Purpose of reviewChimeric antigen receptor (CAR) T cell therapy has been successful in some haematologic malignancies, but the central nervous system (CNS) presents unique obstacles to its use against tumours arising therein. This review discusses recent improvements in the delivery and design of these cells to improve the efficacy and safety of this treatment against malignant gliomas.Recent findingsThe immunosuppressive environment of the CNS affects the functionality of CAR T cells, but recent developments using metabolic manipulation and cytokine delivery have shown that the performance of CAR T cells can be improved in this environment. Emerging techniques can improve the delivery of CAR T cells to the CNS parenchyma, which is normally well protected from peripheral immune cells. The implementation of novel antigens and CAR-expression regulation strategies will improve the specificity and efficacy of these cells. Finally, although autologous T cells have historically been the standard, recent developments have made the use of allogeneic T cells or natural killer (NK) cells more clinically feasible.SummaryThe discoveries highlighted in this review will aid the development of CAR cells that are safer, more resilient against immunosuppressive signals in the CNS, and able to specifically target intracranial tumour cells.
Background While immunotherapy is profoundly efficacious in certain cancers, its success is limited in cancers with lower mutational burdens, such as gliomas. 1 Therefore, investigating neoantigens beyond those from somatic mutations can expand the repertoire of immunotherapy targets. Recent studies detected alternative-splicing (AS) events in various cancer types that could potentially translate into tumor-specific proteins. 2,3 Our study investigates AS within glioma to identify novel MHC-I-presented neoantigens through an integrative transcriptomic and proteomic computational pipeline, complemented by an extensive spatiotemporal analysis of the AS candidates. Methods Bulk RNA-seq of high tumor purity TCGA-GBM/ LGG (n=429) were analyzed through a novel systematic pipeline, and tumor-specific splicing junctions (neojunctions) were identified in silico by cross-referencing with bulk RNA-seq of GTEx normal tissue (n=9,166). Two HLA-binding prediction algorithms were subsequently incorporated to predict peptide sequences with a high likelihood for HLA presentation. Investigation of the tumor-wide clonality and temporal stability of the candidates was performed on extensive RNA-seq data from our spatially mapped intratumoral samples and longitudinally collected tumor tissue RNA-seq. Proteomic validation was conducted through mass-spec analysis of the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM repository (n=99). Results Our analysis of TCGA-GBM/LGG bulk RNA-seq identified 249 putative neojunctions that translate into 222 cancerspecific peptide sequences encoding 21,489 tumor-specific nmers (8-11 amino acids in length, figure 1). Both prediction algorithms concurrently identified 271 n-mers likely to bind and be presented by HLA*A0101, HLA*A0201, HLA*A0301, HLA*A1101, or HLA*A2402. We confirmed the expression of 17 out of 74 HLA*A0201-binding candidates in RNA-seq of two HLA*A0201+ patient-derived glioma cell lines with a subset of candidates found tumor-wide (figure 2). Analysis of CPTAC-GBM mass-spec data detected 42 tumor-specific peptides generated specifically from 23 GBM-specific splicing events (figure 3). 4 candidates were selected for downstream immunogenicity analysis and were selected based on high in silico HLA-presentation scores and detection in RNA sequencing and mass spectrometry. In vitro sensitization of healthy donor-derived CD8+ T-cells was performed with the 4 candidates along with an influenza positive control condition and a no peptide negative control condition. 1 of the 4 neoantigen candidates, mutant-RPL22, was shown to elicit a CD8+ Tcell-specific immune response based on IFNg ELISA assay analysis (figure 4). Conclusions Tumor-specific neojunctions identified in our unique integrative pipeline present novel candidate immunotherapy targets for gliomas and offer a new avenue in neoantigen discovery across cancer types. Abstract 80 Figure 1 Identification of top HLA-presented neoantigens Neoantigen sequences were predicted from glioma-specific neojunctions identified from our nove...
BACKGROUND: While immunotherapy is profoundly efficacious in certain cancers, its success is limited in cancers with lower mutational burden, such as gliomas. Therefore, investigating neoantigens beyond those from somatic mutations can expand the repertoire of immunotherapy targets. Recent studies detected alternative-splicing (AS) events in various cancer types that could potentially translate into tumor-specific proteins. Our study investigates AS within glioma to identify novel MHC-I-presented neoantigen targets through an integrative transcriptomic and proteomic computational pipeline, complemented by an extensive spatiotemporal analysis of the AS candidates. METHODS: Bulk RNA-seq of high tumor purity TCGA-GBM/LGG (n=429) were analyzed through a novel systematic pipeline, and tumor-specific splicing junctions (neojunctions) were identified in silico by cross-referencing with bulk RNA-seq of GTEx normal tissue (n=9,166). Two HLA-binding prediction algorithms were subsequently incorporated to predict peptide sequences with high likelihood for HLA-presentation. Investigation of the tumor-wide clonality and temporal stability of the candidates was performed on extensive RNA-seq data from our spatially mapped intratumoral samples and longitudinally collected tumor tissue RNA-seq. Proteomic validation was conducted through mass-spec analysis of the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM repository (n=99). RESULTS: Our analysis of TCGA-GBM/LGG bulk RNA-seq identified 249 putative neojunctions that translate into 222 cancer-specific peptide sequences which confer 21,489 tumor-specific n-mers (8-11 amino acids in length). Both prediction algorithms concurrently identified 271 n-mers likely to bind and be presented by HLA*A0101, HLA*A0201, HLA*A0301, HLA*A1101, or HLA*A2402. We confirmed the expression of 15 out of 58 HLA*A0201-binding candidates in RNA-seq of two HLA*A0201+ patient-derived glioma cell lines with a subset of candidates found tumor-wide. Analysis of CPTAC-GBM mass-spec data detected 23 tumor-specific peptides with 5 containing detected n-mers highly predicted to be HLA-presented. CONCLUSION: Tumor-specific neojunctions identified in our unique integrative pipeline present novel candidate immunotherapy targets for gliomas and offer a new avenue in neoantigen discovery across cancer types. Citation Format: Darwin Kwok, Takahide Nejo, Aidan Du, Maggie Colton, James Woo, Joseph Costello, Hideho Okada. Integrative transcriptomic and proteomic analysis reveals immunogenic and spatially-conserved HLA-binding neoantigen targets derived from tumor-specific alternative splicing events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4170.
BACKGROUND: The efficacy of immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Recent studies revealed increased alternative splicing (AS) in various cancer types that potentially translate into targetable neoantigens. We developed a novel comprehensive in silico pipeline for detecting tumor-specific AS events (neojunctions), and successfully identified tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicited CD8+ T-cell-mediated immune responses. METHODS: Our computational pipeline first identified recurring splice junctions in high purity TCGA LGG/GBM bulk RNA-seq samples (n=429, PSR > 10%) and not in GTEx normal tissue bulk RNA-seq data (n=9166, PSR < 1%). We subsequently employed two independent algorithms to predict the peptide processing likelihood and the HLA-binding affinity of ASN candidates before validating neojunction and ASN expression in RNA-seq data from patient-derived glioma cell lines (n=68) and spatially-mapped glioma samples (n=535), and in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) GBM mass spectrometry data (n=99). In vitro sensitization of healthy donor-derived CD8+ T-cells against our top ASN candidates followed by 10x VDJ scRNA-seq was performed to identify ASN-specific TCR sequences. We transduced these TCRs into Jurkat76s and co-cultured them with ASN-pulsed T2 cells and ASN and HLA-expressing COS7 cells to determine whether the TCR recognized the presented HLA:ASN. RESULTS: Our analysis identified 249 putative neojunctions that translated into 222 cancer-specific peptides sequences. Iterating across these peptides yielded 17,562 glioma-specific n-mer sequences between 8 to 11 amino acids in length. Both prediction algorithms concurrently identified 636 n-mers likely to be presented by demographically common HLA haplotypes. Through transcriptomic and proteomic validation with patient-derived samples and cell lines, we identified 8 ASNs that were tumor-wide, 4 of which were predicted to be HLA*A0201-presented. In vitro sensitization of healthy-donor derived CD8+ T-cells against these 4 ASNs resulted in an expansion of reactive CD8+ T-cells against ASNs derived from neojunctions within S100A6, RPL22, and GNAS. Subsequent 10x VDJ scRNA-seq on the expanded CD8+ population identified mutRPL22-reactive TCR clonotypes with neoantigen-mediated increases in IFNG and GZMB signatures. When transduced and expressed in Jurkat76 cells, one particular TCR clonotype demonstrated recognition and immunogenic activation against mutRPL22-pulsed T2 cells and COS7 cells endogenously expressing both HLA*A0201 and mutRPL22. CONCLUSION: Our unique integrative pipeline detected novel tumor-wide splice-derived neoantigen candidates, and ASN-reactive TCRs identified through our pipeline offer a new avenue for TCR-based therapies across cancer types. Citation Format: Darwin W. Kwok, Takahide Nejo, Nicholas Stevers, Lee H. Chen, Kaori Okada, Chibo Hong, Gary K. Chan, Akane Yamamichi, Aidan Du, Maggie Colton, James Woo, Joseph Costello, Hideho Okada. Targetable public neoantigens are generated by tumor-wide cancer-specific splicing events in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3152.
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