Der chimäre Antigen Rezeptor (CAR) mit Spezifität für das Tumorstammzell-Antigen NY-Eso-1 hat eine höhere Aktivierungsschwelle für T-Zellen als der T-Zell Rezeptor (TCR)

In der adoptiven Immuntherapie von Tumorerkrankungen wird T-Zellen eine definierte Spezifität durch die Expression eines Antigen-spezifischen rekombinanten T-Zell Rezeptors (TCR) oder eines chimären Antigen-Rezeptors (CAR) verliehen. Die adoptive Zelltherapie mit CAR und TCR modifizierten T-Zellen erzielte Tumorremissionen, beispielsweise CAR T-Zellen bei der Therapie leukämischer Erkrankungen und TCR T-Zellen in der Behandlung solider Tumore und Myelomen. Die Bindedomäne der CARs zur Erkennung der Tumorzellen ist von einem Antikörper abgeleitet, der TCR erkennt MHC präsentiertes Antigen durch die variable TCR α- und β-Kette. In dieser Arbeit wurde ein Vergleich der T-Zell Aktivierung durch CAR und TCR durchgeführt. Als Modell Antigen wurde NY-Eso-1 verwendet, da NY-Eso-1 von Tumorzellen vieler solider und hämatologischer Erkrankungen exprimiert wird. Um bei der unterschiedlichen Struktur des CAR und TCR eine Vergleichbarkeit herzustellen, wurden CARs verwendet, deren Antikörper-abgeleitete Bindedomäne wie der verwendete TCR das HLA-A2 präsentierte NY-Eso-1(157-165) Antigen erkennt. CAR und TCR erkennen spezifisch das Antigen, jedoch mit unterschiedlicher Affinität; die CARs binden mit einer etwa 30fach oder 350fach höheren Affinität als der TCR. Sowohl der CAR als auch der TCR induzieren eine Antigen-abhängige T-Zell Aktivierung, erkenntlich an der Sekretion proinflammatorischer Zytokine und der zytolytischen Aktivität der modifizierten T-Zellen. Trotz höherer Affinität ist die Antigen-abhängige Aktivierungsschwelle der CAR T-Zellen höher als die der TCR T-Zellen. Die CD28 Kostimulation im CD28CD3ζ CAR verändert nicht die Aktivierungsschwelle, auch eine Steigerung der Affinität der CAR Bindedomäne verändert nicht die Aktivierungsschwelle. Jedoch verstärken sowohl die CD28 Kostimulation als auch die höhere Affinität die Effektorfunktionen der T-Zelle. Eine Trennung des ζ Primärsignals und der CD28 Kostimulation auf zwei koexprimierte CAR Moleküle verringert die T-Zell Aktivierung im Vergleich zum CAR mit kombinierter CD28CD3ζ Signaldomäne. Unsere Ergebnisse zeigen, dass T-Zellen gegen intrazelluläre Antigene sowohl durch einen TCR als auch einen CAR gerichtet werden können, jedoch sind CAR T-Zellen trotz höherer Affinität weniger sensitiv in der Erkennung geringer Antigenmengen als TCR modifizierte T-Zellen und bieten dadurch eine höhere Selektivität für Zielzellen mit hoher Antigendichte. Bei geringer Antigenexpression, wie sie auf NY-Eso-1 exprimierenden Tumorzellen vorkommen kann, ist jedoch eine TCR gerichtete T-Zell Therapie gegenüber CAR T-Zellen zu bevorzugen.The chimeric antigen receptor (CAR) with specificity for the tumor stem cell associated antigen NY-Eso-1 has a higher activation threshold than the NY-Eso-1 specific T cell receptor (TCR) In adoptive immunotherapy T cells are engineered to express an antigen-specific receptor with defined specificity for a tumor-associated antigen (TAA). T cells can be modified with a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). Current studies with either CAR or TCR modified T cells showed lasting and complete tumor regression; CAR redirected T cells exhibited success in the therapy of leukemia, for example, and TCR redirected T cells showed efficacy in the therapy of solid tumors or myeloma. The CAR recognizes the respective antigen by an antibody derived binding domain while the TCR recognizes the antigen by the variable regions of the α and β chains. In contrast to the TCR, the CAR recognizes the antigen in a MHC independent manner and is restricted to the recognition of cell surface antigens. We here compared CAR and TCR driven T cell activation. For a site-by-site comparison, we engineered a “TCR-like” CAR which recognizes the same MHC presented peptide as the recombinant TCR. As model antigen we used the tumor-associated antigen NY-Eso-1 which is presented by the HLA-A2 on solid and hematologic tumor cells. Both the anti-NY-Eso-1 TCR and anti-NY-Eso-1 CAR induced antigen-dependent T cell activation indicated by secretion of proinflammatory cytokines and cytolysis. The binding affinity of the two used CARs is 30-fold and 350-fold higher than the affinity of the TCR. Although the CAR has a higher binding affinity, the CAR modified T cells showed a higher activation threshold than the TCR redirected T cells. The integration of the costimulatory domain CD28 into the CAR and the increase of binding affinity had no impact on the activation threshold of CAR modified T cells; albeit, both CD28 costimulatory domain and improved affinity increased the magnitude in T cell effector functions. Splitting the primary ζ signal and the CD28 costimulatory domain onto two receptors lowered the T cell activation against target cells compared to the CD28CD3ζ CAR with one polypeptide chain. Our data show that both TCR and CAR redirect a T cell response towards intracellular antigens presented in the MHC context. Despite higher binding affinity, CAR T cells are less sensitive in recognizing the antigen than TCR modified T cells. Therefore CAR modified T cells are more selective in recognizing target cells with high antigen density levels while TCR redirected T cells are more effective in targeting tumor cells with low antigen amounts, like NY-Eso-1 positive tumors

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Addition of Multimodal Immunotherapy to Combination Treatment Strategies for Children with DIPG: A Single Institution Experience

Background: The prognosis of children with diffuse intrinsic pontine glioma (DIPG) remains dismal despite radio- and chemotherapy or molecular-targeted therapy. Immunotherapy is a powerful and promising approach for improving the overall survival (OS) of children with DIPG. Methods: A retrospective analysis for feasibility, immune responsiveness, and OS was performed on 41 children treated in compassionate use with multimodal therapy consisting of Newcastle disease virus, hyperthermia, and autologous dendritic cell vaccines as part of an individualized combinatorial treatment approach for DIPG patients. Results: Patients were treated at diagnosis (n = 28) or at the time of progression (n = 13). In the case of 16 patients, histone H3K27M mutation was confirmed by analysis of biopsy (n = 9) or liquid biopsy (n = 9) specimens. PDL1 mRNA expression was detected in circulating tumor cells of ten patients at diagnosis. Multimodal immunotherapy was feasible as scheduled, until progression, in all patients without major toxicity. When immunotherapy was part of primary treatment, median PFS and OS were 8.4 m and 14.4 m from the time of diagnosis, respectively, with a 2-year OS of 10.7%. When immunotherapy was given at the time of progression, median PFS and OS were 6.5 m and 9.1 m, respectively. A longer OS was associated with a Th1 shift and rise in PanTum Detect test scores. Conclusions: Multimodal immunotherapy is feasible without major toxicity, and warrants further investigation as part of a combinatorial treatment approach for children diagnosed with DIPG

Randomized Controlled Immunotherapy Clinical Trials for GBM Challenged

Simple Summary: Although multiple meta-analyses on active specific immunotherapy treatment for glioblastoma multiforme (GBM) have demonstrated a significant prolongation of overall survival, no single research group has succeeded in demonstrating the efficacy of this type of treatment in a prospective, double-blind, placebo-controlled, randomized clinical trial. In this paper, we explain how the complexity of the tumor biology and tumor-host interactions make proper stratification of a control group impossible. The individualized characteristics of advanced therapy medicinal products for immunotherapy contribute to heterogeneity within an experimental group. The dynamics of each tumor and in each patient aggravate comparative stable patient groups. Finally, combinations of immunotherapy strategies should be integrated with first-line treatment. We illustrate the complexity of a combined first-line treatment with individualized multimodal immunotherapy in a group of 70 adults with GBM and demonstrate that the integration of immunogenic cell death treatment within maintenance chemotherapy followed by dendritic cell vaccines and maintenance immunotherapy might provide a step towards improving the overall survival rate of GBM patients. Immunotherapies represent a promising strategy for glioblastoma multiforme (GBM) treatment. Different immunotherapies include the use of checkpoint inhibitors, adoptive cell therapies such as chimeric antigen receptor (CAR) T cells, and vaccines such as dendritic cell vaccines. Antibodies have also been used as toxin or radioactive particle delivery vehicles to eliminate target cells in the treatment of GBM. Oncolytic viral therapy and other immunogenic cell death-inducing treatments bridge the antitumor strategy with immunization and installation of immune control over the disease. These strategies should be included in the standard treatment protocol for GBM. Some immunotherapies are individualized in terms of the medicinal product, the immune target, and the immune tumor-host contact. Current individualized immunotherapy strategies focus on combinations of approaches. Standardization appears to be impossible in the face of complex controlled trial designs. To define appropriate control groups, stratification according to the Recursive Partitioning Analysis classification, MGMT promotor methylation, epigenetic GBM sub-typing, tumor microenvironment, systemic immune functioning before and after radiochemotherapy, and the need for/type of symptom-relieving drugs is required. Moreover, maintenance of a fixed treatment protocol for a dynamic, deadly cancer disease in a permanently changing tumor-host immune context might be inappropriate. This complexity is illustrated using our own data on individualized multimodal immunotherapies for GBM. Individualized medicines, including multimodal immunotherapies, are a rational and optimal yet also flexible approach to induce long-term tumor control. However, innovative methods are needed to assess the efficacy of complex individualized treatments and implement them more quickly into the general health system

Individualized Multimodal Immunotherapy for Adults with IDH1 Wild-Type GBM: A Single Institute Experience

Synergistic activity between maintenance temozolomide (TMZm) and individualized multimodal immunotherapy (IMI) during/after first-line treatment has been suggested to improve the overall survival (OS) of adults with IDH1 wild-type MGMT promoter-unmethylated (unmeth) GBM. We expand the data and include the OS of MGMT promoter-methylated (meth) adults with GBM. Unmeth (10 f, 18 m) and meth (12 f, 10 m) patients treated between 27 May 2015 and 1 January 2022 were analyzed retrospectively. There were no differences in age (median: 48 y) or Karnofsky performance index (median: 80). The IMI consisted of 5-day immunogenic cell death (ICD) therapies during TMZm: Newcastle disease virus (NDV) bolus injections and sessions of modulated electrohyperthermia (mEHT); subsequent active specific immunotherapy: dendritic cell (DC) vaccines plus modulatory immunotherapy; and maintenance ICD therapy. There were no differences in the number of vaccines (median: 2), total number of DCs (median: 25.6 × 106), number of NDV injections (median: 31), and number of mEHT sessions (median: 28) between both groups. The median OS of 28 unmeth patients was 22 m (2y-OS: 39%), confirming previous results. OS of 22 meth patients was significantly better (p = 0.0414) with 38 m (2y-OS: 81%). There were no major treatment-related adverse reactions. The addition of IMI during/after standard of care should be prospectively explored

From telomere to telomere: The transcriptional and epigenetic state of human repeat elements

Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome

From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.

Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome