A Newly Developed Anti-L1CAM Monoclonal Antibody Targets Small Cell Lung Carcinoma Cells

Abstract

Few effective treatments are available for small cell lung cancer (SCLC), indicating the need to explore new therapeutic options. Here, we focus on an antibody-drug conjugate (ADC) targeting the L1 cell adhesion molecule (L1CAM). Several publicly available databases reveal that (1) L1CAM is expressed at higher levels in SCLC cell lines and tissues than in those of lung adenocarcinoma and (2) the expression levels of L1CAM are slightly higher in SCLC tissues than in adjacent normal tissues. We conducted a series of in vitro experiments using an anti-L1CAM monoclonal antibody (termed HSL175, developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. Our HSL175-DT3C conjugates theoretically kill cells only when the conjugates are internalized by the target (L1CAM-positive) cells through antigen-antibody interaction. The conjugates (an ADC analog) were effective against two SCLC-N (NEUROD1 dominant) cell lines, Lu-135 and STC-1, resulting in decreased viability. In addition, L1CAM silencing rendered the two cell lines resistant to HSL175-DT3C conjugates. These findings suggest that an ADC consisting of a humanized monoclonal antibody based on HSL175 and a potent anticancer drug would be effective against SCLC-N cells.

Keywords: ASCL1; L1 cell adhesion molecule (L1CAM); NEUROD1; antibody–drug conjugate (ADC); small cell lung cancer (SCLC).

Figure 1

Small cell lung carcinoma (SCLC) cell lines and tissues express L1CAM mRNA. (A) L1CAM and SYP mRNA expression in SCLC and lung adenocarcinoma (LUAD) cell lines. The RNA-seq data of 29 SCLC and 36 LUAD cell lines are presented. (B) Correlation between L1CAM and SYP mRNA expression in SCLC cell lines (n = 52). Pearson’s correlation coefficient (R) = 0.541. (C) L1CAM mRNA expression in normal lung, SCLC, LUAD, and LUSC tissues. Expression data from 7 normal lung and 79 SCLC tissues were obtained from a previous report [13]. Expression data from 534 LUAD and 502 LUSC samples were derived from the Cancer Genome Atlas database [14,15]. *** p < 0.001.

Figure 2

Two SCLC cell lines (Lu-135 and STC-1) express L1CAM. (A) Phase contrast images of Lu-135 and STC-1 cells with L1CAM silencing. Cells were reverse-transfected with NC siRNA, L1CAM siRNA #1, or L1CAM siRNA #2 (10 nM each) and cultured for 48 h. Scale bars: 200 μm (left; low magnification) and 50 μm (right; high magnification). (B) Conventional RT-PCR for the expression of L1CAM, SYP, NEUROD1, and ACTB mRNA in Lu-135 and STC-1 cells. Cells were treated as described in (A). (C) Western blot analysis of the cells treated as described in (A). Of note, NCI-H69 was used as a positive control for ASCL1. (D) Western blot analysis of HuL cells and SCLC-N cells for L1CAM. (E) Correlation between L1CAM and NEUROD1 or ASCL1 mRNA expression in SCLC cell lines (n = 52). (F) Regulated Gene Ontology results for the top 100 protein-coding DEGs in L1CAM-silenced Lu-135 cells compared with control cells.

Figure 3

Lu-135 cells are highly sensitive to HSL175-DT3C conjugates. (A) Effects of HSL175-DT3C conjugates on the viability of Lu-135 cells. Cells were transfected with NC siRNA, L1CAM siRNA #1, or L1CAM siRNA #2 (10 nM each), cultured for 48 h, and then incubated with HSL175-DT3C conjugates (0–10 μg/mL each) for another 72 h. Results are presented as mean ± SD. *** p < 0.001. (B) Phase contrast images of Lu-135 cells. Cells were transfected with NC siRNA, L1CAM siRNA #1, or L1CAM siRNA #2 (10 nM each), cultured for 48 h, and then incubated with HSL175-DT3C conjugates (1 μg/mL each) for another 72 h. Scale bars: 200 μm (left; low magnification) and 50 μm (right; high magnification). (C) Effects of HSL175-DT3C conjugates on the viability of Lu-135 cells. Cells were cultured with control IgG-DT3C conjugates or HSL175-DT3C conjugates (0.1 μg/mL each) for 96 h. Results are presented as mean ± SD. *** p < 0.001. (D) Effects of HSL175-DT3C conjugates on levels of the apoptosis marker cleaved PARP in Lu-135 cells. Cells were cultured with control IgG-DT3C conjugates or HSL175-DT3C conjugates (1 μg/mL each) for 72 h.

Figure 4

HSL175-DT3C conjugates are also effective against STC-1 cells. (A) Effects of HSL175-DT3C conjugates on the viability of STC-1 cells. Cells were transfected with NC siRNA, L1CAM siRNA #1, or L1CAM siRNA #2 (10 nM each), cultured for 48 h, and then incubated with HSL175-DT3C conjugates (0–10 μg/mL each) for another 72 h. Results are presented as mean ± SD. *** p < 0.001. (B) Phase contrast images of STC-1 cells. Cells were transfected with NC siRNA, L1CAM siRNA #1, or L1CAM siRNA #2 (10 nM each), cultured for 48 h, and then incubated with HSL175-DT3C conjugates (1 μg/mL each) for another 48 h. Scale bars: 200 μm (left; low magnification) and 50 μm (right; high magnification). (C) Effects of HSL175-DT3C conjugates on the viability of STC-1 cells. Cells were cultured with control IgG-DT3C conjugates or HSL175-DT3C conjugates (1 μg/mL each) for 96 h. Results are presented as mean ± SD. *** p < 0.001. (D) Effects of HSL175-DT3C conjugates on levels of the apoptosis marker cleaved PARP in STC-1 cells. Cells were cultured with control IgG-DT3C conjugates or HSL175-DT3C conjugates (1 μg/mL each) for 48 h.