RESUMO
The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.
Assuntos
Mesotelioma Maligno , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Anticorpos Monoclonais , Linfócitos T , Imunoterapia Adotiva , Ensaios Antitumorais Modelo de Xenoenxerto , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas de Membrana/genéticaRESUMO
The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Mesotelioma/genética , Anticorpos Monoclonais/genética , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Glicosilação , Humanos , Lectinas/química , Lectinas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Proteínas de Membrana/imunologia , Mesotelioma/imunologia , Mesotelioma/patologia , Mesotelioma Maligno , Ácido N-Acetilneuramínico/metabolismo , Peptídeos/genética , Peptídeos/imunologia , Polissacarídeos/genética , Polissacarídeos/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologiaRESUMO
The absence of highly specific markers for malignant mesothelioma (MM) has served an obstacle for its diagnosis and development of molecular-targeting therapy against MM. Here, we show that a novel mucin-like membrane protein, sialylated protein HEG homolog 1 (HEG1), is a highly specific marker for MM. A monoclonal antibody against sialylated HEG1, SKM9-2, can detect even sarcomatoid and desmoplastic MM. The specificity and sensitivity of SKM9-2 to MM reached 99% and 92%, respectively; this antibody did not react with normal tissues. This accurate discrimination by SKM9-2 was due to the recognition of a sialylated O-linked glycan with HEG1 peptide. We also found that gene silencing of HEG1 significantly suppressed the survival and proliferation of mesothelioma cells; this result suggests that HEG1 may be a worthwhile target for function-inhibition drugs. Taken together, our results indicate that sialylated HEG1 may be useful as a diagnostic and therapeutic target for MM.