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BCR-ABL kinase is the major oncogenic driver of chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKIs), which are highly potent in targeting BCR-ABL, are currently used as first-line treatment. Although TKIs are effective, drug resistance caused by the emergence of drug-selected secondary mutations in BCR-ABL remains a major problem for relapse, especially in patients with compound mutations. In this study, we aimed to investigate potential neoepitopes derived from mutated BCR-ABL and to generate neoepitope-specific TCRs for adoptive T cell therapy. Two candidate peptides derived from the E255V and the T315I mutation (designated ABL-E255V and ABL-T315I) were selected for study based on their in silico predicted binding affinity to HLA-A2. By immunizing transgenic mice that express a diverse human T cell receptor (TCR) repertoire restricted to HLA-A2, we detected CD8+ T cell responses against the ABL-E255V, but not the ABL-T315I peptide. From immune responding mice, two E255V-specific TCRs were isolated. Human CD8+ T cells were engineered to express the specific TCRs for characterization, in which one TCR was identified as a therapeutic candidate due to its superior avidity and lack of detectable off-target reactivity. Importantly, we demonstrated that the ABL-E255V neoepitope was naturally processed and presented. In summary, our results demonstrate that HLA-A2 + CML cells harboring the E255V mutation can be targeted by specific TCRs, which may benefit patients who are highly resistant to available TKIs due to compound mutations.

Recent findings demonstrated proinflammatory functions of interleukin (IL)-9–producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as Cd40 ligand and Cd96, whereas expression of Cd25 and Cd83 was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9⁺ compared with IFN-γ⁺ T cells. Although Il9 was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96low cells. Transfer of CD96low Th9 cells into recombination activating gene 1-deficient (Rag1 −/−) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96high Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96high Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9–associated inflammations such as IBD.

Diffuse midline glioma is the leading cause of solid cancer-related deaths in children with very limited treatment options. A majority of the tumors carry a point mutation in the histone 3 variant (H3.3) creating a potential HLA-A*02:01 binding epitope (H3.3K27M26-35). Here, we isolated an H3.3K27M-specific T cell receptor (TCR) from transgenic mice expressing a diverse human TCR repertoire. Despite a high functional avidity of H3.3K27M-specific T cells, we were not able to achieve recognition of cells naturally expressing the H3.3K27M mutation, even when overexpressed as a transgene. Similar results were obtained with T cells expressing the published TCR 1H5 against the same epitope. CRISPR/Cas9 editing was used to exclude interference by endogenous TCRs in donor T cells. Overall, our data provide strong evidence that the H3.3K27M mutation is not a suitable target for cancer immunotherapy, most likely due to insufficient epitope processing and/or amount to be recognized by HLA-A*02:01 restricted CD8+ T cells.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope F S GEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors in vivo after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

Tumor infiltrating myeloid cells play contradictory roles in the tumor development. Dendritic cells and classical activated macrophages support anti-tumor immune activity via antigen presentation and induction of pro-inflammatory immune responses. Myeloid suppressor cells (MSCs), for instance myeloid derived suppressor cells (MDSCs) or tumor associated macrophages play a critical role in tumor growth. Here, treatment with sodium oleate, an unsaturated fatty acid, induced a regulatory phenotype in the myeloid suppressor cell line MSC-2 and resulted in an increased suppression of activated T cells, paralleled by increased intracellular lipid droplets formation. Furthermore, sodium oleate potentiated nitric oxide (NO) production in MSC-2, thereby increasing their suppressive capacity. In primary polarized bone marrow cells, sodium oleate (C18:1) and linoleate (C18:2), but not stearate (C18:0) were identified as potent FFA to induce a regulatory phenotype. This effect was abrogated in MSC-2 as well as primary cells by specific inhibition of droplets formation while the inhibition of de novo FFA synthesis proved ineffective, suggesting a critical role for exogenous FFA in the functional induction of MSCs. Taken together our data introduce a new unsaturated fatty acid-dependent pathway shaping the functional phenotype of MSCs, facilitating the tumor escape from the immune system.

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRAS G12V - and RAC2 P29L -derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRAS G12V - and RAC2 P29L -derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2 P29L -derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven ‘reverse immunology’ pipeline for the identification of cancer-specific neo-splicetopes.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac . Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Using HLA-A 0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed that PanCoVac is processed and presented by HLA-A 0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines .

Correspondence to Dr Gerald Willimsky; gerald.willimsky@charite.de Our discussion on the targetability of neoantigens in diffuse midline glioma (DMG) is important since (1) DMG as a pediatric high-grade glioma is a devastating disease and a major cause of childhood cancer deaths and (2) adoptive T cell therapy has been shown to be very efficient when targeting ubiquitously expressed CD19 as a B cell lineage-antigen, lacking however bulk evidence about the clinical feasibility of the approach for targeting single amino acid mutations. Since the K27M mutation in H3.3 is a tumor driver in DMG,1 such an approach would be extraordinarily effective and precise, as exclusively cancer cells will be targeted by T cells. [...]we are afraid that false hope is given to children and parents participating for example in clinical trials already set up (NCT02960230) or to be started in the future (NCT04749641, NCT05478837), that altogether aim to target the H3.3K27M26-35 epitope in HLA-A*02:01-positive patients. [...]the statement in the letter to the editor by Chheda et al that the endogenous presentation of the H3.3K27M epitope in HLA-A*02:01 may not be robust is an understatement. In four additional tumor cell lines (one of which was the same U87MG cells as used by Chheda et al3) we even did not achieve T cell recognition when the mutant H3.3 was overexpressed 15–20-fold (online supplemental figures 1B and 2B in Immisch et al).2 More strikingly, using the H3.3 mutant-specific antibody RM192 in western blotting, we detected mutant H3.3 naturally expressed from the heterozygous mutant allele in midline glioma cell lines (figure 2B in Immisch et al)2 as well as overexpressed in tumor cell lines (figure 2B and online supplemental figure 2A in Immisch et al).2 Mutant H3.3K27M protein could also be detected by immunohistochemistry in a series of midline gliomas.6 There is no doubt that the sensitivity to detect mutant H3.3 by H3.3K27M26-35-specific T cells would be by far higher than that of immunodetection in western blotting or histology by a mutant H3.3-specific antibody, if the respective T cell epitope would be naturally processed and presented. Since we accompanied our T cell recognition assays with cell lines overexpressing CDK4R24L and found recognition by the respective CDK4-specific TCR14/352 in all cases, we can exclude any deficiencies in APM or HLA-A*02:01 expression that could have caused the lack of neoantigen presentation to T cells.

BackgroundPersonalized immunotherapy of pancreatic ductal adenocarcinoma (PDAC) through T-cell mediated targeting of tumor-specific, mutanome-encoded neoepitopes may offer new opportunities to combat this disease, in particular by countering recurrence after primary tumor resection. However, the sensitive and accurate calling of somatic mutations in PDAC tissue samples is compromised by the low tumor cell content. Moreover, the repertoire of immunogenic neoepitopes in PDAC is limited due to the low mutational load of the majority of these tumors.MethodsWe developed a workflow involving the combined analysis of next-generation DNA and RNA sequencing data from matched pairs of primary tumor samples and patient-derived xenograft models towards the enhanced detection of driver mutations as well as single nucleotide variants encoding potentially immunogenic T-cell neoepitopes. Subsequently, we immunized HLA/human T-cell receptor (TCR) locus-transgenic mice with synthetic peptides representing candidate neoepitopes, and molecularly cloned the genes encoding TCRs targeting these epitopes.ResultApplication of our pipeline resulted in the identification of greater numbers of non-synonymous mutations encoding candidate neoepitopes with increased confidence. Furthermore, we provide proof of concept for the successful isolation of HLA-restricted TCRs from humanized mice immunized with different neoepitopes, several of which would not have been selected based on mutanome analysis of PDAC tissue samples alone. These TCRs mediate specific T-cell reactivity against the tumor cells in which the corresponding mutations were identified.ConclusionEnhanced mutanome analysis and candidate neoepitope selection increase the likelihood of identifying therapeutically relevant neoepitopes, and thereby support the optimization of personalized immunotherapy for PDAC and other poorly immunogenic cancers.

Background Immune escape is one of the hallmarks of cancer and several new treatment approaches attempt to modulate and restore the immune system’s capability to target cancer cells. At the heart of the immune recognition process lies antigen presentation from somatic mutations. These neo-epitopes are emerging as attractive targets for cancer immunotherapy and new strategies for rapid identification of relevant candidates have become a priority. Methods We carefully screen TCGA data sets for recurrent somatic amino acid exchanges and apply MHC class I binding predictions. Results We propose a method for in silico selection and prioritization of candidates which have a high potential for neo-antigen generation and are likely to appear in multiple patients. While the percentage of patients carrying a specific neo-epitope and HLA-type combination is relatively small, the sheer number of new patients leads to surprisingly high reoccurence numbers. We identify 769 epitopes which are expected to occur in 77629 patients per year. Conclusion While our candidate list will definitely contain false positives, the results provide an objective order for wet-lab testing of reusable neo-epitopes. Thus recurrent neo-epitopes may be suitable to supplement existing personalized T cell treatment approaches with precision treatment options.