Cancer Cell, Volume 18 Supplemental Information The Therapeutic Effect of Anti-HER2/neu Antibody Depends on Both Innate and Adaptive Immunity SaeGwang Park, Zhujun Jiang, Eric D. Mortenson, Liufu Deng, Olga Radkevich-Brown, Xuanming Yang, Husain Sattar, Yang Wang, Nicholas K. Brown, Mark Greene, Yang Liu, Jie Tang, Shengdian Wang, and Yang-Xin Fu CONTENTS: Supplemental Figures Figure S1. Lymphocytes are increased after antibody. Figure S2. Efficacy of anti-her2/neu antibody is associated with T cells Figure S3. The some drugs suppress lymphocyte proliferation. Figure S4. The combination of anti-neu antibody plus Ad-LIGHT immunotherapy eradicates established neu+ solid tumors.
A B CD8 Tunnel 2 ** migg α-neu migg % in total cells 1 ** α-neu 0 CD8 NK 50μm Figure S1, related to Fig. 2. Lymphocytes are increased after antibody. A, WT BALB/c mice were inoculated s.q. with 5x10 5 TUBO cells (n=5/group) and treated with anti-neu (150 μg) or control Ig (150 μg) with or without the administration of a CD8-depleting antibody (clone 53.6.7, 200 μg) on days 14 and 21. *p<0.05 compared to anti-neu plus anti-cd8 antibody treated mice. Data from one of two experiments shown. B, Comparison of different anti-cd8 antibodies on DC depletion. Female BALB/c mice were injected with 200 μg of different anti- CD8 antibodies (clone YTS 169.4.2, 53.6.72 and 2.43.1) by i.p. Two days later, spleens were collected and digested with collagenase to get single cell suspension. Then these splenocytes were stained with indicated antibodies and analyzed by FACS Caliber. 2
A Gated CD11C + cells % CD8 + DC 9.4% 0.2% 3.6% 2.7% CD8 B Tumor volume(mm 3 ) 1200 1000 800 600 400 200 0 CD4 Control α-neu + α-cd8 migg + α-cd8 α-neu migg * 5 15 25 35 45 Days after tumor inoculation YTS 169.4.2 53.6.72 Average TIL C 45 35 25 15 5 P>0.03 Pre-Tx Post-Tx 2.43.1 Her2 + Her2 45 35 25 15 5 p=0.25 Pre-Tx Post-Tx Figure S2, related to Fig. 3. Increase of immune responses by anti-neu antibody is associated with T cells. A, WT BALB/c mice were inoculated s.q. with 5x10 5 TUBO cells (n=5/group) and treated with anti-neu (150 μg) or control Ig (150 μg) with or without the administration of a CD8-depleting antibody (clone 53.6.7, 200 μg) on days 14 and 21. *p<0.05 compared to anti-neu plus anti-cd8 antibody treated mice. Data from one of two experiments shown. B, Comparison of different anti-cd8 antibodies on DC depletion. Female BALB/c mice were injected with 200 μg of different anti-cd8 antibodies (clone YTS 169.4.2, 53.6.72 and 2.43.1) by i.p. Two days later, spleens were collected and digested with collagenase to get single cell suspension. Then these splenocytes were stained with indicated antibodies and analyzed by FACS Caliber. C. Increase of tumor infiltrating leukocytes in Herceptin treated breast cancer. Residual HER2 + tumors were removed 3 weeks after a 18-week treatment of
Taxotere with or without trastuzumab. We compared pre- and post-treatment breast tissue biopsies from patients with HER2 + (all treated with trastuzumab and Taxotere) and HER2 - (treated with Taxotere alone) breast tumors obtained from University of Chicago hospital s tumor specimen bank. Each sample was analyzed at 40X power, and the number of TIL in 10 fields was evaluated to calculate average for each sample. After antibody treatment, most lymphocytes surrounding tumor cells were CD8 + T cells (15.3 +/- 1.3 per high power field), while few CD4 + T cells infiltrated and surrounded tumor cells (1.3 +/- 1.2). 4
Figure S3, related to Figure 5. The some drugs suppress lymphocyte proliferation. WT BALB/c mice were treated with 150 μg of anti-neu with or without 60 mg/kg PTX. Cell populations were evaluated in (A) the spleen three days after treatment. (B) Total numbers of cells were also analysed on day three after treatment. (C) The proliferation of Lymphocytes three days after last treatment of PTX was calculated by staining Ki67 (PE-anti-ki67, BD). One of two representative experiments is shown.
Figure S4, related to Fig. 6. The combination of anti-neu antibody plus Ad-LIGHT immunotherapy eradicates established neu + solid tumors. A, TUBO-bearing WT BALB/c mice (n=6/group) were treated with a low dose of anti-neu antibody (50 μg) on days 18 and 25. 1x10 10 viral particle of Ad-LIGHT or control Ad-LacZ were injected intratumorally on day 18. **, p<0.005 compared to other groups. B, Tumor-free mice from the anti-neu antibody + Ad- LIGHT group described in (A) were re-challenged with 5x10 6 TUBO and 4T1 tumor cells s.c. at least 30 days after complete rejection of primary tumors. C and D, TUBO-bearing neu Tg mice (n=5/group) were treated with 100 µg of neu antibody on days 18 and 25 and 1x10 10 vp of Ad- LIGHT or control Ad-Null intra-tumorally on days 18 and 21. Average tumor volume (C) and survival (D) are shown. *, p < 0.05; **, p < 0.005. All experiments were done three times. 6