NRXN1 as a novel potential target of antibody-drug conjugates for small cell lung cancer

Abstract

Small cell lung cancer (SCLC) is a high-grade malignancy, and treatment strategies have not changed for decades. In this study, we searched for novel targets for antibody-drug conjugate (ADC) therapy for SCLC. We identified transmembrane proteins overexpressed specifically in SCLC with little or no expression in normal tissues and decided to focus on the cell adhesion molecule neurexin-1 (NRXN1). The cell surface overexpression of NRXN1 was confirmed using flow cytometry in SCLC cell lines (SHP77 and NCI-H526). The combination of a primary anti-NRXN1 monoclonal antibody and a secondary ADC exhibited anti-tumor activity in SCLC cell lines. Moreover, the knockout of NRXN1 in SHP77 cells resulted in a loss of the anti-tumor activity of NRXN1-mediated ADC therapy. Thus, NRXN1 could be a novel target for ADC therapy for the treatment of SCLC that is worth further research.

Keywords: NRXN1; antibody-drug conjugates; cell adhesion molecule; novel molecular targets; small cell lung cancer.

Figure 1. NRXN1 expression in cell lines and surgical specimens of lung tissue including non-SCLC and normal tissues.

(A) The relative expressions in cell lines were examined using qRT-PCR with the SYBR green dye assay; data for NRXN1 is shown. The log₂-scale relative gene expression is indicated on the y-axis. Error bars, SD. (B) Flow cytometry of NRXN1 on cell lines. Cell surface NRXN1 was assessed using FITC-conjugated anti-rabbit polyclonal antibody following rabbit anti-NRXN1 (Black trace) or IgG isotype control (gray-filled) antibodies. FCM, flow cytometry. (C) Percentage of NRXN1-positive cells examined for each cell line using flow cytometry. Cells were stained with rabbit anti-NRXN1 polyclonal antibody followed by FITC conjugated goat anti-rabbit IgG. A one-way analysis of variance (ANOVA) followed by the Tukey test was performed. (^* P < 0.05; ^** P < 0.01; ^*** P < 0.0001; Tukey test). Error bars, SD. (D) Relative expression of NRXN1 in surgical specimens including normal lung by qRT-PCR using the SYBR green dye assay. The y-axis shows the NRXN1 expression levels relative to normal lung tissue. The horizontal bars indicate the median gene expression levels for each group. Error bars, SD. Ad, adenocarcinoma. NL, normal lung. SCLC, small cell lung cancer. Sq, squamous cell carcinoma.

Figure 2. NRXN1 expression in normal multiple organs in humans.

The relative expressions of NRXN1 in normal multiple organs of human tissue were examined using qRT-PCR with the SYBR green dye assay. The y-axis shows the NRXN1 expression levels relative to normal lung tissue. The y-axis is set to the same scale as that of Figure 1D so that the expression intensities of both surgical specimens and normal organs can be visually compared. Error bars, SD.

Figure 3. In vitro growth inhibition of NRXN1-targeted ADC.

(A–F) In vitro growth inhibition of anti-NRXN1 monoclonal antibody only, secondary ADC only, isotype control antibody (IgG) followed by secondary ADC, and anti-NRXN1 monoclonal antibody followed by secondary ADC on incubation with (A) SHP77, (B) SHP77 KO, (C) HEK293, (D) HEK293-NRXN1, (E) NCI-H526, and (F) PDC. All the assays were performed in triplicate. A two-way ANOVA followed by the Tukey test was performed to assess the difference between IgG with the second ADC group and anti-NRXN1 mAb with the second ADC group. A P value < 0.05 was considered significant (^* P < 0.05; ^** P < 0.01; ^*** P < 0.0001). Error bars represent the SD of the mean. mAb, Monoclonal antibody. ns, Not significant.

Figure 4. Apoptosis assay of NRXN1-targeted ADC at IC₅₀ dose calculated by growth inhibition curves.

Late apoptotic cells were quantified by Cy7-conjugated annexin-V and PI using flow cytometry. Results were analyzed using a one-way ANOVA followed by the Dunnett multiple comparisons test (^* P < 0.05; ^** P < 0.01; ^*** P < 0.0001 versus no-treatment control group; Dunnett test). Error bars represent the SD of the mean. mAb, monoclonal antibody.