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Cited by 18Glioma Diagnosis and TreatmentImmunotherapy and Immune ResponsesCancer Immunotherapy and Biomarkers

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T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas

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Kilian, Michael, Friedrich, Mirco, Sanghvi, Khwab, Green, Edward, Pusch, Stefan, Kawauchi, Daisuke, Löwer, Martin, Sonner, Jana K., Krämer, Christopher, Zaman, Julia, Jung, Stefanie, Breckwoldt, Michael O., Willimsky, Gerald, Eichmüller, Stefan B., von Deimling, Andreas, Wick, Wolfgang, Sahm, Felix, Platten, Michael, Bunse, Lukas·2022·Clinical Cancer Research
DOI 10.1158/1078-0432.ccr-21-1881PMC PMC9401455

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Abstract

Purpose: Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. Experimental Design: Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor–targeting guide RNAs. Results: We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas. Conclusions: The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.

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Crossref · 10.1158/1078-0432.ccr-21-1881

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Figure 1. Identification of CICR215W/Q point mutations as potential target for targeted immunotherapy. A, Experimental overview. Peptides harboring potential glioma-related neoepitopes were used for vaccination of A2.DR1 MHC-transgenic mice. B, Representative ELISpot assay of splenocytes isolated from CICR215W vaccinated mice restimulated for 48 hours with the indicated peptide. C, Quantification of data shown in B, n = 6 mice. D, Representative flow cytometric analysis of splenocytes from CICR215W vaccinated mice restimulated for 24 hours with the indicated 27-mer peptide and intracellularly stained for IFNγ production. E, Quantification of data shown in D, n = 6 mice. NS, not significant. F, The Cancer Genome Atlas (TCGA) analysis of the LGG dataset (n = 516); frequent mutations in the peptide position 215 are shown. G, Proportion of patients with CIC215-mutated tumors in the TCGA oligodendroglioma (n = 208) and Heidelberg oligodendroglioma (n = 181) cohorts. C and E, Data are represented as individual values and mean ± SEM. Statistical significance was determined by paired two-tailed Student t test.
Figure 1. Identification of CICR215W/Q point mutations as potential target for targeted immunotherapy. A, Experimental overview. Peptides harboring potential glioma-related neoepitopes were used for vaccination of A2.DR1 MHC-transgenic mice. B, Representative ELISpot assay of splenocytes isolated from CICR215W vaccinated mice restimulated for 48 hours with the indicated peptide. C, Quantification of data shown in B, n = 6 mice. D, Representative flow cytometric analysis of splenocytes from CICR215W vaccinated mice restimulated for 24 hours with the indicated 27-mer peptide and intracellularly stained for IFNγ production. E, Quantification of data shown in D, n = 6 mice. NS, not significant. F, The Cancer Genome Atlas (TCGA) analysis of the LGG dataset (n = 516); frequent mutations in the peptide position 215 are shown. G, Proportion of patients with CIC215-mutated tumors in the TCGA oligodendroglioma (n = 208) and Heidelberg oligodendroglioma (n = 181) cohorts. C and E, Data are represented as individual values and mean ± SEM. Statistical significance was determined by paired two-tailed Student t test.CC BY-NC-ND · PubMed Central
Figure 2. Establishment of an A2.DR1 MHC-humanized glioma model. A, Experimental overview. P0 A2.DR1 pups were electroporated with plasmids encoding for CRISPR-Cas9 and 3 guide RNAs (gRNA) targeting p53, Nf1, and Pten. Growing tumors were excised, passaged in NSG mice, and used to generate a cell line that was reimplanted into adult A2.DR1 mice. MRI and H&E histology are shown for respective tumor stages. B, Pie chart depicting the proportions of mutations that are shared by the parental tumor and ex vivo passaged cell line or that are newly acquired after in vitro passage. C, Verification of the CRISPR-mediated functional knockout of p53 in the A2.DR1 glioma cell line. D, Representative immunofluorescence images of A2.DR1 glioma orthotopically injected into the brain of adult A2.DR1 mice, 20 days after injection. E, MHC expression of the A2.DR1 glioma cell line, incubated for 24 hours with recombinant murine IFNγ. FMO, fluorescence minus one control. F, Immunofluorescence image of CicR215W overexpressing A2.DR1 glioma cells.
Figure 2. Establishment of an A2.DR1 MHC-humanized glioma model. A, Experimental overview. P0 A2.DR1 pups were electroporated with plasmids encoding for CRISPR-Cas9 and 3 guide RNAs (gRNA) targeting p53, Nf1, and Pten. Growing tumors were excised, passaged in NSG mice, and used to generate a cell line that was reimplanted into adult A2.DR1 mice. MRI and H&E histology are shown for respective tumor stages. B, Pie chart depicting the proportions of mutations that are shared by the parental tumor and ex vivo passaged cell line or that are newly acquired after in vitro passage. C, Verification of the CRISPR-mediated functional knockout of p53 in the A2.DR1 glioma cell line. D, Representative immunofluorescence images of A2.DR1 glioma orthotopically injected into the brain of adult A2.DR1 mice, 20 days after injection. E, MHC expression of the A2.DR1 glioma cell line, incubated for 24 hours with recombinant murine IFNγ. FMO, fluorescence minus one control. F, Immunofluorescence image of CicR215W overexpressing A2.DR1 glioma cells.CC BY-NC-ND · PubMed Central
Figure 3. Discovery and validation of CICR215W-reactive TCRs. A, T cells pooled from 5 individual T-cell lines generated from CICR215W vaccinated mice were subjected to an IFNγ-secretion assay, sorted for IFNγ-producing CD4+ T cells and subsequently used for single-cell VDJ sequencing using the 10x Genomics platform. IFNγ staining by flow cytometry is shown. B, Left, clonotype distribution of A; right, color-coded abundance of clones in individual mice determined by TCRβ deep sequencing. C, Validation of α-CICR215W-TCR-transduced (CT2) CD4+ T cells. Left, coexpression of GFP as reporter for TCR expression in α-CICR215W-TCR-transduced T cells as determined by flow cytometry. Mock, transduction control. Right, IFNγ production of GFP+ α-CICR215W-TCR-transduced T cells after restimulation with irradiated splenocytes pulsed with the indicated peptides. D, IFNγ production of TCR-transduced T cells restimulated with irradiated splenocytes pulsed with the indicated peptide. n = 3 biological replicates. E, NFAT-reporter assay of CT-2 TCR- and NFAT-reporter transduced T cells, restimulated for 4 hours with irradiated splenocytes pulsed with the respective peptide, measured by luciferase signal. n = 3 technical replicates. F, IFNγ secretion of CT2 transduced T cells cocultured with DCs loaded with lysate from CicR215W or CicWT A2.DR1 glioma cells. n = 4 biological replicates. MFI, mean fluorescence intensity. D–F, Data are represented as individual values (D, F) and mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey test. RLU, relative light unit.
Figure 3. Discovery and validation of CICR215W-reactive TCRs. A, T cells pooled from 5 individual T-cell lines generated from CICR215W vaccinated mice were subjected to an IFNγ-secretion assay, sorted for IFNγ-producing CD4+ T cells and subsequently used for single-cell VDJ sequencing using the 10x Genomics platform. IFNγ staining by flow cytometry is shown. B, Left, clonotype distribution of A; right, color-coded abundance of clones in individual mice determined by TCRβ deep sequencing. C, Validation of α-CICR215W-TCR-transduced (CT2) CD4+ T cells. Left, coexpression of GFP as reporter for TCR expression in α-CICR215W-TCR-transduced T cells as determined by flow cytometry. Mock, transduction control. Right, IFNγ production of GFP+ α-CICR215W-TCR-transduced T cells after restimulation with irradiated splenocytes pulsed with the indicated peptides. D, IFNγ production of TCR-transduced T cells restimulated with irradiated splenocytes pulsed with the indicated peptide. n = 3 biological replicates. E, NFAT-reporter assay of CT-2 TCR- and NFAT-reporter transduced T cells, restimulated for 4 hours with irradiated splenocytes pulsed with the respective peptide, measured by luciferase signal. n = 3 technical replicates. F, IFNγ secretion of CT2 transduced T cells cocultured with DCs loaded with lysate from CicR215W or CicWT A2.DR1 glioma cells. n = 4 biological replicates. MFI, mean fluorescence intensity. D–F, Data are represented as individual values (D, F) and mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey test. RLU, relative light unit.CC BY-NC-ND · PubMed Central
Figure 4. Adoptive transfer of α-CICR215W-TCR T cells targeting CicR215W mutated A2.DR1 gliomas. A, Experimental overview: Activated A2.DR1 T cells were transduced with the α-CICR215W-TCR TCR coexpressing GFP and subsequently enriched and expanded for intraventricular injection in A2.DR1 glioma-bearing mice. B,In vivo T-cell proliferation of α-CICR215W-TCR T cells in A2.DR1 mice vaccinated with the CICR215W peptide or MOG measured by Cell Trace Far Red dilution. C, Kaplan–Meier curve of A2.DR1 glioma-bearing mice after 2 intraventricular adoptive transfers (AT) of α-CICR215W-TCR T cells and α-Flu-TCR T cells. n = 10 for Flu and n = 11 for CIC. D, Abundance of CD45+, CD3+ T cells in the tumor of CICR215W-reactive T cells or Flu-reactive T cell–treated mice. E, FACS gating for Ly6Chigh monocyte-derived myeloid cells. F, Abundance of Cxcl9 and MHCII expressing Ly6Chigh monocyte-derived macrophages. D and E,n = 5 for CIC and n = 6 for Flu. G, Kaplan–Meier curve of irradiated (total dose 12 Gy) A2.DR1 glioma-bearing mice after 2 intraventricular ATs of α-CICR215W-TCR T cells and α-Flu-TCR T cells. n = 13 for Rad, n = 12 for Flu (irrelevant control), and n = 12 for CIC. B, D, and F, Data are represented as mean ± SEM, statistical significance was determined by unpaired two-tailed Student t test. C and G, Statistical significance was determined by log-rank Mantel–Cox test. NS, not significant.
Figure 4. Adoptive transfer of α-CICR215W-TCR T cells targeting CicR215W mutated A2.DR1 gliomas. A, Experimental overview: Activated A2.DR1 T cells were transduced with the α-CICR215W-TCR TCR coexpressing GFP and subsequently enriched and expanded for intraventricular injection in A2.DR1 glioma-bearing mice. B,In vivo T-cell proliferation of α-CICR215W-TCR T cells in A2.DR1 mice vaccinated with the CICR215W peptide or MOG measured by Cell Trace Far Red dilution. C, Kaplan–Meier curve of A2.DR1 glioma-bearing mice after 2 intraventricular adoptive transfers (AT) of α-CICR215W-TCR T cells and α-Flu-TCR T cells. n = 10 for Flu and n = 11 for CIC. D, Abundance of CD45+, CD3+ T cells in the tumor of CICR215W-reactive T cells or Flu-reactive T cell–treated mice. E, FACS gating for Ly6Chigh monocyte-derived myeloid cells. F, Abundance of Cxcl9 and MHCII expressing Ly6Chigh monocyte-derived macrophages. D and E,n = 5 for CIC and n = 6 for Flu. G, Kaplan–Meier curve of irradiated (total dose 12 Gy) A2.DR1 glioma-bearing mice after 2 intraventricular ATs of α-CICR215W-TCR T cells and α-Flu-TCR T cells. n = 13 for Rad, n = 12 for Flu (irrelevant control), and n = 12 for CIC. B, D, and F, Data are represented as mean ± SEM, statistical significance was determined by unpaired two-tailed Student t test. C and G, Statistical significance was determined by log-rank Mantel–Cox test. NS, not significant.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Kilian, M., Friedrich, M., Sanghvi, K., Green, E., Pusch, S., Kawauchi, D., Löwer, M., Sonner, J. K., Krämer, C., Zaman, J., Jung, S., Breckwoldt, M. O., Willimsky, G., Eichmüller, S. B., von Deimling, A., Wick, W., Sahm, F., Platten, M., & Bunse, L.
Affiliations
  • German Cancer Research Center (DE)
  • Johannes Gutenberg University Mainz (DE)
  • 4DKTK Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Corresponding author
Bunse, Lukas
ORCID
Venue
Clinical Cancer Research
Publisher
American Association for Cancer Research (AACR)
Published
2021-11-15
Volume
28
Issue
2
Pages
378-389
ISSN
  • 1078-0432 (ISSN-L)
  • 1557-3265
DOI
10.1158/1078-0432.ccr-21-1881
PMC
PMC9401455
OpenAlex
W3213468635
Cited by
18
References
55
Citations by year
2026: 1 · 2025: 5 · 2024: 2 · 2023: 6 · 2022: 3 · 2021: 1
FWCI
1.69
Type
journal article
Language
en
Open access
Licence
Locations
  • Clinical Cancer Research (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
German Research Foundation (404521405)
Topics
  • Health Sciences › Medicine › Oncology › Glioma Diagnosis and Treatment
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
Keywords
glioma, MHC class II restriction, T cell receptor transgenic T cells, adoptive T cell therapy, tumor suppressor mutations, adoptive immunotherapy
MeSH terms
Receptors, Chimeric Antigen, Animals, Disease Models, Animal, Glioma, Immunotherapy, Neoplasm Recurrence, Local, T-Lymphocytes, Immunotherapy, Adoptive, Mice

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