Fig 2: ROR1 expression in CRC tissues and CRC cells(A) Western blot of ROR1 expression in human CRC tissues (T) and their adjacent normal tissues (N). Total proteins were used as loading control. (B, C) The intensity of each lane was calculated by Image J software and analyzed by paired t-test. Statistics results showed that P<0.001. (D) Flow cytometry of ROR1 expression in the immortalized normal colon cells and CRC cells.

Fig 3: Binding analysis of antibodies generated through fishing with recombinant CD22, ROR1, and FCRL5 proteins. (A) Dose–response ELISA of one representative antibody (human IgG1) per protein. Each antibody was analyzed for binding to the specific protein used for fishing (target protein) and a non-related protein carrying the same tag (non-target protein). Binding was detected using an HRP-labeled anti-human antibody and a luminescent substrate. (B) Flow cytometry analysis of one representative antibody (human IgG1 or scFv) per target showing the binding to target cells, that is, chronic lymphocytic leukemia (CLL) cells (anti-FCRL5) or Raji cells (anti-CD22 and anti-ROR1), with or without a prior blocking step with a commercial polyclonal antibody of the same specificity as the tested antibody. Binding was detected using an APC-labeled anti-human antibody (anti-CD22 and anti-ROR1) or an AF647-labeled anti-tag antibody (anti-FCRL5).

Fig 4: Frequency, based on NGS analysis, of discovered clones in the different strategies including the input phage pool. Median frequency is shown with red bars. (A) Antibodies, previously discovered through direct screening with corresponding specificities, CD23 (n = 107), CD72 (n = 132), and CD200, CD21, CD32, or HLA-DR (n = 34). (B) Antibodies, discovered through fishing, binding FCRL5, ROR1, or CD22 (n = 17). For statistical analysis, Friedman’s test with Dunn’s multiple comparison was done using GraphPad Prism.

Fig 5: ROR1 expression on CRC tissue microarray by immunohistochemistry staining(A) The IHC staining of ROR1 expression in breast tumor (1) and its adjacent normal tissue (2). (B) Detection of ROR1 expression in CRC tissues. Positive ROR1 staining was shown in brown color and the nucleus counterstained with hematoxylin was shown in blue color. The magnification was ×200 in B1, ×400 in B2. (C) Different levels of ROR1 expression detected by TMA-IHC analysis. (1) Score 0 indicates that none or little cells exhibit ROR1 expression; (2) score 1 indicates that more than 25% of tumor cells exhibit weak ROR1 expression; (3) score 2 indicates more than 50% of tumor cells have weak expression or more than 25% of tumor cells have moderate ROR1 expression; (4) score 3 indicates more than 75% of tumor cells have moderate expression or more than 50% of tumor cells have strong ROR1 expression. (D) The proportion of negative (score 0), weak (score 1), moderate (score 2) and strong (score 3) staining for ROR1 protein in CRC patients. (E) The scores of ROR1 in CRC tissues at different stages were analyzed by two tail t-test (P < 0.001). (F) The scores of ROR1 in CRC tissues with different status of lymph node metastasis were analyzed by two tail t-test (P < 0.001). LN, lymph node.