(D) Biodistribution of Cy5.5\IgG and Cy5.5\T4H11 in tumors and vital organs (heart, liver, spleen, lung and kidney) of mice at 12?h post injection. corresponding antibody shifted to 37? for 3?h. MOL2-13-1855-s002.tif (1.1M) GUID:?71CA1C7A-A990-47FA-91E6-B81F25E0243C Fig. S3. Kinetic analysis of candidate anti\DDR1 monoclonal antibodies to recombinant human DDR1 ECD by SPR. Names of antibodies are as follows: R5\E12\C3, T2\C8\G12, T3\D11\H5, R1\A6\H8, T1\C10\C2, Y4\D4\F7, Y4\D4\G11 and T4\C2\C5. Each antibody was assayed in a 2\fold serial dilution with concentrations of 2?nm, 4?nm, 8?nm, 16?nm, 32?nm and 64?nm. MOL2-13-1855-s003.tif (1.2M) GUID:?51EA8506-AFF6-4352-915C-4C32652FF5F9 Fig. S4. Representative images of T4H11 staining for DDR1 expression in human normal tissues. 1, cerebrum; 2, cerebellum; 3, heart; 4, liver; 5, spleen; 6, lung; 7, kidney; 8, spinal cord; 9, nerve; 10, lymph node; 11, adrenal gland; 12, skeletal muscle; 13, Cathepsin Inhibitor 1 smooth muscle; 14, ovary; 15, testis, 16, stomach; 17, esophagus; 18, small intestine; 19, colon; 20, nerve; 21, salivary gland; 22, thyroid gland. Magnification, 10. Black scale bar: 250?m. MOL2-13-1855-s004.tif (6.7M) GUID:?BBFFF30E-0BE6-435E-BBA3-B8027EAD578F Fig. S5. Binding ability of antibody to recombinant proteins. Cathepsin Inhibitor 1 T4H11 was detected for Cathepsin Inhibitor 1 DDR1 family member cross\reactivity by ELISA. DDR1 ECD (dot, black) or DDR2 ECD (square, red) was coated onto an ELISA plate. T4H11 was applied at the indicated concentrations. Error bars represent the standard error of the mean (SEM). MOL2-13-1855-s005.tif (1.4M) GUID:?C681896D-93A4-464C-A100-E8C6D2D7AE5B Fig. S6. Antibody binding Rabbit Polyclonal to KCNT1 ability for living cells. Cells expressing DDR1 Cathepsin Inhibitor 1 at the surface (HT\29) were incubated with T4H11 over a range of concentrations prior to staining with Alexa Fluor 488\labeled goat anti\mouse IgG (H+L) secondary antibody. Mean fluorescence intensity (MFI) of Alexa Fluor 488 signal was measured by FCM. MOL2-13-1855-s006.tif (1.3M) GUID:?E99041AC-1C8F-46C5-9C12-BBAAB08F1873 Fig. S7. Inhibition of cell proliferation by T4H11\DM4 and control IgG\DM4. Cell viability was measured at 72?h after treatment with T4H11\DM4 (solid square; red) or IgG\DM4 (solid circle; black) at several concentrations in HT\29 colon cancer cells using CCK\8 assay. Cell viability was profoundly inhibited by T4H11\DM4. The IC50 value of T4H11\DM4 and IgG\DM4 were 4.57??2.07?nm and more than 1000?nm, respectively. Error bars represent the standard error of the mean (SEM). MOL2-13-1855-s007.tif (1016K) GUID:?C78609B7-4054-44AC-AF13-85C707AAB7F4 Fig. S8. antitumor efficacy of T4H11\DM4 and IgG\DM4 against HT\29 xenografts. Antitumor efficacy of T4H11\DM4 and IgG\DM4 in HT\29 xenograft models (potency of T4H11\DM4 in colon cancer cell lines with different cell surface expression levels of DDR1. MOL2-13-1855-s012.docx (15K) GUID:?87E04F7B-0B52-42FE-8A7F-8EEBDBE74D54 Abstract DDR1 has been identified as a cancer\associated receptor tyrosine kinase that is highly expressed in several malignancies relative to normal tissues. Clinically approved multi\kinase inhibitors, such as nilotinib, inhibit DDR1\mediated tumor growth in xenograft models, suggesting DDR1 might be a potential target for cancer treatments. Here, we employed an antibody\based strategy with a novel anti\DDR1 antibody\drug conjugate (ADC) for colon carcinoma treatment. We developed T4H11\DM4, an ADC targeting DDR1 which carries the tubulin inhibitor payload DM4. Immunohistochemical analysis of a tissue microarray containing Cathepsin Inhibitor 1 100 colon cancer specimens revealed that DDR1 was highly expressed in 81% of tumor tissues. Meanwhile, high expression of DDR1 was associated with poor survival in patients. efficacy of T4H11\DM4 and the levels of DDR1 expression on the cell surface was observed. Tumor cell proliferation was caused by the induction of mitotic arrest, indicating that the antitumor effect was mediated by DM4. In addition, T4H11\DM4 was efficacious in oxaliplatin\resistant colon cancer models. In exploratory safety studies, T4H11\DM4 exhibited no overt toxicities when multi\doses were administered at 10?mgkg?1 into BALB/c nude mice or when a single dose up to 50?mgkg?1 was administered into BALB/c mice. Overall, our findings highlight the potential of DDR1\targeted ADC and may facilitate the development of a new effective therapeutic strategy for colon cancer. experiments showed that 7rh benzamide could slow tumor growth and induce a 50% suppression of tumor size in subcutaneous xenografts of gastric carcinoma (Hur and with an acceptable safety profile, suggesting anti\DDR1 ADC is a promising strategy for colon carcinoma.