradiated (200 rad) and then injected systemically with 3 consecutive intravenous injections (week 0) of 1 × 107 T-bodies with αHER2-4D5, αCD24 CAR, or irrelevant specificity. During week 6, 5 mice in the control (irrelevant) group that initially received TNP-specific T-bodies and had a large tumor burden were treated with cyclophosphamide, followed by 3 consecutive intravenous injections of 1 × 107 fresh HER2-4D5 specific T-bodies (green line) 1 day after preconditioning. Eight weeks after the first T-body administration, all surviving mice were preconditioned again with irradiation and re-treated the next day with the CD24-specific T-bodies (yellow line). The part of the experiment testing initial treatment was repeated 3 times with similar results; the part testing re-treatment was performed once. Results shown are from the single experiment that continued to re-treatment. Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Expression of target antigens on Wapac xenografts. The level and frequency of expression of Her2/neu and CD24 in Wapac-4 and Wapac-5 xenografts from different generations were determined by immunofluorescence using either (A) cells or (B) fixed sections. (A) Cells dissociated from xenograft tissues were fluorescently labeled with the specific antibodies or anti-DNP as isotype control (as detailed in Materials and Methods). To separate between the human and murine cells, double staining was performed with anti-human EpCAM APC-labeled antibody and anti-human CD24, SWA-11. The percentage of human cells expressing surface Her2/neu and CD24 and staining intensity is shown. (B) Paraffin sections were stained with an anti-human Her2/neu antibody and a Cy5-labeled secondary antibody. CD24 and CD44 were detected by a Pacific Blue–conjugated anti-human CD24 and Alexa 488–labeled anti-human CD44 antibody. Stained sections were mounted in Immunoselect Antifading Mounting Medium containing PI (Dianova, Hamburg, Germany). Slides were recorded using a Carl Zeiss LSM710 microscope (Carl Zeiss, Oberkochen, Germany) and analyzed for ErbB2, CD24, and CD44 expression using ImageJ version 1.41 (National Institutes of Health). Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 T-body treatment of Wapac xenografts. Groups of SCID mice bearing palpable orthotopically transplanted (A) Wapac-4 and (B) Wapac-5 xenografts were preconditioned (200 rad irradiation) and a day later were injected systemically (intravenously) for 3 alternate days with 1 × 107 T-bodies of TNP, Her2/neu, or CD24 specificity. Mice were monitored for tumor growth. Surviving mice were recorded as dead when tumor volume reached 2 cm3. The survival data shown represent a single experiment. The differences between the survival of Wapac-4– and Wapac-5–bearing mice treated with CD24 specific T-bodies versus the Sp6 control were significant (P < .05 and P < .0005, respectively). The differences between the survival of Wapac-4– and Wapac-5–bearing mice treated with Her2/neu-specific T-bodies versus the Sp6 control were significant only in Wapac-5–bearing mice (P < .0005). Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Pattern of metastatic spread of orthotopically transplanted Capan-1 xenograft. (A) Luciferase-labeled Capan-1 human PAC cells were injected into the murine pancreas, and luminescence images were taken by IVIS 13 weeks after transplantation. Images shown are of the primary tumor and distant metastases in the liver and left inguinal lymph node. (B) The same mouse after death and removal of the peritoneum. Black arrows indicate liver metastases, yellow arrows indicate primary tumor with high local invasiveness to surrounding tissues, and the blue arrow indicates inguinal lymph node metastasis. Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Soluble Her2/neu does not block CAR-redirected T-cell activation. T-bodies (104 cells per well) with the high-affinity CAR Her2-714, the moderate affinity 743 CAR, or without (w/o) CAR, respectively, were coincubated with the Her2/neu+ Sk-Ov-3 tumor cell line (5 × 104 cells) for 48 hours in the presence of added soluble Her2/neu-Fc fusion protein at increasing concentrations. Specific cytotoxicity of T-bodies toward tumor cells and interferon gamma secretion were determined and set at 100% in the absence of added Her2 protein; alterations in cytotoxicity and interferon gamma levels are shown. Gastroenterology 2012 143, 1375-1384.e5DOI: (10.1053/j.gastro.2012.07.017) Copyright © 2012 AGA Institute Terms and Conditions