MAGE-A10 Protein Expression in Advanced High Grade Serous Ovarian Cancer Is Associated with Resistance to First-Line Platinum-Based Chemotherapy.

Ovarian cancer has a dismal prognosis. Standard treatment following surgery relies on platinum-based chemotherapy. However, sizeable percentages of patients are unresponsive. Identification of markers predicting the response to chemotherapy might help select eligible patients and spare non-responding patients from treatment-associated toxicity. Cancer/testis antigens (CTAs) are expressed by healthy germ cells and malignant cells of diverse histological origin. This expression profile identifies them as attractive targets for cancer immunotherapies. We analyzed the correlations between expression of MAGE-A10 and New York esophageal-1 cancer (NY-ESO-1) CTAs at the protein level and the effectiveness of platinum-based chemotherapy in patients with advanced-stage high-grade serous ovarian carcinoma (HGSOC). MAGE-A10 and NY-ESO-1 protein expression was analyzed by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded samples from 93 patients with advanced-stage HGSOC treated at our institutions between January 1996 and December 2013. The correlation between the expression of these markers and response to platinum-based chemotherapy, evaluated according to RECIST 1.1 criteria and platinum sensitivity, measured as platinum-free interval (PFI), progression free (PFS), and overall survival (OS) was explored. The MAGE-A10 protein expression predicted unresponsiveness to platinum-based chemotherapy (p = 0.005), poor platinum sensitivity (p < 0.001), poor PFS (p < 0.001), and OS (p < 0.001). Multivariate analysis identified MAGE-A10 protein expression as an independent predictor of poor platinum sensitivity (p = 0.005) and shorter OS (p < 0.001). Instead, no correlation was observed between the NY-ESO-1 protein expression and response to platinum-based chemotherapy (p = 0.832), platinum sensitivity (p = 0.168), PFS (p = 0.126), and OS (p = 0.335). The MAGE-A10 protein expression reliably identified advanced-stage HGSOC unresponsive to platinum-based chemotherapy. Targeted immunotherapy could represent an important alternative therapeutic option in these cancers.

Phase 1 Clinical Trial Evaluating the Safety and Anti-Tumor Activity of ADP-A2M10 SPEAR T-Cells in Patients With MAGE-A10+ Head and Neck, Melanoma, or Urothelial Tumors.

Background: ADP-A2M10 specific peptide enhanced affinity receptor (SPEAR) T-cells are genetically engineered autologous T-cells that express a high-affinity melanoma-associated antigen (MAGE)-A10-specific T-cell receptor (TCR) targeting MAGE-A10-positive tumors in the context of human leukocyte antigen (HLA)-A*02. ADP-0022-004 is a phase 1, dose-escalation trial to evaluate the safety and anti-tumor activity of ADP-A2M10 in three malignancies (https://clinicaltrials.gov: NCT02989064). Methods: Eligible patients were HLA-A*02 positive with advanced head and neck squamous cell carcinoma (HNSCC), melanoma, or urothelial carcinoma (UC) expressing MAGE-A10. Patients underwent apheresis; T-cells were isolated, transduced with a lentiviral vector containing the MAGE-A10 TCR, and expanded. Patients underwent lymphodepletion with fludarabine and cyclophosphamide prior to receiving ADP-A2M10. ADP-A2M10 was administered in two dose groups receiving 0.1×109 and >1.2 to 6×109 transduced cells, respectively, and an expansion group receiving 1.2 to 15×109 transduced cells. Results: Ten patients (eight male and two female) with HNSCC (four), melanoma (three), and UC (three) were treated. Three patients were treated in each of the two dose groups, and four patients were treated in the expansion group. The most frequently reported adverse events grade ≥3 were leukopenia (10), lymphopenia (10), neutropenia (10), anemia (nine), and thrombocytopenia (five). Two patients reported cytokine release syndrome (one each with grade 1 and grade 3), with resolution. Best response included stable disease in four patients, progressive disease in five patients, and not evaluable in one patient. ADP-A2M10 cells were detectable in peripheral blood from patients in each dose group and the expansion group and in tumor tissues from patients in the higher dose group and the expansion group. Peak persistence was greater in patients from the higher dose group and the expansion group compared with the lower dose group. Conclusions: ADP-A2M10 has shown an acceptable safety profile with no evidence of toxicity related to off-target binding or alloreactivity in these malignancies. Persistence of ADP-A2M10 in the peripheral blood and trafficking of ADP-A2M10 into the tumor was demonstrated. Because MAGE-A10 expression frequently overlaps with MAGE-A4 expression in tumors and responses were observed in the MAGE-A4 trial (NCT03132922), this clinical program closed, and trials with SPEAR T-cells targeting the MAGE-A4 antigen are ongoing.

5-aza-2'-deoxycytidine (DAC) treatment induces the MAGE-A10 expression and improves the cytotoxicity of MAGE-A10-specific CTLs in lung cancer cells.

BACKGROUND: MAGE-A10 is a subtype of the Melanoma-associated antigen A (MAGE-A), a class of tumor antigens that are extensively expressed in various histological types of tumors and represents an attractive target for tumor immunotherapy. Epigenetic-modifying drugs can enhance the expression of tumor antigens and improve the cytotoxicity of antigen-specific T cells. 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor (DNMTI) considered an epigenetic-modifying drug, could enhance the expression of MAGE-A10 in cancer cells. METHODS: Human lung cancer cell lines (H1975 and A549) and primary lung cancer cells (L228, L329 and L419) were used. 5-aza-2'-deoxycytidine was used to induce the expression of MAGE-A10 in tumor cells. MAGE-A10 antigenic peptide (sequence: SLLKFLAKV) was used to induce differentiation of MAGE-A10-specific cytotoxic T lymphocytes (CTLs). Interferon-γ release assay was used to detect the capacity of MAGE-A10 peptide to induce CTLs. Cell Counting Kit-8 (CCK-8) analysis was performed to detect the cytotoxicity of MAGE-A10-specific CTLs. Real-time PCR and western blot analysis was used to detect the mRNA and protein levels, respectively. Immunohistochemistry was performed to detect the protein expression in cancer and adjacent normal tissue. Kaplan-Meier plotter online database was used to analyze the overall survival (OS), post-progression survival (PPS), and first progression (FP). RESULTS: The lysis rate of MAGE-A10-specific CTLs in L419 and H1975 were found to be 65.9% and 80.5%, respectively. Both L419 and H1975 showed significantly higher lysis rate in group 1 than in group 3 (6.7, 26.7%), group 2 (0, 0%) and group 4 (0, 0%) (P=0.0003, P≤0.0001, P≤0.0001, respectively). Online data mining using Kaplan-Meier plotter suggested that high expression of MAGE-A10 was significantly and negatively associated with OS (Plogrank =2.1e-05) and PPS (Plogrank =0.0057), and FP (Plogrank =3.2e-12). CONCLUSIONS: High-level expression of MAGE-A10 improved the anti-tumor immune cytotoxicity of MAGE-A10-specific CTLs in lung cancer cell lines and primary lung cancer cells. However, MAGE-A10 gene expression was negatively associated with prognosis according to the survival analysis. Thus, we hypothesize that high-level of MAGE-A10 expression in vivo may inhibit the differentiation of MAGE-A10-specific CTLs.

Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate.

Circulating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive in vitro testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity ("X-scan"). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.

Suppression of MAGE-A10 alters the metastatic phenotype of tongue squamous cell carcinoma cells.

MAGE-A10 is a member of the MAGE protein family (melanoma associated antigen) which is overexpressed in cancer cells. Although MAGE-A10 has been characterized for some time and is generally associated to metastasis its function remains unknown. Here we describe experiments using as models oral squamous cell carcinoma (OSCC) cell lines displaying increasing metastatic potential (LN1 and LN2). These cell lines were transduced with lentivirus particles coding for short hairpin against MAGE-A10 mRNA. Repression of MAGE-A10 expression in LN2 cells altered their morphology and impaired growth of LN1 and LN2 cell lines. Furthermore, repression of MAGE-A10 expression increased cell-cell and cell matrix adhesion. Furthermore shMAGEA10 cells were shown to assemble aberrantly on a 3D culture system (microspheroids) when compared to cells transduced with the control scrambled construct. Cell migration was inhibited in knocked down cells as revealed by two different migration assays, wound healing and a phagokinetic track motility assay. In vitro invasion assay using a leiomyoma tissue derived matrix (myogel) showed that shMAGEA10 LN1 and shMAGEA10 LN2 cells displayed a significantly diminished ability to penetrate the matrices. Concomitantly, the expression of E-cadherin, N-cadherin and vimentin genes was analyzed. shMAGEA10 activated the expression of E-cadherin and repression N-cadherin and vimentin transcription. Taken together the results indicate that MAGE-A10 exerts its effects at the level of the epithelial-mesenchymal transition (EMT) presumably by regulating the expression of adhesion molecules.