Min Tang

Supplementary Figure 2. DLL3-positive cells were generated and characterized in vitro to identify binding epitopes of 1-B12. (A) Schematic illustration of full-length and truncated human DLL3 proteins expressed in 293T cells for epitope mapping. All constructs expressing truncated DLL3 included an N-terminal Flag for convenient detection. (B) Assessment of the transduction efficiency of DLL3-ECD. (C) The secretion of IFN-γ by DLL3-CAR-T cells after a 24-hour coculture with 293T cells expressing either full-length or truncated DLL3 was measured using an ELISA assay.

Supplementary Figure 6. The affinity of Humanized VHHs was determined by flow cytometry (FACS, fluorescence-activated cell Sorting) for the half-effective concentration (median effective concentration; EC50)

Supplementary Figure 7. Determination of binding affinity for 1-B12 and its humanized antibody using KD values. (A) KD value of 1-B12-Fc, (B) KD value of 1-B12-HM1-Fc, (C) KD value of 1-B12-HM3-Fc.

Supplementary Figure 3. Schematic of retroviral vector constructs encoding VHH for transduction into T cells. Optimized CAR-T cell construct: The VHH domain is directly linked to the CD8 hinge and transmembrane (TM) domain, followed by the 4-1BB costimulatory domain (CSD) and CD3ζ molecule in tandem downstream.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by a high relapse rate, limited treatment options, and a poor prognosis. Delta-like ligand 3 (DLL3) has emerged as a promising target for SCLC. Notably, chimeric antigen receptor T (CAR-T) cells that use the variable domain of heavy-chain–only antibodies (VHH) demonstrate superior efficacy compared with their ScFv counterparts. However, the therapeutic effectiveness of anti-DLL3 VHH CAR-T cells is yet to be fully explored. To leverage the therapeutic potential of VHHs, we first immunized alpacas and screened for anti-DLL3 VHHs using yeast display. Then, 1-B12 and 5 identified the positive clones and compared them based on their affinity, specificity, and cytotoxicity in CAR-T cell models. Moreover, 1-B12 was selected as the humanized sequence due to its higher affinity, greater specificity, and stronger cytotoxicity. Finally, the functionality of the four humanized VHH-CAR-T cells from the 1-B12 sequence was evaluated through in vitro assays that measured cytokine production and cytotoxicity, followed by in vivo studies to assess their antitumor efficacy. The anti-DLL3 VHHs exhibited strong affinity, specificity, and cytotoxicity in CAR-T cell models. Notably, the HM-CAR-T cells exhibited robust cytokine secretion and cytotoxic activity against tumor cells. Moreover, these HM-CAR-T cells demonstrated significant antitumor efficacy in vivo. This study highlights effective strategies for developing DLL3-specific VHHs and their application in CAR-T therapy, which supports their clinical potential as a promising immunotherapeutic approach for cancers that express DLL3.

Supplementary Figure 9. The relative proliferation ratio of T cells upon repeated stimulation against NCI-H446-DLL3 cells. The relative proliferation rate of cells expressing VHH-1-B12 and its humanized antibody after repeated stimulation with NCI-H446-DLL3 cells compared to the T3 positive control and Flag negative control.

Supplementary Figure 12. Representative IHC images of DLL3 expression in small-cell lung cancer. Staining of DLL3 high-expression (upper) and low-expression (lower) samples with commercial antibodies and VHH-derived antibodies

Supplementary Table 3. The amino acid sequences of 1-B12, HM1, HM2, HM3 and HM4

Supplementary Table 2. Antibody sequences used in this study

Supplementary Figure 11. The proportions of CD3, CD4, and CD8-positive cells in the spleen and tumor tissues of mice (A) Schedule of the mouse experiment (Created with BioRender.com), (B) The proportions of CD3, CD4, and CD8-positive cells in the spleen tissues of mice, (C) The proportions of CD3, CD4, and CD8-positive cells in the tumor tissues of mice.