A phase I/II escalation trial design T-RAD : Treatment of metastatic lung cancer with mRNA-engineered T cells expressing a T cell receptor targeting human telomerase reverse transcriptase (hTERT)

Funding Information: This research was funded the South-Eastern Norway Regional Health Authority (Grant number 2017075), the Research Council of Norway (Grant number: 244388), and the Radium Hospital Research Foundation. Publisher Copyright: Copyright © 2022 Maggadóttir, Kvalheim, Wernhoff, Sæbøe-Larssen, Revheim, Josefsen, Wälchli, Helland and Inderberg.Background: Adoptive cellular therapy (ACT) with genetically modified T cells aims to redirect T cells against resistant cancers through introduction of a T cell receptor (TCR). The Radium-4 TCR was isolated from a responding patient in a cancer vaccination study and recognizes the enzymatic component of human Telomerase Reverse Transcriptase (hTERT) presented on MHC class II (HLA-DP04). hTERT is a constitutively overexpressed tumor-associated antigen present in most human cancers, including non-small-cell lung cancer (NSCLC), which is the second most common type of cancer worldwide. Treatment alternatives for relapsing NSCLC are limited and survival is poor. To improve patient outcome we designed a TCR-based ACT study targeting hTERT. Methods: T-RAD is a phase I/II study to evaluate the safety and efficacy of Radium-4 mRNA electroporated autologous T cells in the treatment of metastatic NSCLC with no other treatment option. Transient TCR expression is applied for safety considerations. Participants receive two intravenous injections with escalating doses of redirected T cells weekly for 6 consecutive weeks. Primary objectives are safety and tolerability. Secondary objectives include progression-free survival, time to progression, overall survival, patient reported outcomes and overall radiological response. Discussion: Treatment for metastatic NSCLC is scarce and new personalized treatment options are in high demand. hTERT is a tumor target applicable to numerous cancer types. This proof-of-concept study will explore for the first time the safety and efficacy of TCR mRNA electroporated autologous T cells targeting hTERT. The T-RAD study will thus evaluate an attractive candidate for future immunotherapy of solid tumors.Peer reviewe

A full scale comparative study of methods for generation of functional Dendritic cells for use as cancer vaccines

<p/> <p>Background</p> <p>Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC). Recently, Dauer and co-workers presented a modified protocol for differentiation of human monocytes into mature DCs within 48 hours (Fast DC). Here we report a functional comparison of the two strategies for generation of DCs from human monocytes with adaptions for large-scale clinical use.</p> <p>Methods</p> <p>The Elutra Cell Selection System was used to isolate monocytes after collection of leukapheresis product. The enriched monocytes were cultured in gas permeable Teflon bags with IL-4 and GM-CSF for 24 hours (Fast DC) or 5 days (Standard DC) to obtain immature DCs. The cells were then transfected with mRNA from the leukemia cell line Jurkat E6 by electroporation and incubated for additional 24 h or 2 days in the presence of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and PGE₂) to obtain mature DCs.</p> <p>Results</p> <p>Mature Fast DC and Standard DC displayed comparable levels of many markers expressed on DC, including HLA-DR, CD83, CD86, CD208 and CCR7. However, compared to Standard DC, mature Fast DC was CD14^highCD209^low. Fast DC and Standard DC transfected with Jurkat E6-cell mRNA were equally able to elicit T cell specifically recognizing transfected DCs in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.</p> <p>Conclusion</p> <p>Our results indicate that mature Fast DC are functional antigen presenting cells (APCs) capable of inducing primary T-cell responses, and suggest that these cells may be valuable for generation of anti-tumor vaccines.</p

Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

Background Human telomerase reverse transcriptase (hTERT) is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. Results By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. Conclusion The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression

Exploring the Impact of mRNA Modifications on Translation Efficiency and Immune Tolerance to Self-Antigens

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1’ glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms’ tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH2CH3) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens

Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia

Background Leukemia is a clonal disorder characterized by uncontrolled proliferation of haematopoietic cells, and represents the most common form of cancer in children. Advances in therapy for childhood leukemia have relied increasingly on the use of high-dose chemotherapy often combined with stem-cell transplantation. Despite a high success rate and intensification of therapy, children still suffer from relapse and progressive disease resistant to further therapy. Thus, novel forms of therapy are required. Methods This study focuses on dendritic cell (DC) vaccination of childhood leukemia and evaluates the in vitro efficacy of different strategies for antigen loading of professional antigen-presenting cells. We have compared DCs either loaded with apoptotic leukemia cells or transfected with mRNA from the same leukemia cell line, Jurkat E6, for their capacity to induce specific CD4+ and CD8+ T-cell responses. Monocyte-derived DCs from healthy donors were loaded with tumor antigen, matured and co-cultured with autologous T cells. After one week, T-cell responses against antigen-loaded DCs were measured by enzyme-linked immunosorbent spot (ELISPOT) assay. Results DCs loaded with apoptotic Jurkat E6 cells or transfected with Jurkat E6-cell mRNA were both able to elicit specific T-cell responses in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets. Conclusion The results indicate that loading of DCs with apoptotic leukemia cells or transfection with tumour mRNA represent promising strategies for development of cancer vaccines for treatment of childhood leukemia