
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38252815
202313397
10.1073/pnas.2313397121
research-articleResearch ArticlebiochemBiochemistry407
Biological Sciences
Biochemistry
Increased expression of SSEA-4 on TKI-resistant non–small cell lung cancer with EGFR-T790M mutation
Chen Nai-Yu a b 1
Lin Chih-Wei c d 1
Lai Ting-Yen a
Wu Chung-Yi a
Liao Pei-Chi c
Hsu Tsui-Ling a
Wong Chi-Huey chwong@sinica.edu.tw
a e 2 https://orcid.org/0000-0001-8225-6808

aGenomics Research Center, Academia Sinica, Taipei 11529, Taiwan
bInstitute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221, Taiwan
cInstitute of Biochemistry and Molecular Biology, China Medical University, Taichung 406040, Taiwan
dGraduate Institute of Biomedical Sciences, China Medical University, Taichung 406040, Taiwan
eDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037
2To whom correspondence may be addressed. Email: chwong@sinica.edu.tw.
Contributed by Chi-Huey Wong; received September 4, 2023; accepted December 22, 2023; reviewed by Ronald T. Raines, Steven Sucheck, Lai-Xi Wang, and Pan-Chyr Yang

1N.-Y.C. and C.-W.L. contributed equally to this work.

22 1 2024
30 1 2024
22 7 2024
121 5 e231339712104 9 2023
22 12 2023
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

Tyrosine kinase inhibitors (TKIs) of epithelial growth factor receptor (EGFR) have been used for the treatment of non–small cell lung cancer (NSCLC). However, patients under this treatment often develop drug resistance, especially those with T790M mutation in EGFR. To identify new targets for the treatment of NSCLC with TKI resistance and EGFR-T790M mutation, we found that the stage-specific embryonic antigen-4 (SSEA-4) is highly expressed in this type of NSCLC cells and the increased expression of SSEA-4 or the associated enzyme β3GalT5 correlates with poor survival of NSCLC. We demonstrated that an anti-SSEA-4 monoclonal antibody, especially that with a well-defined Fc-glycan, is highly effective against TKI-resistant NSCLC with EGFRT790M mutation in vitro and in vivo.

Non–small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme β3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

lung cancer
SSEA-4 expression
TKI resistance
EGFR T790M
antibody therapy
AS | Genomics Research Center, Academia Sinica (GRC) 501100022281 N/A Nai-Yu ChenChih-Wei LinTing-Yen LaiChung-Yi WuTsui-Ling HsuChi-Huey Wong
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pmcNon–small cell lung cancer (NSCLC) is a life-threatening disease, accounting for 85% of all lung cancer cases that caused 1.8 million deaths worldwide in 2020 (1). It is a disease often with poor early prognosis and high mortality, and most current therapeutic approaches have encountered drug resistance (2). The tyrosine kinase domain of epidermal growth factor receptor (EGFR) has been the major target for treatment of NSCLC, and several generations of tyrosine kinase inhibitors (TKIs) have been approved for the treatment of NSCLC patients especially those with exon 19 deletion or exon 21 L858R mutation in EGFR (3, 4). However, patients under TKI therapy often developed drug resistance due to secondary mutations and among them the most common (~60%) is EGFR-T790M mutation (5). EGFR-T790 has been designated as a “gatekeeper” residue and is important for regulating inhibitor specificity in the adenosine triphosphate (ATP) binding pocket. The binding of TKI to the ATP-binding pocket of EGFR-T790M is weakened after treatment of NSCLC patients with TKI and as a result TKI resistance is developed (5). TKIs of third generation have been developed and shown to be effective in targeting aberrant activation of EGFR and inhibiting tumor growth in patients with resistance to first-generation TKIs. However, patients receiving this treatment often developed resistance again, especially those carrying C797S or other mutations in addition to T790M mutation. Therefore, NSCLC with TKI-resistance and EGFR-T790M mutation represents a major challenge in lung cancer treatment and points to an urgent medical need to develop new therapeutic strategies for the disease.

Altered glycolipids generated by aberrant glycosylation have been recognized as possible anticancer targets (6–8), especially many tumor-associated carbohydrate antigens discovered to date are highly sialylated or fucosylated, and often found on the surface of cancer cells and their stem cells (9–12). Among these cancer-associated glycoconjugates, SSEA-4 (sialyl-Gb5) is commonly found on human embryonic stem cells (13–15), then disappears after stem cell differentiation, but often appears again together with the other two globo-series GSLs (glycosphingolipids) on the cell surface of many epithelial cancers [e.g., ovarian, gastric, prostate, lung, breast, renal, and pancreatic cancers (16–20) and their stem cells (21–23)]. However, there is little or no such glycolipid found on normal cells or tissues (18, 24). In addition, previous studies showed that SSEA-4 played a major role in mediating the invasion of MCF-7 human breast cancer cells through activation of focal adhesion kinase (FAK) and cSrc (25). SSEA-4 positive cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in a decreased cellular adhesion (26). In our recent studies, SSEA-4 was found to compete with FK-506 for binding to FKBP4 during its transport to the cell surface in breast cancer cells (27). On the other hand, galactin-8 interacted with SSEA-4 to inhibit its transport to the cell surface (28). In addition, knockdown of β1,3-galactosyltransferase 5 (β3GalT5), a key enzyme involved in the galactosylation of Gb4 (globoside-4) to form Gb5b (SSEA-3), was found to trigger cancer cell apoptosis and suppress metastasis (29). We also showed that the globo-series GSLs are linked to a complex of FAK-CAV1-AKT-RIP, and AKT of the complex is linked to the EGFR-mediated kinase signaling pathway. Knockdown of β3GalT5 resulted in the dissociation of RIP from the complex and the dissociated RIP then interacted with FADD to cause activation of CASP3 and cancer cell apoptosis (28). All these studies suggest that globo-series GSLs are unique markers on cancer cells and play important roles in cancer progression and could be new targets for development of cancer therapies. This is further supported by the ongoing phase 3 global trial of a Globo-H vaccine against triple-negative breast cancer (NCT03562637), the phase 2 trials of a second-generation Globo-H-CRIM197 vaccine for the treatment of NSCLC (NCT05442060) and esophageal cancer (NCT05376423) (28, 30, 31). Since most types of cancers expressed higher levels of SSEA-4 than the other two globo-series GSLs, and SSEA-4 positive cancer cells exhibited high tumorigenic capacity with an increased risk of disease-related mortality and poor prognosis (24, 32–34), the potential of SSEA-4 targeted cancer therapy could be explored, especially with use of vaccines and monoclonal antibodies (mAbs) (35, 36). Given the fact that the globo-series GSLs are cancer-specific, and SSEA-4 is highly expressed on TKI-resistant NSCLC with EGFR-T790M mutation, we evaluated SSEA-4 as a target for the treatment of this subtype of cancer in this study.

Results

Expression of SSEA-4 in Lung Cancer.

SSEA-4 is one of the three globo-series GSLs which are exclusively expressed on the surface of many cancers, but the expression level and role of SSEA-4 in lung cancer progression remain unclear. In the biosynthesis of globo-series GSLs (Fig. 1A), SSEA-3 is first generated from Gb4 by β3GalT5 and converted to SSAE-4 and Globo-H by ST3Gal2 and FUT1/FUT2, respectively. To understand the expression of SSEA-4 in lung cancer cells, we analyzed SSEA-4 and the other two globo-series GSLs (SSEA-3 and Globo-H) by flow cytometry. Cell lines with more than 15% of total cells positive in flow cytometry are labeled as SSEA-4 positive. The result (Fig. 1B and SI Appendix, Fig. S1 and Table S1) showed that among the lung cancer cell lines, the level of SSEA-4 expression is generally higher than the other globo-series GSLs. We further analyzed the expression level of SSEA-4 among 13 NSCLC cell lines including the wild-type EGFR cell lines H23, H460, EKVX, and H226; the TKI-resistant cell lines CL68 (Del19/T790M), CL97 (G719A/T790M), PC9IR (Del19), HCC827IR (Del19), H1975 (L858R/T790M), and H1975-CS (L858R/T790M/C797S); and the TKI-sensitive cancer cell lines PC9 (Del19), H3255 (L858R), and HCC827 (Del19). The result (Fig. 1 B–D) (SI Appendix, Table S1) showed that the expression of globo-series glycans was relatively low on NSCLC cells with wild-type EGFR (H23, H460, EKVX, H226). All lung cancer cells with EGFR mutation had high SSEA-4 expression except H3255, and eight of the nine cancer cell lines with EGFR mutation were SSEA-4 positive, four were SSEA-3 positive, and three were Globo-H positive. Moreover, the expression levels of SSEA-3 and SSEA-4 were both increased in the lung cancer cell lines with EGFR mutation such as HCC827 and H1975. Importantly, SSEA-4 was observed in all four TKI-resistant lung cancer cell lines with EGFR-T790M mutation (CL68, CL97, H1975, and H1975-C797S abbreviated as H1975-CS), and all three globo-series GSLs were expressed on H1975 and H1975-CS cells. Besides, the level of SSEA-4 expression in CL68 and H1975 cell lines is higher than Globo-H as shown in antibody staining (Fig. 1 E–G). This finding indicates that among the three globo-series GSLs, SSEA-4 is more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and SSEA-4 in this subpopulation may be a good target for development of new treatments.

Fig. 1. Expression of globo-series glycolipids on NSCLC cell lines. (A) Schematic diagram of the biosynthesis of globo-series GSLs. Globo-series GSLs are synthesized in a sequential manner by specific glycosyltransferases. SSEA-3, the precursor of SSEA-4 and Globo-H, is synthesized from Gb4 by β3GalT5. Then, ST3Gal2 and FUT-1/2 catalyze the transfer of sialic acid and fucose, respectively, to SSEA-3 to form SSEA-4 and Globo-H. (B–D) The expression levels of SSEA-3, SSEA-4, and Globo-H are presented as geometric means of fluorescence intensity. SSEA-4, SSEA-3, and Globo-H were stained by MC813-70, MC631, and VK9 antibodies and the corresponding isotype control. Representative data are shown (n = 3). (E and F) Opera Phenix Image of CL68 lung cancer cells stained with FICT-conjugated VK9 (green, targeting Globo-H) only or with a mixture of APC-conjugated MC813–70 (red, targeting SSEA-4) and FICT-conjugated VK9 in a 1:50 ratio. (G) Opera Phenix Image of H1975 lung cancer cells stained with FICT-conjugated VK9 (green, targeting Globo-H) only or with a mixture of APC-conjugated MC813–70 (red, targeting SSEA-4) and FITC-conjugated VK9 in a 1:50 ratio. Cell nuclei were stained with Hoechst dye (blue).

Clinical Significance of β3GalT5 and ST3Gal2 Associated with SSEA-4 Biosynthesis in NSCLC.

Since SSEA-4 was highly expressed on the surface of lung cancer cell lines, we were curious about the role of SSEA-4 in patients with lung cancer. Therefore, we analyzed the clinical mRNA sequence data of LUAD tissues with EGFR mutation (n = 66) and of normal adjacent tissues (n = 637) obtained from The Cancer Genome Atlas (TCGA) research network and the KM-Plotter database to investigate the relationship of the mRNA levels of β3GalT5 and ST3Gal2 and the survival rate. The Kaplan–Meier survival analysis (Fig. 2 A–C) showed a significantly lower overall survival (OS) for those with high expression of β3GalT5 or ST3Gal2, or a combination of both in LUAD patients. Besides, a significantly higher mRNA level of β3GalT5 was observed in LUAD patients with EGFR mutation (Fig. 2D), but there was no significant difference in ST3Gal2 mRNA expression between LUAD patients with or without EGFR mutation (Fig. 2E). To study the relationship between the mRNA levels of the enzymes involved in Globo H synthesis and survival, the OS for lung cancer patients with the mRNA level of FUT1 or FUT2 or in combination with β3GalT5 was investigated and the result was shown in SI Appendix, Fig. S2 A–D. The LUAD patients with higher FUT2 expression had poor OS compared to those with lower expression of FUT2 or higher expression of FUT1. Kaplan–Meier survival analysis (SI Appendix, Fig. S2D) showed that β3GalT5 combined with FUT2 expression significantly reduced the OS rate in LUAD patients. The mRNA level of FUT2 is significantly increased in LUAD patients with EGFR mutation compared to FUT1 or normal LUAD tissues (SI Appendix, Fig. S2 E and F). Taken together, these findings indicate that increased expression of β3GalT5 or β3GalT5 + ST3Gal2 or β3GalT5 + FUT2 measured by the corresponding mRNAs correlates with poor clinical outcome in LUAD patients with EGFR mutation.

Fig. 2. Clinical significance of SSEA-4 biosynthesis enzymes (β3GalT5 and ST3Gal2) measured as mRNA levels in LUAD patients with or without EGFR mutation. (A) Kaplan–Meier analysis of β3GalT5 mRNA in 719 LUAD patient samples, showing the probability of OS in months with high (red) or low (black) expression of β3GalT5 mRNA (HR = 1.61, P = 2.4e-04). (B) Kaplan–Meier analysis of the ST3Gal2 mRNA level in 672 LUAD patient samples, indicating the OS in months with high (red) or low (black) expression of ST3Gal2 mRNA (HR = 1.73, P = 3.5e-05) or (C) β3GalT5 + ST3Gal2 mRNAs (HR = 1.88, P = 1.5e-06). (D) The TCGA database showed that β3GalT5 mRNA was overexpressed in 66 LUAD patients and correlated with EGFR mutation, but (E) ST3Gal2 mRNA expression was not significantly different between those with or without EGFR mutation.

TKI-Resistant NSCLC with EGFR-T790M Mutation Is Associated with SSEA-4 Expression.

When comparing the expression of SSEA-4 on lung cancer cell lines, we observed that SSEA-4 expression was associated with TKI-resistance and EGFR-T790M mutation. The result in Fig. 3 A and B showed that SSEA-4 expression was increased significantly in the TKI-resistant cell lines H1975 (EGFR L858R/T790M, gefitinib resistant) and CL68 (EGFR Del19/T790M, gefitinib resistant) with EGFR-T790M mutation compared to the TKI-sensitive cell lines H3255 (EGFR L858R, gefitinib sensitive) and PC9 (EGFR Del19, gefitinib sensitive) without EGFR mutation at T790. To further understand the increased SSEA-4 expression in TKI-resistant NSCLC with EGFR-T790M mutation, we first determined the level of β3GALT5 mRNA by qRT-PCR and found that the β3GalT5 level is significantly increased in NSCLC cell lines with TKI-resistance and EGFR-T790M mutation (e.g., H1975 and CL68), compared to TKI-sensitive lung cancer cells (e.g., H3255 and PC9) (Fig. 3 C and D). In addition, SSEA-4 expression is decreased in TKI-resistant HCC827IR and PC9IR cell lines with exon-19 deletion compared to TKI-sensitive HCC827 and PC9 cell lines (SI Appendix, Fig. S3 A–D). These observations suggest that the increased expression of β3GALT5 mRNA significantly correlates with TKI resistance and EGFR-T790M mutation.

Fig. 3. TKI resistance and EGFR-T790M mutation trigger SSEA-4 expression. Flow cytometric analysis of SSEA-4 expression and the mRNA expression levels of globo-series glycan enzymes in TKI-resistant NSCLC cell lines with EGFR-T790M mutation. (A) Expression of SSEA-4 in H3255 (L858R, TKI sensitive) and H1975 (L858R/T790M, TKI resistance); (B) Expression of SSEA-4 in PC9 (Del19, TKI sensitive) and CL68 (Del19/T790M, TKI resistance). (C) Relative β3GalT5 mRNA level in H3255 and H1975. (D) Relative β3GalT5 mRNA levels in PC9, and CL68 (E) DNA sequencing of the genome coding sequences of H1975 and H1975 (EGFR-T790). The arrowhead revealed a T-to-C transition. (F) Expression of SSEA-4 in H1975 and H1975 (EGFR T790). (G) Relative β3GalT5 mRNA levels of H1975 and H1975 (EGFR T790). (H) Relative ST3Gal2 mRNA levels of H1975 and H1975 (EGFR T790). (I and J) SSEA4 expression was evaluated in H1975 and H3255 cells treated with MK-2206 and SC79. MFI, means fluorescence intensity. (K) Western blot analysis showed inhibition of the EGFR–PI3K–Akt pathway by 170 nM AZD9291 in TKI-resistant H1975 cells with EGFR-T790M mutation, resulted in the reduction of pEGFR (Phospho-EGFR, Tyr1086), pAKT, and β3GalT5 expression, and suppression of EGFR downstream signaling. Error bars shown in all graphs are SDs of the means (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

To better understand the correlation between TKI-resistance/ EGFR-T790M and SSEA-4 expression, we used the TKI-resistant H1975 cell line with T790M mutation as an experimental model and changed the amino acid from methionine at position 790 back to threonine (T790M to T790) using adenosine base editing (ABE) method (37). The sensitivity of the mutated clones H1975/T790 (Fig. 3E) to gefitinib was tested (SI Appendix, Fig. S4A), and the result showed that the mutant become gefitinib sensitive. This engineered cell line H1975/T790 was then used for comparison with parental H1975/T790M in SSEA-4 expression, and the result of flow cytometry showed a decrease in SSEA-4 expression from T790M to T790 (Fig. 3F). We also examined the mRNA expression of globo-series enzymes and found that the expression level of β3GalT5 or ST3Gal2 is significantly decreased in the H1975/T790 group compared to H1975/T790M (Fig. 3 G and H). This study suggested that EGFR-T790M mutation drives the expression of SSEA-4 in TKI-resistant NSCLC cells.

Activation of EGFR signaling leads to the regulation of multiple pathways associated with RAS/RAF/MAPK, AKT and JAK/STAT3 et al. The EGFR-mediated signal transduction plays an integral role in cell migration, proliferation, and survival, while mutation of EGFR is associated with tumor growth. Previous reports (38) indicated that EGFR-T790M mutation is closely associated with TKI resistance, and TKI resistance promotes tumor growth in lung cancer by activating the AKT signaling pathway (39, 40).

Since up-regulating the AKT signaling pathway is related to TKI resistance (40), we used AKT inhibitor (MK-2206) and AKT activator (SC79) to treat cells and investigate the expression of SSEA-4 to understand the relationship of T790M-AKT-SSEA-4 and the role of AKT signaling in SSEA-4 synthesis. The results (Fig. 3I) showed that the AKT inhibitor reduces the expression of SSEA-4 in H1975 cells with EGFR-T790M, while AKT activator induced SSEA-4 expression in TKI-sensitive cell line H3255 with EGFR L858R mutation (Fig. 3J). We also found that the expression of globo-series glycans in TKI-resistant H1975 (L858R/T790M) and CL68 (Del19/T790M) cell lines with T790M mutation was decreased by treatment with AZD9291, a covalent TKI targeting T790M-mediated TKI resistance in lung cancer, in a dose-dependent manner (Fig. 3K and SI Appendix, Fig. S4 B–H). Based on the western blot analysis, the inhibition of the EGFR–PI3K–Akt pathway by AZD9291 in H1975 cells with EGFR T790M mutation and the resulting reduction of pEGFR (Phospho-EGFR at Tyr1086), pAKT, and β3GalT5 expressions indicated a relationship between SSEA-4 expression and TKI-resistance/EGFR-T790M in NSCLC cells. Under AZD9291 treatment, pEGFR and pAKT were inhibited in a dose-dependent manner, and β3GalT5 was also inhibited after 24 h treatment with 170 nM AZD9291.

To examine the potential relationship of the EGFR-AKT downstream signaling pathways in TKI-resistant NSCLC with EGFR-T790M mutation and SSEA-4 expression, we used H1975 (EGFR-T790M) and H1975/T790 clones to perform RNA-seq assay. The heatmap (SI Appendix, Fig. S5) displayed a heatmap representing the differential expression of genes associated with globo-series glycans, and the decreased expression levels of β3GalT5 and ST3Gal2 in H1975/T790 (clone 18 and clone 19) shown in the heatmap are consistent with our findings (Fig. 3 G and H). Besides, the ingenuity pathway analysis (IPA) of H1975 EGFR-T790M and H1975 EGFR-T790 clones and the transcription factor analysis of these two cell lines with JASPAR database indicated a linkage to the β3GalT5-LTR and ST3Gal2 promoters as the mRNA expressions of HNF4A, GFI1, and HIC1 associated with EGFR-AKT signaling are different in these two cell lines. Based on these studies, we speculated that the expression of SSEA-4 in NSCLC is driven by TKI-resistance and EGFR-T790M mutation through activation of the downstream signaling of the EGFR-AKT pathway, which then promotes the expression of β3GalT5 and ST3Gal2 enzymes to synthesize SSEA-4 (Fig. 4).

Fig. 4. Schematic illustration of SSEA-4 expression influenced by TKI resistance and EGFR-T790M mutation in EGFR and its signaling pathway. It is proposed that EGFR-T790M will activate the downstream signaling of AKT or STAT3 which is up-regulated in TKI-resistant cells. The solid line denotes a direct functional interaction of the transcription factor on the genes.

SSEA-4 Antibody against TKI-Resistant NSCLC Cells with EGFR-T790M Mutation.

To evaluate SSEA-4 as a target for the treatment of TKI-resistant NSCLC with EGFR-T790M mutation, NSCLC cells were first incubated with humanized anti-SSEA-4 mAb (chMC813-70) and rabbit complements, and the cell cytotoxicity was then measured by the lactate dehydrogenase (LDH) assay. The result (Fig. 5 A–C) of complement-dependent cytotoxicity (CDC) assay showed 75% cytotoxicity against H1975 (L858R/T790) cells, 86% cytotoxicity against CL68 (Del19/T790M) cells, and 26% cytotoxicity against CL97 (G719A/T790M). With this promising result, we next modified the Fc-glycans of the chimeric humanized antibody (chMC813-70) at position Asn-297 to α2,6-sialyl complex-type biantennary N-glycan (SCT) and the 3F-sialylated derivative (FSCT) as previously reported to maximize the interaction with FcγIIIA on NK cells to further enhance the effector functions of antibody-dependent cellular cytotoxicity (ADCC) (41, 42). The result showed that both chMC813-70-SCT and chMC813-70-FSCT are more effective than chMC813-70 against H1975, CL68, and CL97 cells (Fig. 5 D–G). The half-maximum effective concentrations (EC50) of chMC813-70-FSCT were: 0.002 mg/mL for H1975, 0.037 mg/mL for CL68, and 0.125 mg/mL for CL97; and the EC50 values of chMC813-70-SCT were: 0.002 mg/mL for H1975, 0.016 mg/mL for CL68, and 0.205 mg/mL for CL97, compared to 0.066 mg/mL for H1975, 0.08 mg/mL for CL68, and 1.592 mg/mL for CL97 with chMC813-70 (Fig. 5G). These results are consistent with our previous study that chMC813-70-SCT or FSCT with a higher affinity to FcγIIIA receptor than chMC813-70 can be used as better therapeutic antibodies and as affinity ligands for isolation of a subpopulation of natural killer (NK) cells with high-expression of FcγIIIA receptor. This subpopulation of NK cells was further expanded and used for treatment of pancreatic cancer, and the efficacy in cancer cell killing assay was found to increase by sevenfold compared to the unseparated NK cells (42).

Fig. 5. SSEA-4 is a therapeutic target for NSCLC with TKI resistance and T790M mutation. (A–C) Lung cancer cell lines (H1975, CL68, and CL97) were treated with MC813-70 and rabbit complement to observe cell lysis induced by SSEA-4 mAbs. The CDC activity of lung cancer cells was detected by the LDH release assay in the presence of 0.1 to 40 μg/mL of antibody as described in Materials and Methods. The data are shown as mean ± SD. (D–G) ADCC assays of chMC813-70, glycoengineered chMC813-70-SCT, and the stable 3-F sialyl derivative (chMC813-70-FSCT). Experiments were performed under the effector-to-target (E:T) ratio of 6:1 with lung cancer EGFR-T790M cells (H1975, CL68, and CL97).

SSEA-4 Antibody Inhibits Lung Tumor Growth In Vivo.

To further assess the efficacy of chMC813-70 mAb in reducing the growth of lung cancer xenograft tumors in nude mice, H1975 (L858R/T790M) and CL68 (Del19/T790M) were injected via s.c. into nude mice (N = 6). Each mouse received i.p. injections of chMC813-70 mAb at doses of 200 μg and 25 μg, twice a week, while the control group received PBS injections in parallel. The result showed that chMC813-70 mAb effectively reduced the growth of tumors and the volumes of H1975 tumors with high SSEA4 expression were significantly reduced (Fig. 6 A and B). The results showed both 43% tumor growth inhibition and significant tumor weight loss (Fig. 6C), while there was no significant difference in mouse body weight in different groups (Fig. 6D). A similar result was shown in CL68 (SI Appendix, Fig. S6 A–C). These results demonstrated that like pancreatic cancer (41, 42), SSEA-4 is a therapeutic target for TKI-resistant NSCLC with EGFRT790M mutation.

Fig. 6. Humanized anti-SSEA-4 mAb (chMC813-70) reduces the growth of TKI-resistant NSCLC cells with T790M mutation in xenograft models. (A) BALB/c nude mice were injected with H1975 cells (106 per mouse). Treatment with vehicle control or chMC813-70 at a given dose was initiated after 4 or 6 d, respectively. Antibody was administered at a dose of 25 μg or 200 μg per mouse, twice per week with intraperitoneal injection for 8 times. Tumor volume was measured at different time points and shown as mean ± SD. (B) Tumor size in mice receiving different treatments was measured using calipers. (C) Tumor weight expressed as statistics of H1975 mice receiving different treatments. (D) Average body weights of H1975 tumor-bearing mice receiving different treatments. Six mice were used in the control group and each treatment group. Error bars shown in all graphs are SDs of the means (n = 6).

Internalization of SSEA4 Antibody in H1975.

It is well known that antibody selection is important for the development of antibody-drug conjugations (ADCs). To investigate the potential of SSEA-4 antibody for ADC, we first evaluated the internalization potential of SSEA-4 antibody chMC813-70 in H1975 and the results showed that the antibody can be internalized within 1 h. We then evaluated the ADC-mediated cancer cell death through SSEA-4 targeting in vitro using antibody chMC813-70 conjugated with the potent tubulin inhibitor Val-Cit-PAB-MMAE attached to the thiol group through a cleavable linker. The cytotoxic activity was tested against H1975 and CL68 lung cancer cell lines and compared to the unconjugated chMC813-70 as control. The lung cancer cells were incubated in growth medium with 100 nM of each reagent for 72 h. As shown in Fig. 7, the SSEA4-ADC exhibited cytotoxicity against H1975 and CL68. These results demonstrated that antibody chMC813-70 can be used for ADC against SSEA-4-bearing TKI-resistant NSCLC cells with EGFR-T790M mutation, further supporting the therapeutic potential of targeting SSEA-4 with ADC in cancer treatment.

Fig. 7. Internalization of SSEA-4 antibody chMC813-70 and in vitro cytotoxicity of ADC against lung cancer cells. (A) Internalization of chMC813-70 at different time points in H1975 cells. H1975 cancer cells were stained with chMC813-70 conjugated with Fluor 488 in 10 μg/mL (Green). The nucleus was stained with Hoechst 33342 in 0.1 μg/mL (blue). Scale bar (10 μm) was used in images. (B) The ADC chMC813-70-MMAE and unconjugated chMC813-70 were tested against H1975 and CL68 lung cancer cell lines. Lung cancer cells (5 × 103cells) were incubated with 100 nM of each ADC in growth medium, and cell viability was determined for 72 h. Error bars shown in graphs are SDs of the means (n = 3).

Discussion

Activating mutations of EGFR play an oncogenic role in lung cancer cells. NSCLC patients with EGFR mutations, especially exon 19 deletion or exon 21-L858R mutation, have been treated with inhibitors of the kinase domain of EGFR such as gefitinib and erlotinib. However, patients eventually develop drug resistance to such inhibitors due to various mechanisms, including secondary mutation (T790M) that accounts for 60% of the patients with drug resistance, activation of alternative pathways, and phenotypic transformation (43, 44). To identify new targets for diagnosis and treatment of NSCLC with TKI resistance, we found that SSEA-4 was highly expressed in all mutant EGFR cancer cell lines, particularly in TKI-resistant cells with EGFR-T790M mutation. We also found that the expression of SSEA-4 is increased with increasing expression of β3GalT5 mRNA in TKI-resistant NSCLC with EGFR mutation. However, when the TKI-resistant H1975 cells with EGFR-T790M mutation were mutated back to EGFR T790, the expression levels of SSEA-4 as well as β3GalT5 and ST3Gal2 mRNA were decreased. In addition, clinical data showed that higher expression of the enzyme β3GalT5 or β3GalT5 combined with ST3Gal2 correlated with a poor survival rate in LUAD patients. All these observations indicate that SSEA-4 is a therapeutic target for TKI-resistant NSCLC with EGFR-T790M mutation.

Previous reports indicated that EGFR-T790M is associated with PI3K-AKT signaling, and EGFR-TKI resistance promotes lung cancer tumor growth by activating the AKT signaling pathway which is linked to the globo-series GSL signaling as described above. In this study, we found that the level of β3GalT5 expression is higher in TKI-resistant/EGFR-T790M lung cancer cells and we were curious whether there are specific transcription factors activating the β3GalT5-LTR promoter. The IPA of H1975 EGFR-T790M and H1975 EGFR-T790 clones and the transcription factor analysis based on the JASPAR database indicated a linkage to the β3GalT5-LTR and ST3Gal2 promoters (45) as the mRNA expressions of HNF4A, GFI1, and HIC1 are different in these two cell lines by IPA analysis. It was shown that the transcription factors HNF4A (46) and HIC1 (47) are directly regulated by EGFR-AKT signaling while GFI1 may be involved but is not directly regulated by EGFR. Therefore, the differential expression of SSEA-4 in TKI-sensitive cells (H3255 and PC9) or H1975 EGFR-T790 clones and TKI-resistant cells with T790M mutation could involve activation of specific transcription factors that regulate β3GalT5 or ST3Gal2 promoter, such as HNF4A, HIC1, and GFI1 (Fig. 4). However, more studies are required to confirm this relationship and to better understand the mechanism. A previous study suggested that Gb4, the precursor of SSEA-3, may interact with EGFR and trigger downstream signaling events (48).

It is known that antibody’s effector functions can be enhanced by either engineering the variable region of antibodies to increase affinity to the target or by removing the core fucose or remodeling the glycans of the antibody Fc portion (49–51). Our lab (12, 41, 42, 52, 53) and the Wang lab (54) have developed methods to engineer the Fc-glycan to modulate Fc interaction with receptors, thereby influencing the effector functions. Our recent studies showed that among the various glycans linked to Fc-Asn297, the humanized antibody glycoform with Fc-SCT or Fc-FSCT has the best binding affinity to FcγIIIA (mainly on NK cell) and FcγIIA (on macrophage and dendritic cell) (53), thereby exhibiting the optimal ADCC, ADCP and vaccinal effect, respectively. In this study, we found that the humanized anti-SSEA4 antibody chMC813-70, especially the homogeneous glycoform chMC813-70-SCT or chMC813-70-FSCT is highly effective against SSEA-4 positive and TKI-resistant NSCLC with EGFR-T790M mutation. Our cell-based assay demonstrated that the humanized antibody chMC813-70 exhibited potent CDC activity against SSEA-4 positive cancer cells, and the ADCC activity of homogeneous chMC813-70 with Fc-SCT or Fc-FSCT was enhanced through optimal engagement with Fc receptors on immune cells, including NK cells, macrophage and dendritic cells (55, 56). The enhanced cell killing activity was further supported in the animal study showing that the tumor volume was significantly reduced after treatment with such anti-SSEA-4 antibodies.

In conclusion, the survival rate of cancer patients remains poor and the drug-resistant problem in NSCLC treatment remains a major challenge. This study revealed that SSEA-4 is highly expressed on TKI-resistant NSCLC cells with EGFR-T790M mutation, and high expression of β3GalT5 or β3GalT5 + ST3Gal2, the key enzymes associated with SSEA-4 biosynthesis, significantly reduced the OS rate of TKI-resistant NSCLC patients with EGFR-T790M mutation. Based on these findings, we have demonstrated in the study in vitro and in vivo that the monoclonal antibody chMC813-70 against SSEA-4 and its homogeneous glycoform with Fc-SCT or Fc-FSCT can be used for the treatment of drug-resistant NSCLC with T790M mutation. In addition, this study demonstrated that chMC813-70 is a promising candidate for development of ADC (55, 56) for the treatment of SSEA4 positive and TKI-resistant NSCLC with EGFR-T790M mutation. This study showed that antibody chMC813-70 can be rapidly endocytosed in H1975 cells, an observation similar to our recent study on breast cancer cells using the same antibody for site-specific drug conjugation to the glycan moiety to obtain an ADC with a drug-to-antibody ratio of 5 (55). Although the efficacy of ADC in this study is not as high as expected, perhaps due to the differences in conditions and conjugation (through a thiol group of wild-type chMC813-70) (57), this study suggests the potential of the antibody for ADC development. Further optimization of ADC to improve efficiency against SSEA-4 positive cancers is ongoing. In summary, this study provides a new direction for tumor recurrence prediction and identifies SSEA-4 as target for the treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Materials and Methods

CL68 and CL97 cell lines were established from patients with informed consent and approval of the institutional review board (National Taiwan University Hospital Research Ethics Committee).

Flow Cytometry.

Cells (5 × 105) were stained with 1 μg Alexa Fluor 488–conjugated anti-SSEA-3 mAb (MC-631, BioLegend), anti-SSEA-4 mAb (MC813-70, BioLegend), or anti-Globo-H mAb (VK9, a gift from Philip O. Livingston, Memorial Sloan–Kettering Cancer Center, New York) in 100 μL FACS buffer (PBS solution with 1% FBS) on ice for 30 min before analysis with FACS Calibur system (BD Biosciences). Cell lines in which more than 15% of total cells were positive in flow cytometry are labeled positive.

qRT-PCR.

Total RNA was isolated using the RNeasy Mini RNA Isolation kit (Qiagen) and reverse-transcribed to cDNA by using a High-Capacity cDNAReverse Transcription kit (ThermoFisher Scientific). The cDNA reaction was diluted 1:10 in H2O and used in Fast SYBR Green real-time PCR reactions (ThermoFisher Scientific) for detection of glycosyltransferase genes. cDNA was examined for the expression of β3GalT5 (forward primer: 5′-AGCGGA AACGAA AGAGGTGGAC-3′; reverse primer: 5′-CCTGAGGACAAA AGCGATGGAC-3′), ST3Gal2 (forward primer: 5′-GCCTTCTTCAAGTATATCCACGA-3′; reverse primer: 5′-CGGTT-GTTCTCCCAGTAG-3′); FUT1 (forward primer: 5′-CCGG​TTTGGTAATCA-GATGG-3′; reverse primer: 5′-CTCAAGTCCGCG-TACTCCTC-3′), FUT2 (forward primer: 5′-ATCATGACCATTGGGACGTT-3′; reverse primer: 5′-GTGCTT-GAGTAAGGGGGACA-3′). All reactions were run in duplicate or triplicate on a QS7 machine (Applied BioSystems) according to the manufacturer’s instructions. Values were normalized to GAPDH and then compared to control cells.

CDC Assay.

The assay was performed according to the procedure reported (18). SSEA-4 antigen high and low cell lines were used for cytotoxicity assay. The CDC activity of anti–SSEA-4 mAbs was measured by LDH-release assay using CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega). Cells (1 × 104) were plated in each well of 96-well plates and were washed with PBS twice after overnight growth. The cells were then incubated with 1 to 2 μg SSEA-4 mAbs or isotype control in 50 μL phenol red-free RPMI with rabbit complement or human complement (20%) (Life Technologies). After incubation in a 5% CO2 incubator at 37 °C for 2 to 4 h, the degree of cell lysis was determined by measuring the amount of LDH released into the culture supernatant. Maximum LDH release was determined by lysing the cells with the lysis solution provided by the commercial source. The percentage of specific lysis was calculated according to the equation: % lysis = (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100.

ADCC Reporter Bioassay.

An ADCC reporter bioassay (Promega G7014) was performed using engineered Jurkat cells expressing the human FcγIIIa-V158 receptor according to the reported procedure (58). Multiple cross-linking of target cells with Jurkat cells by antibodies leads to Jurkat cell luciferase production, which can be quantified to determine the Jurkat cell activation. Target cells were seeded at 12,500 cells per well in a 96-well white plate and incubated at 37 °C, 5% CO2 overnight. After 12 to 16 h, serial dilutions of antibodies were first added to target cells; 75,000 Jurkat cells were then added to the wells containing target cells and antibodies. The effector cell/target cell (E/T) ratio was 6:1. After 6 h of incubation at 37 °C and 5% CO2, the plate was allowed to cool to RT for 15 min, followed by an addition of a luciferase substrate. Five minutes after the luciferase substrate addition, the luminescence was measured using CLARIOstar (BMG Labtech). The fold of induction was calculated by dividing the relative light unit (RLU) (induced-background) by RLU (no antibody control-background). GraphPad Prism 6 software was used for the data analysis.

Tumor Xenografts in Nude Mice.

Lung tumor cell lines H1975 or CL68 were collected by trypsinization, counted, and, respectively, injected at 1 × 106 cells/site or 5 × 106 cells/site subcutaneously on the flanks of BALB/c nude mice (BioLASCO Taiwan). Treatment with vehicle control or chMC813-70 mAb at a given dose was initiated after 4 or 6 d, respectively. ChMC813-70 mAb (25 μg/mouse, 200 μg/mouse) or vehicle was injected intraperitoneally twice weekly. The control group and the treatment group consisted of 6 mice each. Tumors were measured twice weekly with calipers. Tumor volumes were estimated using the modified ellipsoid formula V = 1/2 (length × width2).

Antibody Internalization Assay.

A total of 1 × 104 cells were seeded on chamber coverslips (Ibidi, cat# 80826) containing 0.2 mL of cell culture medium overnight at 37 °C. After removing the medium, cells were placed on ice for 10 min to inhibit intracellular transport. Cells were then incubated with 10 μg/mL of Fluor 488–conjugated SSEA-4 antibody (Invitrogen, Cat# 53-8843-42) in prewarmed Dulbecco’s Modified Eagle Medium (DMEM) medium to allow antibody internalization at different times for Nikon confocal microscopy analysis and image processing.

Antibody Conjugation.

The chMC-813-70 IgG1 antibodies were treated with tris(2-carboxyethyl) phosphine hydrochloride (TCEP; 2.3 equivalents) at 25 °C for 90 min. To the reduced mAb was added the MC-Val-Cit-PAB-MMAE (1.2 equivalents/SH group) in ice-cold dimethylacetamide (DMAC) (5% v/v). After 40 min, the reactions were quenched with excess N-acetylcysteine (NAC) (8 equivalents). The mixture was placed on ice for 30 min before buffer exchange by elution through Sephadex G25 and concentrated by centrifugal ultrafiltration (57). The conjugates were sterile filtered through a 0.2-μm filter under sterile conditions and stored at −80 °C before analysis and testing.

Supplementary Material

Appendix 01 (PDF)

Click here for additional data file.

This work was supported by Academia Sinica. We thank the National RNAi Core Facility at Academia Sinica for providing ABE reagents and related services. We thank Dr. Michael Hsiao’s guidance on the mouse experiment.

Author contributions

N.-Y.C., C.-W.L., and C.-H.W. designed research; N.-Y.C., C.-W.L., T.-Y.L., C.-Y.W., and P.-C.L. performed research; N.-Y.C., C.-W.L., and T.-L.H. analyzed data; and N.-Y.C., C.-W.L., and C.-H.W. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data will be accessible upon publication and are included in the article and/or SI Appendix.

Supporting Information

Reviewers: R.T.R., Massachusetts Institute of Technology; S.S., The University of Toledo; L.-X.W., University of Maryland; and P.-C.Y., College of Medicine, National Taiwan University Hospital.
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