Supplementary figure 1. Systemic delivery of anti-cd47 antibody controls tumor growth in

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Donald Crawford
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1 T u m o r v o lu m e (m m 3 ) P e rc e n t s u rv iv a l P e rc e n t s u rv iv a l Supplementary data a T im e a fte r tu m o r in o c u la tio n (d ) b c * * * T im e a fte r tu m o r in o c u la tio n (d ) T im e a fte r tu m o r in o c u la tio n (d ) Supplementary figure 1. Systemic delivery of anti-cd47 antibody controls tumor growth in syngenic mice. (a) Balb/c mice (n=5/group) transplanted s.c. with 5x1 6 A2 cells were treated intraperitoneally with 4μg of anti-mcd47or RatIg on days 7, 9, 11 and 13. Survival curve is shown. (b-c) C57/BL6 mice (n=1/group) transplanted s.c. with 5x1 5 MC38 cells were treated intraperitoneally with 4μg of anti-mcd47 or RatIg control on days 7, 9, 11 and 13;Tumor size (b) and survival curve (c) are shown. One representative experiment out of two independent experiments is depicted.***p <.1

Supplementary figure 2. The anti-tumor effect of systemic anti-cd47 antibody depends on CD8 + T cells. (a) Balb/c mice (n=5/group) transplanted s.c. with 5x1 6 A2 cells were treated intraperitoneally with 4μg of anti-mcd47or RatIg on days 7, 9, 11 and 13.

2 Supplementary figure 2. The anti-tumor effect of systemic anti-cd47 antibody depends on CD8 + T cells. (a) Balb/c mice (n=5/group) transplanted s.c. with 5x1 6 A2 cells were treated intraperitoneally with 4μg of anti-mcd47or RatIg on days 7, 9, 11 and 13. CD8 + T cells were depleted with 3μg anti-cd8 antibody since day 6. (b) C57/BL6 mice (n=1/group) transplanted s.c. with 5x1 5 MC38 cells were treated intraperitoneally with 4μg of anti-mcd47 or RatIg control on days 7, 9, 11 and 13; CD8 + T cells were depleted with 3μg anti-cd8 antibody since day 6. One representative experiment out of two independent experiments is depicted.*p <.5, **p <.1***, p <.1

3 Supplementary figure 3. Tumor associated macrophage is not essential in response of anti-cd47 treatment. (a) The single cell suspensions from established MC38 tumors were sorted into CD45 + CD11c -/+ CD11b + Ly6c - F4/8 + (Macrophage) and CD45 + CD11c + CD11b -/+ Ly6C - F4/8 - (DC) populations. mrna level of mafb and Zbtb46 in different cell subsets were quantified by real-time PCR assay. Representative data are reported as mean copy numbers ±s.e.m. after intrasample normalization to the levels of reference gene hprt in two independent experiments. (b) WT C57BL/6 mice (n=5/group) were transplanted s.c. with 5x1 5 MC38 cells and treated intratumorally with 5μg of anti-cd47 or RatIg on days 1 and 13.Macrophages were deleted intratumorally with 1μg of anti-csf1 or RatIg on days 1 and 16. One representative experiment out of two independent experiments is depicted. **p <.1***, p <.1

4 IF N - s p o ts /2 x 1 4 c e lls IF N - s p o ts /1 6 ce lls a b 6 4 * * * * * * * * * * 2 5 M a c ro p h a g e D C w /o T U B O Supplementary figure 4. DCs are the major antigen presenting cells in response of anti- CD47 treatment. (a) BALB/c nude mice (n=6) were injected s.c. with 2x1 6 A2-HA and treated with 5 μg of either anti-mcd47 or ratig on day 1. Mice were sacrificed 4 days after the antibody treatment and DLNs were collected. 4x1 3 DC or macrophages were isolated and incubate with 2x1 4 CL4 CD8 + T cells. 48 hours later, IFN-γ-producing cells were enumerated by ELISPOT assay. Result was expressed as number of spots per 2x1 4 CD8 + T cells. (b) MMTV-Her2/Neu transgenic mice (n=4/group) were orthotopic injected with 1 5 breast cancer cell line TUBO into the mammary fat pads. When tumors were established, mice were treated with 5 μg of either anti-mcd47 or ratig on day 1. Mice were sacrificed 4 days after the antibody treatment. 3x1 4 Tumor infiltrating DCs and macrophages were isolated and co-cultured with isolated 3x1 5 CD8 + T cells TUBO vaccined mice. 48 hours later, IFN-γ-producing cells were enumerated by ELISPOT assay. Data are reported as means ±s.e.m. One representative experiment out of two independent experiments is depicted.**p <.1, ****p <.1(unpaired Student's t test).

5 Tumor volume (mm 3 ) 5 RatIg 4 Anti-CD47 Anti-IFNAR + ratig 3 Anti-IFNAR1 + anti-cd ** Time after tumor inoculation (d) Supplementary figure 5. IFNAR1 is required for anti-cd47 mediated tumor inhibition. Balb/c mice (n=4)were injected s.c. with 3x1 6 A2 cells and treated intratumorally with 5μg of anti- CD47 or isotype control rat-ig on days 11 and 14. 5μg anti-ifnar1 was administered intratumorally on days and 2 after anti-cd47 treatment. **p <.1. One representative experiment out of two independent experiments is depicted.

6 a 1% Serum No Serum DC AnnexinV b Macrophage AnnexinV Supplementary figure 6. Serum deprivation induces apoptotic cell death of BMDCs. Day6 BMDCs (a) and BMDMs (b) were harvested and co-cultured with MC38-OTI cells at the ratio of 1:1 in the presence of fresh 5ng/ml GM-CSF (a) or 1ng/ml M-CSF(b) overnight. Apoptosis of DC or macrophage was assessed by FACS analysis after incubation with Annexin-V APC.

7 Table 1. Reduced memory protection after chemotherapy Chemotherapy Growth After Rechallenge Antibody dose Drug Dosage Schedule Growth/Total(%) 5 μg * 2 None 1/9 (11) 5 μg * 2 PTX 4mg/kg* 3 3 d after 4/5 (8) 5 μg * 2 PTX 4mg/kg * 1 1 d before 1/5 (2) 5 μg * 2 CTX 6mg/kg * 3 3 d after 3/6(5) 5 μg * 2 CTX 6mg/kg * 1 1 d before /4 ()

Tumor targeting antibodies bridge innate to adaptive immunity. Yang-Xin Fu MD PhD Professor of Pathology and Immunology UT Southwestern

Tumor targeting antibodies bridge innate to adaptive immunity Yang-Xin Fu MD PhD Professor of Pathology and Immunology UT Southwestern Tumor targeting antibodies are screened and selected to kill tumor