Glioblastoma (GBM) is the most frequent and lethal malignant brain tumor in adults. It emerges with an incidence of 3.2 per 100.000 in the US and 3.91 in Europe. Today, standard treatment after diagnosis consists of surgical removal of tumor tissue, followed by radiation therapy and adjuvant chemotherapy using temozolomide. Even after this rigorous therapy, patients show a median overall survival of only 15.6 months or 20.5 months when the tumor is additionally treated with so-called tumor treating fields. GBM is characterized by molecular heterogeneity within the same patient but also between different patients, which impedes development of novel therapeutics. During the last decades various immunotherapies including (multi-epitope) peptide vaccines, oncolytic viruses or immune checkpoint inhibitors against GBM were tested in small clinical studies, but failed to show a benefit in large studies. A novel kind of immunotherapies that showed great success in hematological tumors so far, is based on chimeric antigen receptors (CAR).
These synthetic receptors can be introduced into immune cells to retarget their function towards tumor cells, independently of the major histocompatibility complex (MHC) that is often down regulated by tumors for immune evasion. A large hurdle for treatment of GBM using immunotherapies such as CAR-T cells, is antigen heterogeneity that limits the effect of therapies against single targets and renders the need for discovery of novel targets to enable treatment of a wide variety of patients with high success.
Analyzing publicly available data and performing RT-qPCR experiments with RNA isolated from GBM tissue of a local cohort of patients, overexpression of two candidate GBM antigens, namely TMEM158 and PTPRZ1 were observed. Overexpression of both antigens in GBM in comparison to normal brain tissue and low-grade gliomas (only TMEM158) was revealed. In addition, a negative correlation between expression and patient survival was detected, as well as a correlation between TMEM158 and CD44 expression, the latter being a marker for GBM stem cells and the mesenchymal GBM subtype. Induction of chemoresistance by TMEM158 seems likely for GBM, since this was already discovered for several other tumor entities. Protein expression of TMEM158 was confirmed by Western blot analysis of different GBM cell lines.
Since cell surface expression of a target protein is a prerequisite for targeting by a CAR-therapy, the expression of TMEM158 on cells from GBM cell lines was analyzed by flow cytometry. For this analysis a fluorescence-labeled peptide, based on sequence information of a known naturally occurring TMEM158 ligand (BINP) was designed. Binding to T98G and U-87 MG was observed, while only very low binding to the neuroblastoma cell line SH-SY5Y was seen in flow cytometry. Partial knockdown of TMEM158 was achieved using DsiRNAs, followed by Western blot (antibody staining) and flow cytometry (peptide staining), confirming the specificity of binding detectable by both methods.
A recombinant fusion protein, consisting of the extracellular part of TMEM158 and a human Fc-antibody fragment was produced in 293T cells by transient transfection of an expression vector. The expected size of the protein produced was confirmed by Western blot. Furthermore, binding of the BINP-peptide to the recombinant protein was analyzed and compared to a scrambled BINP-peptide. In these experiments specific binding of the BINP-peptide was observed, also indicating the functionality of the recombinant protein.
Next, CAR-constructs were designed using the original sequence information from BINP as binding domain and additional variants with amino acid exchanges at different positions. Significant cytotoxicity of all BINP-CAR-T cells was observed against T98G, which showed highest binding of BINP when analyzed by flow cytometry. A BINP-CAR version in which phenylalanine 11 was exchanged with alanine (BINP-F11A-CAR) showed significantly higher cytotoxicity against T98G than the BINP-CAR containing the original BINP sequence (BINP-WT-CAR). Against the U-87 MG cell line, only a version of the BINP-CAR containing an RGD- (arginine-glycine-aspartic acid) motif showed significant cytotoxicity. RGD-motifs are known to bind integrins like αVβ3, which was abundantly present on this cell line, as it was confirmed by flow cytometry within this work. Using this BINP-RGD-CAR version, targeting of both antigens at the same time seems possible. No significant cytotoxicity of the different CAR versions was observed against the TMEM158- and αVβ3-low cell line SH-SY5Y.
In conclusion, overexpression of TMEM158 and PTPRZ1 and their negative influence on survival of patients, as found in recent literature, was confirmed for glioblastoma. Significantly higher expression of TMEM158 in GBM in comparison to low-grade gliomas as well as the correlation with CD44 hint at an association of TMEM158 with the aggressive phenotype of GBM. For all of these reasons, targeting of TMEM158 appears to be very feasible. Cytotoxicity of the produced BINP-CAR-T cells, which are the first CAR-T cells targeting TMEM158 so far, was demonstrated against GBM cells.
Additional to cytotoxicity of the CAR-T cells, other in vitro assays and in vivo models should be utilized to determine more aspects of CAR-T cell function, in the future. For example, proliferation, cytokine release, invasion of tumor tissue, and inactivation of CAR-T cells by the tumor milieu should be quantified. To estimate how many patients could benefit from a therapy against it, percentage of patients and distribution within the tumors should be determined.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:88144 |
| Date | 17 November 2023 |
| Creators | Bach, Christoph |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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