Abstract Glioblastoma is a lethal brain tumor notable for limited spontaneous induction of CD8+ T cell responses. While viral-vector vaccines can drive particularly high magnitudes of tumor-reactive T cells, they have not been investigated for the treatment of glioblastoma. Here, we demonstrate that heterologous prime-boost vaccination with the simian adenovirus ChAdOx1 and poxvirus modified vaccinia Ankara (MVA) treats the orthotopic, syngeneic, checkpoint-inhibitor refractory SB28 murine model of glioblastoma, both in the context of the murine tumor antigen, P1A, and a newly identified tumor-associated antigen expressed by SB28. Adjuvant anti-PD-1 and anti-CTLA-4 did not further improve outcomes. Vaccination induced immunoediting of tumor antigen expression and tumor-specific recruitment of antigen-specific T cells to challenged brains, the majority of which had a CD103+CD69+CD8+ tissue resident memory (TRM)-like phenotype. While induction of TRMs by vaccination has been implicated in superior control of other cancers, their role in mediating anti-glioblastoma immunity was unclear. ChAdOx1/MVA-induced brain TRMs displayed superior polyfunctionality compared to circulating and non-TRM brain antigen-specific CD8+ T cells. Long-term surviving mice maintained consistent levels of antigen-specific TRM cells in their brains at days 70 and 175 post-tumor challenge, despite a three-fold decrease in circulating antigen-specific T cells, indicating durable T cell memory in the brain parenchyma. Furthermore, survivors were protected against a second orthotopic, but not subcutaneous, tumor rechallenge, demonstrating tissue-specific immunological memory. Intracranial adoptive transfer of brain-derived antigen-specific TRMs isolated from vaccinated, tumor-bearing mice was sufficient to prolong the survival of naïve mice challenged with tumors, whereas blood-derived antigen-specific non-TRMs and non-antigen-specific brain-derived CD8+ T cells were not. Despite peripheral administration of the vaccines, antigen-specific CD8+ T cells were induced not only in the brains of tumor-free animals, but in many tissues, including the liver, lung, skin, skull bone marrow, and meninges, with distinct phenotypes observed in different tissues. Overall, we demonstrate that ChAdOx1/MVA vaccination is a potent strategy to induce TRMs to mediate anti-glioblastoma immunity, establishing a basis for further clinical investigation of ChAdOx1/MVA vaccination to treat patients with glioblastoma. Citation Format: Emily Elizabeth Steffke, Laila Latifi, Taijun Hana, Ayaka Hara, Morgan Coombs, Jo Spurgeon, Caitlin Huguely, John Hancock, Brita Anderson, James McAuliffe, Vinnycius Pereira-Almeida, Amanda Wicki, Sara Abdel Malak, Laurine Noblecourt, Meili Zhang, Wei Zhang, Dionne Davis, Nicole Briceno, Hua Song, Chen Cam-El Makranz, Hideho Okada, Mark Gilbert, Carol Leung, Benoit Van den Eynde, Masaki Terabe. Viral vector vaccination induces brain resident memory T cells to drive anti-glioblastoma immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4363.

The aggressiveness of glioblastoma may be due to its limited induction of anti-tumor T cells; therefore, vaccines are attractive candidates to drive tumor-specific T cells. Heterologous prime-boost vaccination with chimpanzee adenovirus (ChAdOx1) and modified vaccinia Ankara (MVA) viral vectors can generate high tumor-targeted CD8+ T cell frequencies. Therefore, we tested whether this vaccination strategy induces effective T cell responses against murine glioblastoma. We used the orthotopic syngeneic SB28.P1A glioblastoma model expressing P1A, a mouse tumor antigen. Both prophylactic and therapeutic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of mice with SB28.P1A tumors. Tumors were enriched for P1A-specific CD8+ T cells, with 40% of CD8+ T cells P1A-specific in the tumor-bearing hemisphere, compared to 20% in the contralateral hemisphere and 5% in the blood. Strikingly, 70% of tumor-infiltrating P1A-specific CD8+ T cells were resident memory T (Trm) cells. The adoptive transfer of sorted P1A-specific Trm cells derived from vaccinated tumor-bearing mice into the brains of naïve mice sufficiently protected mice from later challenge with tumors, while P1A-specific non-Trm cells or non-P1A-specific brain-derived CD8+ T cells did not. The capability of ChAdOx1/MVA vaccination to induce antigen-specific Trms in the brain that are sufficient to protect against glioblastoma is promising for translating this therapy into clinical trials. Supported by NIH intramural research program ZIA BC011877 and the Ludwig Institue for Cancer Research. Vaccines and Immunotherapy (VAC)

Tumours face tryptophan (Trp) depletion, but the mechanisms sustaining protein biosynthesis under Trp stress remain unclear. We report that Trp stress increases the levels of the translation repressor EIF4EBP1. Yet, at the same time, EIF4EBP1 is selectively phosphorylated by the metabolic master regulator MTORC1 kinase, preventing EIF4EBP1 from inhibiting translation. MTORC1 activity under Trp stress is unexpected because the absence of amino acids is typically linked with MTORC1 inhibition. EIF4EBP1-sensitive translation in Trp starved cells is sustained by EGFR and RAS signalling to MTORC1. Via this mechanism, Trp stress enhances the synthesis and activity of the aryl hydrocarbon receptor (AHR). This is noteworthy as Trp catabolites are known to activate AHR, and therefore Trp stress was previously considered to inhibit AHR. Trp stress-induced AHR enhances the expression of key regulators of autophagy, which sustains intracellular Trp levels and Trp-charged tRNAs for translation. Hence, Trp stress switches MTORC1 from its established inhibitory function into an enhancer of autophagy, acting through AHR. The clinical potential of this fundamental mechanism is highlighted by the activity of the mTORC1-AHR pathway and an autophagy signature in 20% of glioblastoma patients, opening up new avenues for cancer therapy. ### Competing Interest Statement AS, ST and CO are founders and AS and CO are managing directors of cAHRmeleon Bioscience GmbH. VIK is a Scientific Advisor for Longaevus Technologies. Authors of this manuscript have patents on AHR inhibitors in cancer (WO2013034685, CO); A method to multiplex tryptophan and its metabolites (WO2017072368, CO); A transcriptional signature to determine AHR activity (WO2020201825, AS, ST, CO); Interleukin-4-induced gene 1 (IL4I1) as a biomarker (WO2020208190, AS, ST, LFSP, MTP, CO) Interleukin-4-induced gene 1 (IL4I1) and its metabolites as biomarkers for cancer (WO2021116357, AS, ST, LFSP, CO); a targeted proteomics method to monitor autophagy (EP23182541, AL, JD).

Background Glioblastomas are immunologically 'cold' tumors with sparse cytotoxic T-cell infiltration. Therefore, viral vector vaccines may be a promising approach to boost the induction of glioblastoma-targeted T cells. It has been previously shown that heterologous prime-boost vaccination with chimpanzee-derived adenovirus ChAdOx1 and modified vaccinia Ankara (MVA) vectors can induce a high magnitude of CD8+ T cells specific for cancer-associated antigens and have therapeutic effects against mouse models of cancer.1 Therefore, we aimed to evaluate whether treating mice with ChAdOx1 and MVA vaccines targeting model tumor antigens and endogenous antigens could be beneficial in the prophylactic and therapeutic settings against syngeneic, intracranial murine glioblastoma. Methods Murine glioblastoma cell lines were developed to express model tumour antigens. In addition, endogenous tumor-associated antigens and neoantigens were identified in murine glioblastoma lines using our novel Mouse nEoanTigen pRedictOr (METRO) antigen discovery pipeline. We then created ChAdOx1 and MVA vectors expressing model tumor antigens or endogenous antigen candidates, and we confirmed their immunogenicity via intracellular cytokine staining of ex vivo stimulated peripheral blood mononuclear cells or splenocytes. In the prophylactic tumor setting, mice were vaccinated with ChAdOx1 and MVA vectors expressing endogenous antigens or a model tumor antigen, then challenged with syngeneic intracranial wild-type or model antigen expressing tumors, respectively. In the therapeutic setting, mice bearing intracranial tumors were treated with vaccines in combination with checkpoint inhibitors after confirming tumor formation. Results ChAdOx1 and MVA heterologous prime-boost vaccination generated a high magnitude of antigen-specific CD8+ T cells against a model tumor antigen. Furthermore, we confirmed the immunogenicity of some of the antigens identified by the METRO pipeline. Prophylactic vaccination targeting a model tumor antigen significantly increased the survival time of mice bearing intracranial tumors engineered to express the same antigen. Ongoing studies are investigating the efficacy of this vaccination strategy in the therapeutic setting. Conclusions Our heterologous prime-boost strategy generates a high magnitude of antigen-specific CD8+ T cells that provide protection against the development of orthotopic glioblastoma tumors in mice. It remains to be seen whether these vaccines can provide a therapeutic benefit to mice bearing intracranial tumors, and the effects of vaccination on the remodeling of the tumor microenvironment and tumor-draining lymph nodes are yet to be determined. Preclinical data generated using our vaccine and tumor models may provide proof-of-concept to move these vaccines into clinical trials to treat patients with glioblastoma. Reference 1. McAuliffe J, Chan HF, Noblecourt L, Ramirez-Valdez RA, Pereira-Almeida V, Zhou Y, Pollock E, Cappuccini F, Redchenko I, Hill AV, Leung CSK, Van den Eynde BJ. Heterologous prime-boost vaccination targeting MAGE-type antigens promotes tumor T-cell infiltration and improves checkpoint blockade therapy. J Immunother Cancer . 2021 Sep; 9 (9):e003218. Ethics Approval All animal work was approved by either the University of Oxford Animal Care and Ethical Review Committee and experimental procedures were carried out in accordance with the terms of the UK Animals (Scientific Procedures) Act Project Licenses P0D369534 and PB050649E; or by the National Cancer Institute-Bethesda Animal Care and Use Committee and experimental procedures were carried out in accordance with the terms of Protocol NOB-024.

For inoperable esophageal adenocarcinoma (EAC), identifying patients likely to benefit from recently approved immunochemotherapy (ICI+CTX) treatments remains a key challenge. We address this using a uniquely designed window-of-opportunity trial (LUD2015-005), in which 35 inoperable EAC patients received first-line immune checkpoint inhibitors for four weeks (ICI-4W), followed by ICI+CTX. Comprehensive biomarker profiling, including generation of a 65,000-cell single-cell RNA-sequencing atlas of esophageal cancer, as well as multi-timepoint transcriptomic profiling of EAC during ICI-4W, reveals a novel T cell inflammation signature (INCITE) whose upregulation correlates with ICI-induced tumor shrinkage. Deconvolution of pre-treatment gastro-esophageal cancer transcriptomes using our single-cell atlas identifies high tumor monocyte content (TMC) as an unexpected ICI+CTX-specific predictor of greater overall survival (OS) in LUD2015-005 patients and of ICI response in prevalent gastric cancer subtypes from independent cohorts. Tumor mutational burden is an additional independent and additive predictor of LUD2015-005 OS. TMC can improve patient selection for emerging ICI+CTX therapies in gastro-esophageal cancer.

BACKGROUND:Despite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell viability and functionality within the immunosuppressive tumor microenvironment, where oxygen levels are often low. Hypoxia can reduce CD8 T-cell fitness in several ways and CD8 T cells are mostly excluded from hypoxic tumor regions. Given the challenges to achieve durable reduction of hypoxia in the clinic, ameliorating CD8 T-cell survival and effector function in hypoxic condition could improve tumor response to immunotherapies.METHODS:Activated CD8 T cells were exposed to hypoxia and metformin and analyzed by fluorescence-activated cell sorting for cell proliferation, apoptosis and phenotype. In vivo, metformin was administered to mice bearing hypoxic tumors and receiving either adoptive cell therapy with tumor-specific CD8 T cells, or immune checkpoint inhibitors; tumor growth was followed over time and CD8 T-cell infiltration, survival and localization in normoxic or hypoxic tumor regions were assessed by flow cytometry and immunofluorescence. Tumor oxygenation and hypoxia were measured by electron paramagnetic resonance and pimonidazole staining, respectively.RESULTS:We found that the antidiabetic drug metformin directly improved CD8 T-cell fitness in hypoxia, both in vitro and in vivo. Metformin rescued murine and human CD8 T cells from hypoxia-induced apoptosis and increased their proliferation and cytokine production, while blunting the upregulation of programmed cell death protein 1 and lymphocyte-activation gene 3. This appeared to result from a reduced production of reactive oxygen species, due to the inhibition of mitochondrial complex I. Differently from what others reported, metformin did not reduce tumor hypoxia, but rather increased CD8 T-cell infiltration and survival in hypoxic tumor areas, and synergized with cyclophosphamide to enhance tumor response to adoptive cell therapy or immune checkpoint blockade in different tumor models.CONCLUSIONS:This study describes a novel mechanism of action of metformin and presents a promising strategy to achieve immune rejection in hypoxic and immunosuppressive tumors, which would otherwise be resistant to immunotherapy.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+T cells remain disputed. Using multiomics analysis of CD8+T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunc-tional CD8+T cell states. CD8+T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an ex-panded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-ac-tivating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canon-ical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma high-lighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.

Immunotherapy based on immunecheckpoint blockade (ICB) using antibodies induces rejection of tumours and brings clinical benefit in patients with various cancer types 1 . However, tumours often resist immune rejection. Ongoing efforts trying to increase tumour response rates are based on combinations of ICB with compounds that aim to reduce immunosuppression in the tumour microenvironment but usually have little effect when used as monotherapies 2 , 3 . Here we show that agonists of α2-adrenergic receptors (α2-AR) have very strong anti-tumour activity when used as monotherapies in multiple immunocompetent tumour models, including ICB-resistant models, but not in immunodeficient models. We also observed marked effects in human tumour xenografts implanted in mice reconstituted with human lymphocytes. The anti-tumour effects of α2-AR agonists were reverted by α2-AR antagonists, and were absent in Adra2a -knockout (encoding α2a-AR) mice, demonstrating on-target action exerted on host cells, not tumour cells. Tumours from treated mice contained increased infiltrating T lymphocytes and reduced myeloid suppressor cells, which were more apoptotic. Single-cell RNA-sequencing analysis revealed upregulation of innate and adaptive immune response pathways in macrophages and T cells. To exert their anti-tumour effects, α2-AR agonists required CD4 + T lymphocytes, CD8 + T lymphocytes and macrophages. Reconstitution studies in Adra2a -knockout mice indicated that the agonists acted directly on macrophages, increasing their ability to stimulate T lymphocytes. Our results indicate that α2-AR agonists, some of which are available clinically, could substantially improve the clinical efficacy of cancer immunotherapy.

Summary Therapeutic cancer vaccines, whether based on neoantigens or shared antigens, will likely be given in the clinic together with the standard of care, which often comprises immune checkpoint blockade therapy and chemotherapy. It remains unclear, however, whether vaccines effectively synergize with chemotherapy. Here, we tested the combination of a heterologous prime-boost viral vector vaccine with chemotherapy (CarboTaxol) and anti-PD- 1. We show that this triple combination improves tumor control and survival in different murine tumor models. CarboTaxol, and also cyclophosphamide, acted as an immune adjuvant for the vaccines, enhancing tumor-specific CD8+ T-cell responses, irrespective of the presence of a tumor. These chemotherapies expanded stem-like T cell factor 1 (TCF1)+CD8+ T cells. Inhibition of the transcriptional activity of TCF1/β-catenin with a small molecule inhibitor abolished the immune adjuvant effect of CarboTaxol. This study sheds light on the new immunomodulatory roles of chemotherapies and holds promises for clinical testing of this combination strategy. Highlights The combination of CarboTaxol with viral vector cancer vaccines and anti-PD-1 promotes better tumor control, tumor clearance, and survival CarboTaxol increases TCF1 expression in CD8+ T cells and expands stem-like TCF1+CD8+ T cells CarboTaxol acts as an adjuvant for cancer vaccines irrespective of the presence of a tumor and this effect is mediated by TCF1/β-catenin activity

Table S2 shows a selectivity screen of PF-06840003 in in vitro pharmacological assays.

Clinical characteristics of the patients analyzed for IDO1 expression in tumor-draining lymph nodes.

Kinetics of IDO1 expression during LPS-induced DC maturation in vitro.

Supplementary Table S1 from An Inducible Mouse Model of Melanoma Expressing a Defined Tumor Antigen

Glioblastoma is an extremely aggressive and difficult cancer to treat, which may partly be due to its limited ability to induce T-cell responses. However, combining viral vector vaccines with other therapies to generate tumor-specific T cells may provide a meaningful benefit to patients. Here, we investigated whether heterologous prime-boost vaccination with chimpanzee-derived adenoviral vector ChAdOx1 and modified vaccinia Ankara (MVA) vaccines could generate therapeutically effective CD8+ T-cell responses against a model antigen P1A, a mouse homolog of human tumor-associated Melanoma Antigen GenE (MAGE)-type antigens, expressed by a BGL-1 mouse glioblastoma cell line. We demonstrated that heterologous prime-boost vaccination with ChAdOx1/MVA vaccines targeting P1A generated a high magnitude of CD8+ T cells specific for the P1A35-43 epitope presented by the MHC class I molecule H-2Ld. Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impact the magnitude of antigen-specific CD8+ T-cell responses and may impact protective efficacy. However, the substantial induction of myeloid-derived suppressor cells (MDSCs) by this tumor model presents a significant challenge in the therapeutic setting. Future work will investigate the efficacy of this vaccination strategy on intracranial P1A-expressing BGL-1 models. Citation Format: Emily E. Steffke, James McAuliffe, Vinnycius Pereira Almeida, Amanda Wicki, Laurine Noblecourt, Gary Kohanbash, Hideho Okada, Carol Sze Ki Leung, Masaki Terabe, Benoit J. Van Den Eynde. Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 682.

Supplementary Methods, Figures 1-6 from Tumor-Initiated Inflammation Overrides Protective Adaptive Immunity in an Induced Melanoma Model in Mice

Heme is a well-established pro-inflammatory signaling molecule involved in different pathological settings. Seika et al. (link) elegantly demonstrate that the accumulation of free heme in the colon amplifies the DNA damage, the abnormal proliferation of epithelial cells and can sustain a state of chronic inflammation. This mechanism provides a novel explanation for gastrointestinal syndrome (GIS) and bleeding, two severe gastrointestinal side effects observed during anticancer therapy. Based on this evidence, the Authors propose the depletion of free heme by increasing HO-1 activity and Hx sequestration as a potential therapeutic approach to ameliorate the side effects of common antitumoral therapies (link). In this scenario, although the effects on cancer prevention of many antioxidant compounds and food supplements are debated (4, 5), novel therapeutic approaches could be exploited targeting antioxidant heme proteins to ameliorate the side effects of anti-cancer therapies.Free heme may also exacerbate the inflammatory response during bacterial or viral infections with simultaneous intravascular hemolysis. Thus, the antioxidant effects of bilirubin can be identified as a potential therapeutic tool in a context of high oxidative stress resulting from endotoxemia.

Background Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated with poor disease prognosis and immune suppression. Mechanistically, the respective roles of tryptophan shortage and kynurenine production in suppressing immunity remain unclear. Kynurenine was proposed as an endogenous ligand for the aryl hydrocarbon receptor (AHR), which can regulate inflammation and immunity. However, controversy remains regarding the role of AHR in IDO1/TDO-mediated immune suppression, as well as the involvement of kynurenine. In this study, we aimed to clarify the link between IDO1/TDO expression, AHR pathway activation and immune suppression. Methods AHR expression and activation was analyzed by RT-qPCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO. In vitro differentiation of naïve CD4 + T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing different Ahr alleles or a knockout of Ahr , and cultured in medium with or without tryptophan and kynurenine. Results We confirmed that IDO1/TDO expression activated AHR in HEK-293-E cells, as measured by the induction of AHR target genes. Unexpectedly, AHR was also overexpressed on IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR on tryptophan deprivation. AHR overexpression was not dependent on general control non-derepressible 2 (GCN2), and strongly sensitized the AHR pathway. As a result, kynurenine and other tryptophan catabolites, which are weak AHR agonists in normal conditions, strongly induced AHR target genes in tryptophan-depleted conditions. Tryptophan depletion also increased kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner. Tryptophan deprivation potentiated Treg differentiation from naïve CD4 + T cells isolated from mice bearing an AHR allele of weak affinity similar to the human AHR. Conclusions Tryptophan deprivation sensitizes the AHR pathway by inducing AHR overexpression and increasing cellular kynurenine uptake. As a result, tryptophan catabolites such as kynurenine more potently activate AHR, and Treg differentiation is promoted. Our results propose a molecular explanation for the combined roles of tryptophan deprivation and kynurenine production in mediating IDO1/TDO-induced immune suppression.

While immunotherapy is a critical modality for the treatment of many cancers, many patients (pts) do not respond to checkpoint blockade. Vaccination against tumour antigens has potential to enhance this, and despite the historically limited success of vaccines in monotherapy, effective immune induction is observed. Prime-boost viral vaccination is a highly potent platform, priming immunity with the CHAdOx1 adenoviral vector, followed by MVA vaccinia vector boosting, against MAGE-A3 and NYESO-1. Nonclinical data to support the rationale for enhanced CHAdOx1-MVA efficacy, in combination with chemo- and immunotherapy, has been generated in an in vivo surrogate model ( McAuliffe J et al, 2021 ). The MAGE-A3 antigen is expressed on tumours of a high percentage of cancer patients including non-small cell lung cancer (NSCLC). NYESO-1, expressed on a subset of MAGE-A3+ tumours, has the additional advantage of being highly immunogenic in cancer patients, either spontaneously or in response to peptide vaccination. The safety, tolerability, efficacy and immunogenicity of ChAdOx delivery of MAGE-A3/NYESO-1 antigens, boosted by MVA-MAGE-A3 (MAGE-A3+ pts) or MVA-MAGE-A3 and MVA-NYESO-1 (MAGE-A3+NYESO-1+ pts) are explored in this trial. This is a multi-centre, first-in-human, phase I/IIa, randomised, open label trial, run in the UK, for patients scheduled to receive first-line chemo-immunotherapy containing pembrolizumab as standard of care (SoC) treatment, for Stage III/IV NSCLC (and potentially additional indications) expressing MAGE-A3 +/- NYESO-1 (NCT04908111). Patients receive trial vaccination from Cycle 3 of their SoC treatment. In the initial Safety Run-In Stage, opened in October 2021, all patients receive ChAdOx1-MAGE-A3-NYESO prime vaccination and either MVA-MAGE-A3 single boost (3 evaluable pts) or MVA-MAGE-A3 and MVA-NYESO double boost (3 evaluable pts) vaccinations with SoC. The currently expanding Rolling Recruitment Stage includes a NSCLC Randomised Cohort of approximately 80 pts, randomised 1:1 to addition of vaccination. The primary objective is safety and tolerability, with key secondary objectives of immunogenicity and clinical efficacy. Tertiary objectives include correlative translational analyses. CRUKD/20/001, Release date: 07 Dec 2020. Cancer Research UK Centre for Drug Development. Has not received any funding.