Figure 5.

Binding analysis of sacituzumab

(A) Schematic diagram of domain- or loop-substituted TROP-2 constructs with that from mouse homolog.

(B) Flow cytometry analyses of the key domain of interaction between TROP-2 and sacituzumab. The HA-tagged proteins of wild-type (TROP-2-WT), mouse TROP-2 (mTROP-2), mouse CRD-substituted (TROP-2-mCRD), mouse TY-substituted (TROP-2-mTY), mouse CPD-substituted (TROP-2-mCPD), and two exposed loops of CPD substituted (TROP-2-mRCPD and TROP-2-mACPD) TROP-2 were expressed on HEK-293T cells and incubated with sacituzumab, respectively. The PerCP-conjugated anti-HA tag antibody was used to detect the expression of TROP-2 on the surface of HEK-293T cells, whereas APC-conjugated anti-human IgG antibody was used to detect the binding of sacituzumab with TROP-2 on HEK-293T cells. Untransfected HEK-293T cells and TROP2-WT expressing HEK-293T cells stained with isotype control antibody were used as negative controls.

(C) Western blot analysis of the key domain responsible for the interaction between TROP-2 and sacituzumab. The left panel represents the detection of different variants of TROP-2 expression on HEK-293T cells with anti-HA-tag antibody (left). The right panel represents the detection of the binding of sacituzumab to different TROP-2 variants as indicated using sacituzumab as detection antibody. Lanes 1 to 9 represent untransfected HEK-293T cells, HEK-293T cells transfected with empty vector, TROP-2-WT, TROP-2-mCRD, TROP-2-mTY, TROP-2-mCPD, TROP-2-mRCPD, TROP-2-mACPD and mTROP-2, respectively.

(D) The predicted binding region of sacituzumab on TROP-2 based on the flow cytometry-based binding assay, with RCPD loop highlighted in red (left). The location of RCPD in TROP-2 cis- or trans-assembly is indicated (right). See also Figure S5.