A novel therapeutic strategy to rescue the immune effector function of proteolyticallyinactivated

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1 A novel therapeutic strategy to rescue the immune effector function of proteolyticallyinactivated cancer therapeutic antibodies Supplemental Data File in this Data Supplement: Supplementary Figure 1-4 Figure S1: IdeS expression in BT474 breast cancer cells; Figure S2: Detection of MMPs in cancer cell lysates and culture supernatant; Figure S3: scigg-t was not in the SKBR-3 cell culture medium; Figure S4: Detection of immune cells infiltration in mice by flow cytometry. Legends for supplemental figures Figures for supplemental figures 1-4. Supplemental Data Figure S1. IdeS expression in BT474 breast cancer cells. (A). The IdeS expression cassette. SP, signal peptide (SP), aa1-19, CD8 leader sequence; Flag, flag tag, aa20-27; IdeS (29-339), aa28-338, the IdeS encoding gene with codon usage optimized for mammalian expression. (B). Amino sequence of the IdeS expression cassette. The 19 amino acid residues underlined in blue is the CD8 leader sequence. The 8 amino acid residues underlined in red is the flag tag. (C). DNA sequence of the IdeS expression cassette. Nucleotides underlined in blue is the CD8 leader sequence. Nucleotides underlined in red is the flag tag. BamHI and HindIII restriction sites were added at the 5 and 3 of the target gene for cloning, respectively. Figure S2. Detection of MMPs in cancer cell lysates and culture supernatant. Cancer cell lysates and culture media were prepared from the cell cultures. Cell lysates (A) and cell culture supernatant (B) were harvested for MMP measurement. MMP expression was detected using the Quantibody reverse-phase human MMP array kit according to the manufacturer s instructions (n=4). Figure S3. scigg-t was not in the SKBR-3 cell culture medium. SKBR-3 cells were cultured and treated with trastuzumab at 10 µg/ml for 24 hours. scigg-t was detected by Western blotting with an anti-human Fc antibody under reducing and denaturing conditions. Intact trastuzumab (IgG-T) and single hinge cleaved trastuzumab (scigg-t) were used as loading controls. Trastuzumab in the culture medium without cancer cells was also included as a negative control. Figure S4. Detection of immune cells infiltration in mice by flow cytometry. Mice splenocytes (n=3) were harvested and washed. The cells (1x10 6 /tube) were stained with antibodies: FITC-anti-Mouse CD11b and APC-anti-Mouse Ly-6G for 30 min. The stained cells were washed with 2%BSA-PBS. After that the cells were suspended with 300 ml 2%BSA-PBS

2 and the positive counts were measured using a Guava easycyte HT instrument (A & B). (C), The total mice splenocytes were counted by using Automated Cell Counter (Bio-RAD TC10). TC10 Trypan Blue Dye 0.4% was used for live cell measurement.

3 A B N SPFlagIdeS(29-339) MALPVTALLLPLALLLHAADYKDDDDKADSFSAN QEIRYSEVTPYHVTSVWTKGVTPPAKFTQGEDVFH APYVANQGWYDITKTFNGKDDLLCGAATAGNML HWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFD VKEAIDTKNHQLDSKLFEYFKEKAFPYLSAKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGI FDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKEL TEGKALGLSHTYANVRINHVINLWGADFDSNGNL KAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAK EIKEDNIGAQVLGLFTLSTGQDSWNQTN C C GGATCC-(BamHI) ATGGCCCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCACTGCTGCTGCACGCC GCCGACTACAAGGACGACGACGACAAGGCCGACAGCTTCAGCGCCAACCAGGA AATCCGGTACAGCGAAGTGACCCCCTACCACGTGACCAGCGTGTGGACCAAGG GCGTGACACCCCCTGCCAAGTTTACCCAGGGCGAGGACGTGTTCCACGCCCCCT ATGTGGCTAACCAGGGGTGGTACGACATCACCAAGACCTTCAACGGCAAGGAC GATCTGCTGTGCGGAGCCGCCACAGCCGGCAATATGCTGCACTGGTGGTTCGAC CAGAACAAGGACCAGATCAAGCGCTACCTGGAAGAACACCCCGAGAAGCAGA AGATCAATTTCAACGGCGAGCAGATGTTCGACGTGAAAGAGGCCATCGACACC AAGAATCACCAGCTGGACAGCAAGCTGTTCGAGTACTTCAAAGAGAAGGCATT CCCCTACCTGAGCGCCAAGCACCTGGGCGTGTTCCCAGACCACGTGATCGACAT GTTCATCAACGGCTACCGGCTGAGCCTGACCAACCACGGCCCTACCCCTGTGAA AGAAGGCAGCAAGGACCCCAGAGGCGGCATCTTCGACGCCGTGTTCACCAGAG GCGACCAGAGCAAGCTGCTGACCAGCCGGCACGATTTCAAAGAAAAGAACCTG AAAGAGATCAGCGACCTGATCAAGAAAGAGCTGACCGAGGGCAAGGCCCTGG GCCTGTCTCACACCTATGCCAACGTGCGGATCAACCATGTGATCAACCTGTGGG GAGCCGACTTCGACAGCAACGGCAACCTGAAGGCCATCTACGTGACCGACAGC GACTCCAACGCCAGCATCGGCATGAAGAAATACTTCGTGGGCGTGAACAGCGC CGGCAAGGTGGCCATCTCCGCCAAAGAAATCAAAGAGGACAACATCGGCGCAC AGGTGCTGGGACTGTTCACCCTGAGCACCGGCCAGGACAGCTGGAACCAGACC AACTGAAAGCTT(HindIII) Supplementary Figure 1. IdeS expression in BT474 breast cancer cells.

4 pg/ml (lg) pg/ g protein (lg) A MMP1 MMP2 MMP3 MMP8 MMP9 MMP10 MMP B SKBR-3 SKOV-3 BT474 MCF MMP1 MMP2 MMP3 MMP8 MMP9 MMP10 MMP13 1 SKBR-3 SKOV-3 BT474 MCF7 Supplementary Figure 2. Detection of MMPs in cancer cell lysates and culture supernatant.

5 IgG-T scigg-t Medium SKBR-3 50KD IgG HC 25KD Fc (m) Supplementary Figure 3. scigg-t was not in the SKBR-3 cell culture medium.

6 CD11b (Counts) Ly-6G (Counts) Total splenocytes (n=4-6) A C B Supplementary Figure 4. Detection of immune cells infiltration in mice by flow cytometry.