Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.
Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.
Adoptive therapy with T-cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T-cell reactivity. Here, we propose a sequence of assays to improve selection of TCRs and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against cancer germline antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif toward its cognate peptide, and a TCR's cross-reactivity toward noncognate peptides..
Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-β upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.
Introduction Large-scale sequencing studies have unraveled the mutational landscape of myelofibrosis (MF), demonstrating clonal heterogeneity and importance of genetically defined subgroups in disease prognosis and progression. In order to elucidate the genetics of MF progression and its molecular drivers during JAK inhibition therapy, we performed in-depth genetic studies on longitudinal blood samples from 15 MF patients covering a disease span of 3 to 5 years after initiation of ruxolitinib. Methods Sequential samples from 15 MF patients (PMF n=8; post-ET/PV-MF n=7) accounting for a total of 42 time points representing 58.5 years of ruxolitinib treatment were investigated by whole-exome sequencing (WES). Additionally, we performed targeted deep sequencing of patient-specific mutations in flow-sorted cell fractions to study clonal repartition within the hematopoietic differentiation tree. Finally, we genotyped more than 5000 Lin-CD34+ progenitor cells using a single-cell multiplexed qPCR approach on a micro-fluidic platform (Fluidigm) to infer MF phylogeny. Results WES identified a median of 14 non-silent somatic mutations per patient at initiation of ruxolitinib treatment (=baseline WES; Figure 1A). When comparing mutations between first and last investigated time points, the majority of baseline mutations (162/201=81%) could be detected also at a later disease stage. A total of 39 mutations were lost and 80 new mutations were detected at the last time point. All patients showed at least one gained/ lost mutation in sequential samples. We noted frequent acquisition of mutations in genes of the RAS/RTK pathways in one third of patients. Two patients with a JAK2 V617F mutation achieved a molecular remission at a level of persisting residual disease of 1x10-3 with ruxolitinib therapy. In one of them, a total of 13 mutations were detected at baseline. In the second sample, taken three years later, a completely different set of mutations was identified and at the last time point, four years after initiation of therapy, none of the mutations were detected. This likely represents genetic drift during neutral evolution as a consequence of a rapid expansion after JAK inhibition. All other 13 patients showed only a modest - if any - decrease of 10-20% JAK2/CALR allele burden which was often accompanied with the expansion of JAK2/CALR-wildtype clones due to positive selection and/or freed clonal space under treatment. However, in some patients with durable response to ruxolitinib, we noted opposing dynamics of clones questioning a common origin. The three patients who progressed to leukemia showed a higher number of mutations at baseline and all of them acquired mutations in KRAS or NRAS over time. As one example, MPN18 harbored mutations in ASXL1, ETV6, and SRSF2 at baseline. Thereafter, and in addition to other driver genes (IDH2,KRAS) a second JAK2 Mutation at codon R867 was acquired, which has been reported to confer treatment resistance to JAK Inhibitors (Marty, Blood 2014). Mutation analysis in flow-sorted cell fractions showed a higher allelic mutation load in the myeloid compared to the lymphoid compartment with only few mutations being detected at low allele frequency in lymphocytes. Interestingly, some patients showed evidence of differential expansion among different myeloid cell lineages (Figure 1B). Next, we sorted 480 CD34+ single-cells per sample from 12 time points from 8 patients which allowed identification of subclones at ≥2% frequency based on priori power calculations. Sorting errors (e.g.cell doublets, empty wells)determined the mean cell sorting failure rate to be 12.5%. We employed a heuristic search algorithm to select a phylogenetic tree with Maximum Likelihood under a finite site model of evolution. Loss of heterozygosity (LOH) events were found in 7/8 patients and were not restricted to the JAK2 locus. In some patients, LOH of JAK2 occurred independently in two subclones, a phenomenon of convergent evolution (Figure 1C). We also noted cases with multiple 9pUPDs, of which one got selected during therapy. LOH events gave rise to both, a mutant homozygous but also reversion to a wildtype genotype. Conclusions Comprehensive serial genotyping of MF patients treated with ruxolitinib revealed heterogeneous patterns of clonal composition and evolution. Our data support LOH as a major determination factor for clonal diversification in MF. EM, KY, and MF contributed equally Figure 1 Disclosures Zenz: Abbvie: Consultancy, Honoraria, Other: Travel support; Roche: Consultancy, Other: Travel support; Janssen: Consultancy; Takeda: Consultancy; Gilead: Honoraria. Bullinger:Pfizer: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Bayer: Other: Financing of scientific research; Seattle Genetics: Honoraria; Sanofi: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Le Coutre:Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Ogawa:Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding. Damm:Novartis: Research Funding; AbbVie: Other: Travel support.
BackgroundMelanoma-associated antigen 1 (MAGE-A1) is a cancer-testis antigen with highly selective expression in testis (which is an immune privileged site) and in multiple high unmet medical need cancers. Therefore, it represents an attractive target for T cell receptor (TCR)-based therapies. TK-8001 is a MAGE-A1 directed TCR with optimized affinity and specificity, derived from the huTCR mouse platform,1 introduced by retroviral transduction into autologous patient-derived CD8+ T cells. The anticipated mode of action of TK-8001 is to bind to MAGE-A1-epitope presenting tumor cells and eliminate them via CD8+ cytotoxic activity and interferon-γ release. Preclinical exploration of the TK-8001 TCR has demonstrated potent antitumor activity, even in low-expressing MAGE-1 positive tumor cells, and favorable benchmarking vs. existing MAGE-A1 directed TCRs derived from human donors. This abstract describes the currently launched phase 1/2 trial for TK-8001.MethodsThe IMAG1NE trial (Immunotherapeutic MAGE-A1 directed Neoplasm Elimination) is a phase 1/2, first-in-human, open-label, accelerated titration, two-part clinical trial of TK-8001 (MAGE-A1-directed TCR-transduced autologous CD8+ T cells) in subjects with HLA-A*02:01 genotype and advanced-stage/metastatic, MAGE-A1+ solid tumors that either have no approved therapeutic alternative(s) or are in non-curable state and have received a minimum of two lines of systemic therapy. Major endpoints for the IMAG1NE trial will be safety, pharmacokinetics, pharmacodynamics (e.g. cytokine profiles) as well as preliminary clinical efficacy (degree of tumor mass reduction and duration of response).In Part 1 of the trial, three different doses of TK-8001 will be explored for safety and preliminary clinical efficacy in an accelerated titration design. The starting dose is set at 1x10E8 MAGE-A1 TCR transduced CD8+ T cells followed by two escalation steps. Part 2 of the trial will enroll up to 30 subjects with advanced-stage, MAGE-A1 positive cancer to confirm safety and efficacy.The study is expected to open for enrolment in Q4/2021. For further information please contact T-knife GmbH at info@t-knife.com.ReferencesLi, Liang-Ping, J Christoph Lampert, Xiaojing Chen, Catarina Leitao, Jelena Popović, Werner Müller, and Thomas Blankenstein. Transgenic mice with a diverse human T cell antigen receptor repertoire. Nature Medicine 2010;16: 1029–34.Ethics ApprovalIn progress, expected 11/2021
BackgroundCancer testis antigens (CTAs) are considered attractive targets for T cell receptor (TCR)-based cellular therapies as their expression in healthy adults is considered restricted to the immune-privileged testis. However, low-level expression of some CTAs in healthy tissue has been observed, resulting in significant on-target/off-cancer toxicity. Melanoma associated antigen 1 (MAGE-A1) is a member of the MAGE-A CTA family, whose members are known to influence cellular signaling pathways through their E3 ubiquitin ligase-binding MAGE homology domain. MAGE-A proteins are frequently expressed in different cancer types, have been linked to oncogenic activity and their expression has been associated with poor prognosis.1 Literature data suggest that in healthy tissues MAGE-A1 is detected in testis, only, with one exception suggesting MAGE-A1 RNA expression in cerebellum and cerebrum.2 Therefore, to evaluate MAGE-A1 as a potential target for cellular immunotherapies, an in-depth analysis of MAGE-A1 expression in > 70 different healthy tissue types and > 5,000 cancer biopsies was conducted, aiming to assess if MAGE-A1 represents a valid and safe target.MethodsA MAGE-A1 antibody with high specificity (TK-AbMA1P) was identified and characterized for immunohistochemistry. A large panel of > 70 different healthy tissue types and > 5,000 tumor biopsies was explored and scored for MAGE-A1 expression by tissue microarray. Identified cancer entities with relevant MAGE-A1 expression were further investigated to assess spatial intratumoral MAGE-A1 expression distribution and expression consistency between primary tumor and lymph node/distant metastases.ResultsCharacterization of TK-AbMA1P demonstrated fully paralog-selective staining for MAGE-A1. Analysis of MAGE-A1 expression in over 70 different healthy tissues confirmed strictly selective expression of MAGE-A1 in testis. An extended analysis of various CNS tissues including cerebellum and cerebrum did not reveal any expression in CNS. The analysis of > 5,000 tumor biopsies showed significant MAGE-A1 expression in distinct subgroups of multiple major tumor types with high unmet medical need. Substantial expression was detected for example in non-small-cell lung cancer, various breast cancer subtypes, gastrointestinal and urogenital cancers, among others. Extended analysis of the MAGE-A1 positive tumors demonstrated highly homogenous and consistent spatial intratumoral distribution of MAGE-A1 expression as well as between primary tumor and metastases.ConclusionsThis analysis confirms that MAGE-A1 is a highly selectively expressed CTA and demonstrates relevant expression in various indications with high unmet medical need, suggesting that MAGE-A1 is an ideal target for highly potent TCR-based adoptive cell therapy.ReferencesWeon JL, Potts PR. The MAGE protein family and cancer. Curr Opin Cell Biol 2015;37:1–8.Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM, Phan GQ, Hughes MS, Kammula US, Miller AD, Hessman CJ, Stewart AA, Restifo NP, Quezado MM, Alimchandani M, Rosenberg AZ, Nath A, Wang T, Bielekova B, Wuest SC, Akula N, McMahon FJ, Wilde S, Mosetter B, Schendel DJ, Laurencot CM, Rosenberg SA. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013;36(2):133–51.Ethics ApprovalThis study was approved by the Ethics Commission of the Ärztekammer Hamburg; approval number WF-049/09. Participants gave informed consent before taking part.
Zusammenfassung Mehr als 20 Jahre nach den ersten Versuchen, die gezeigt haben, dass T-Lymphozyten mittels Gentransfer eines chim?ren Antigenrezeptors (CAR) k?nstlich mit einer Spezifit?t gegen Tumorantigene ausger?stet werden k?nnen, erzielt dieser Ansatz bahnbrechende therapeutische Erfolge bei Patienten mit B-Zell-Leuk?mien unter Verwendung von Anti-CD19-CAR-T-Zellen. Die langanhaltende klinische Remission in Patienten mit ansonsten ?beraus schlechter Prognose ist der 2. und 3.?Generation chim?rer Antigenrezeptoren geschuldet, die nun die f?r eine Expansion von T-Lymphozyten in vivo wichtige Kostimulation intrinsisch innerhalb des CAR vorhalten. Der gro?e therapeutische Durchbruch in zuvor unheilbaren h?matologischen Erkrankungen hat zu einer Flut von neuen therapeutischen Ans?tzen in einer Vielzahl von h?matologischen und soliden Tumoren gef?hrt, bei denen diese neue Zelltherapie nun klinisch erprobt wird. Es bleibt abzuwarten, ob sich die guten klinischen Ergebnisse, die bei akuten lymphatischen Leuk?mien (B-ALL) erzielt wurden, auch auf andere h?matologische Erkrankungen und insbesondere auch auf solide Tumoren ausweiten lassen. Nichtsdestotrotz stehen f?r Patienten mit B-Zell-Leuk?mien, bei denen bisherige Standardtherapien ? inklusive einer Stammzelltransplantation ? versagt haben, heute neue Therapieoptionen zur Verf?gung. Diese effizienten Therapieverfahren sollten m?glichst zeitnah auch f?r Patienten in Deutschland zug?ngig gemacht werden. Die Einrichtungen f?r Transfusionsmedizin, H?matologie und Onkologie sowie die nationalen und internationalen Aufsichtsbeh?rden sind gefragt, hier konstruktiv und mit Blick auf den klinischen Bedarf ? jedoch ebenfalls die Sicherheit ? der Patienten eine rasche Verf?gbarkeit in Deutschland zu gew?hrleisten.
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