Journal for ImmunoTherapy of Cancer

BackgroundRecent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific mutations as a key driver of tumor regression and overall survival. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target a patients mutation-derived tumor-specific neoantigens. The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations and control tumor growth by generating neoantigen-specific T cell responses using a pre-clinical mouse tumor model. MethodsWhole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique non-synonymous mutations. The netMHCcons algorithm was used to predict 137 potential neoantigens. We validated 20 immunogenic neoantigens either by peptide immunization followed by ELISPOT or by the presence of CD8+ T cells recognizing the neoantigen peptide following checkpoint inhibitor treatment. Two ADXS-NEO vectors were constructed; Lm20, targeting 20 validated immunogenic neoantigens, and Lm19, targeting most of the non-validated NSMs. ResultsBoth Lm19 & Lm20 significantly slowed tumor growth in C57BL/6 mice compared to control. An accumulation of ADXS-NEO-specific TILs was observed in tumor bearing mice treated with either Lm19 or Lm20. Examination of the tumor microenvironment in Lm19 or Lm20 treated mice revealed a decrease in the frequency and absolute number of Tregs, TAMs, MDSCs, and PD1high exhausted CD8+ T cells as well as an increase in the frequency and absolute number of effector CD8+ T cells, relative to control. ConclusionADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control in mice.

BackgroundCheckpoint inhibitors improve survival in patients with several types of tumors. However, resistance to checkpoint inhibitors creates an opportunity for patients to benefit from novel immunotherapies. The type 2 cytokines IL-4, IL-13 and TSLP have been implicated in suppressing anti-tumor immune responses through T and myeloid cells. Our current study tested whether combined therapeutic blockade of IL-4, IL-13 and TSLP improved anti-tumor immunity alone and in combination with PD1 antagonism. MethodsWe used in vitro experiments with primary cells to identify cell types likely to participate in controlling tumors upon IL-4, IL-13, TSLP and PD1 blockade. Therapeutic blockade in the subcutaneous CT26 model tested in vivo tumor growth inhibition and associated immunological changes. Bioinformatic analysis of human tumor bulk RNA sequencing data probed for survival associations with IL-4/IL-13 and TSLP transcriptional responses. ResultsIn vitro, IL-4 suppressed T cell-mediated tumor growth inhibition and reduced monocyte-derived dendritic cell expression of proteins associated with anti-tumor immunity. In vivo, blocking IL-4, IL-13, TSLP and PD1 improved tumor growth inhibition by creating "hotter" tumors. This was associated with repolarization of CD4 and CD8 T cells and shifts in monocyte, conventional type 1 and type 2 (or monocyte-derived) dendritic cell programs. Transcriptional responses to IL-4/IL-13 and TSLP were associated with poor survival outcomes across patients with several types of cancers. ConclusionTherapeutic blockade of IL-4, IL-13 and TSLP may drive immunological tumor growth inhibition in subsets of cancer patients alone and in combination with checkpoint inhibitors. Improved tumor growth inhibition was likely driven through augmented cytotoxic T cell priming in secondary lymphoid organs and improved reactivation by repolarized monocytes and dendritic cells in tumors.

BackgroundHumanized tumor models could be particularly valuable for cancer immunotherapy research, as they may better reflect human-specific aspects of the interfaces between tumor and immune system of human cancer. However, endogenous antitumor immunity in humanized models is still largely undefined. MethodsWe established a novel autologous humanized mouse tumor model by using NSG mice reconstituted with human immune cells from hematopoietic progenitors and tumors generated from transformed autologous human B cells. We demonstrate growth of solid lymphoid tumors after subcutaneous implantation, infiltration by endogenous human immune cells and immunocompetence of the model. FindingsWe found human T cell subsets described in human cancer, including progenitor exhausted (Tpex), terminally exhausted (Tex-term) and tissue-resident (TRM) cells in tumor-bearing humanized mice with accumulation of Tex-term and TRM in the tumor. In addition, we identified tumor-reactive CD8+ T cells through expression of CD137. This subpopulation of de novo arising human CD137+ CD8+ T cells displayed a highly proliferative, fully activated effector and exhausted-like phenotype with enhanced expression of activation and exhaustion markers like PD-1, CD39, CD160, TIM-3, TIGIT and TOX, the senescence marker CD57 (B3GAT1) and cytolytic effector molecules such as PRF1, GZMH and NKG7. Moreover, these CD137+ CD8+ T cells exhibited tumor-specific clonal expansion and presented signature overlap with tumor-reactive CD8+ T cells described in human cancer. We demonstrate superior anticancer activity of this exhausted-like human CD8+ T cell subset by adoptive transfer experiments using recipients bearing autologous human tumors. Mice adoptively transferred with CD137+ CD8+ T cells showed reduced tumor growth and higher CD8+ T cell tumor infiltration, correlating with control of human tumors. InterpretationWe established an immunocompetent humanized tumor model, providing a tool for immunotherapy research and defined effective anticancer activity of human effector CD8+ T cells with an exhausted-like phenotype, supporting clinical exploration of such cells in adoptive T cell therapies. FundingSwiss Cancer Research foundation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAntitumor immune responses and outcome of immunotherapeutic interventions are not always consistent between mouse models of cancer and data available in humans. This may be due to species-specific differences, therefore models with a potential for better translatability are needed, such as humanized mouse models. However, there is limited data on human antitumor T cell immunity in humanized mice. Added value of this studyIn this study, we established an immunocompetent humanized tumor model that recapitulates hallmarks of human antitumor T cell responses, offering the possibility for further translational investigation of the interface between human tumors and endogenous anticancer immunity. Furthermore, using functional in vitro assays and adoptive transfer, our study demonstrates the key importance of human effector CD8+ T cells with an activated and exhausted-like phenotype in the antitumor immune response. Implications of all the available evidenceThe autologous humanized tumor model provided in this study can serve as a tool to elucidate human-specific immune features. By bridging a gap between syngeneic mouse tumor models and human-specific antitumor immune responses, the model may help open up avenues for greater translatability of preclinical data. Our findings suggest that exhausted-like effector CD8+ T cells can be harnessed for clinical development of adoptive T cell therapies.

BackgroundCancer immunotherapy often triggers immune-related adverse events (irAEs). Analysis of irAEs in large checkpoint inhibitor (CPI) trials has enhanced their management and demonstrated their prognostic value for treatment outcome. However, data on irAEs in non-standard cancer immunotherapies (CITs) are limited, and systematic exploration is lacking. Identifying predictive biomarkers for irAEs in these therapies is still emerging and essential for improving patient care. MethodsWe established a harmonized data mart from 27 early-phase CIT trials, encompassing 14 molecules with diverse mechanisms across various cancer indications. This dataset includes 3,608 patients, both CPI-naive and CPI-experienced, with detailed information on clinical data, tumor characteristics, soluble biomarkers, and genome-wide genotyping. We examined the occurrence of different irAEs and CIT molecules concerning incidence, severity, and onset. A meta-analysis was conducted to assess the association between risk factors and the time to onset of irAEs. Finally, we explored the predictive value of irAEs for clinical outcomes, specifically measured by progression-free survival (PFS). ResultsOur analysis reveals significant variation in irAE incidence and kinetics across CIT molecules. Common irAEs include hepatitis, rash, acute kidney injuries, and hypothyroidism, with hepatitis often severe and others mild. Hepatitis is frequently associated with immunocytokine treatment, while T-cell bispecifics (TCBs) are linked to organ-specific toxicities. Hepatic metastases correlate with hepatitis but inversely with rash; elevated liver enzymes are associated with hepatitis, and high ferritin levels with acute kidney injury risk. Higher myeloid cell counts are associated with reduced rash likelihood. No tumor microenvironment (TME) associations were found, and polygenic risk scores (PGS) show limited utility in our setting. Rash correlates with improved outcomes, whereas hepatitis is associated with a poorer prognosis, independent of baseline prognostic state assessed by the Real World Prognostic score (ROPRO). ConclusionsThese findings highlight the complexity of immune toxicities in early-phase trials, emphasizing the importance of the CIT class, as well as the roles of tumor burden, metastasis sites, and systemic immune state in the development of irAEs. Additionally, the observed association between skin toxicities and improved PFS suggests that skin toxicity could serve as a marker of systemic immune activation across immunotherapy contexts. Key messagesWhat is already known on this topic[bullet] Cancer immunotherapy can induce immune-related adverse events (irAEs); their management and prognostic significance have advanced thanks to data from large checkpoint inhibitor trials. What this study adds[bullet] This study reveals the complexity of irAEs in early-phase pan-immunotherapy trials, highlighting the impact of tumor burden, metastasis sites, and systemic immune state, while identifying skin toxicity as a potential surrogate marker for improved patient outcomes. How this study might affect research, practice or policy[bullet] Our study lays a foundation for pan-immunotherapy irAE research, offering insights for clinicians and drug developers to assess risk profiles and guide the design of future trials for new immunotherapies.

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify {beta} T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human {beta} TCR constant gene segments with murine {beta} TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-{gamma} ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

In cancer, CD8+ T cell responses are dominated by exhausted T cells, which can be reinvigorated using immune checkpoint blockade therapy and can control large tumors. However, it remains unclear which T cell fate best supports long-term immunity following tumor regression or clearance and a period of minimal antigen load. This question is particularly relevant following surgical tumor resection, when tuning the immune system could prevent recurrence. To determine which T cell fate provides durable protection following surgery and metastatic rechallenge, we modulated T cell priming using anti-PD-1, IFN-{beta} or agonistic anti-CD40 and assessed effects on CD8+ T cell differentiation and overall survival. IFN-{beta} and anti-CD40 promoted effector and memory-like T cell states, respectively, whereas anti-PD-1 did not markedly alter T cell differentiation, yet conferred the greatest survival benefit against metastatic tumors. Notably, anti-PD-1 induced epigenetic remodeling, which was detectable upon metastatic recall, consistent with the maintenance of a circulatory intermediate-exhausted T cell state. Thus, while effector and memory precursor-like T cells could be generated with IFN-{beta} and agonistic anti-CD40, only the intermediate-exhausted T cell state driven by anti-PD-1 supported durable anti-tumor immunity. SummaryThis study shows that PD-1 blockade during T cell priming promotes a circulatory intermediate-exhausted CD8 T cell state that uniquely supports durable anti-tumor immunity after surgical resection and metastatic challenge, outperforming effector or memory-like T cell responses generated by IFN-{beta} or CD40 agonist treatment, respectively.

Interleukin-2 (IL-2) remains an attractive cytokine for enhancing antigen-specific CD8 T cell responses in cancer immunotherapy, but systemic toxicity hinders its broad clinical application. To address this, various IL-2-based therapeutics have been engineered with altered IL-2 receptor bias or targeted delivery to tumors, the tumor microenvironment, or immune cell populations. Ideally, IL-2 signals should be selectively delivered to antigen-specific CD8 T cells, boosting their responses and promoting effector differentiation while sparing non-targeted populations. Immuno-STATTM (Selective Targeting and Alteration of T cells) is a fusion protein platform comprising a bivalent peptide-MHC class I complex and an affinity-attenuated IL-2 mutein that co-stimulates TCR and IL-2 signaling in epitope-specific CD8 T cells. Here, we investigated whether a DbGP33-41-targeted Immuno-STAT enhances DbGP33-specific CD8 T cell responses in a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Immuno-STAT treatment selectively expanded DbGP33-specific CD8 T cells with an effector-like phenotype. Non-targeted DbGP276-specific CD8 T cells showed little to no expansion in response to DbGP33-41-targeted Immuno-STAT therapy, underscoring the selectivity of this approach. However, minor changes in phenotypic markers, including increased expression of CD25 and CX3CR1, were observed in non-targeted CD8 T cells, likely reflecting bystander IL-2 signaling. Combining Immuno-STAT with PD-1 blockade augmented DbGP33-specific CD8 T cell responses more effectively than PD-1 blockade alone, with minor effects on the non-targeted DbGP276-specific population. These findings inform the clinical development of Immuno-STAT and other IL-2 therapeutics and highlight the value of coordinated TCR and IL-2 stimulation during chronic antigen exposure, alone or in combination with PD-1 blockade. IMPORTANCEInterleukin-2 (IL-2) is a key cytokine for promoting effector differentiation of antigen-specific CD8 T cells and remains an attractive agent in cancer immunotherapy, but systemic toxicity limits its clinical use. This study addresses a central challenge in IL-2-based immunotherapy: delivering IL-2 to cognate antigen-specific CD8 T cells while minimizing activation of non-targeted populations. Using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection, we show that the Immuno-STAT (Selective Targeting and Alteration of T cells) platform selectively expands targeted virus-specific CD8 T cells and enhances their function while limiting effects on non-targeted populations. We also show that combining Immuno-STAT with PD-1 blockade further enhances targeted virus-specific CD8 T cell responses during chronic LCMV infection. These findings provide mechanistic and preclinical support for integrating T cell receptor (TCR) specificity with IL-2 signaling to advance cancer immunotherapy and guide next-generation IL-2 therapeutics for cancer and chronic infection.

BackgroundCross-presentation of tumor antigens by antigen-presenting cells (APCs) is essential for initiating effective anti-tumor T cell immunity. The presence of cross-presenting immune cells across multiple solid tumors correlates with improved clinical outcomes. Despite the importance of this process, the identities and characteristics of tumor-derived MHC-I antigens that are cross-presented by APCs remain largely undefined, limiting rational design of targeted immunotherapies. MethodsWe performed an immunopeptidomic analysis of cross-presented glioblastoma (GBM) antigens on APCs, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, and splenic dendritic cells, using SILAC labeling and in vitro co-culture systems. Additionally, we also profiled endogenous APC and tumor antigen repertoires. We made selected cross-presented antigen targets into mRNA vaccines and evaluated their immunogenicity in comparison to tumor endogenous antigens in vivo. ResultsWe identified over one thousand putative cross-presented GBM antigens. Comparative analysis of endogenous APC and tumor antigen repertoires revealed that cross-presented antigens possess distinct features and are predominantly shaped by intrinsic antigen processing and presentation pathways within APCs, resulting in limited cross-presentation of tumor-specific epitopes. Two doses of mRNA encoding cross-presented tumor-specific epitopes delayed tumor growth and elicited robust antigen-specific T cell responses. ConclusionOur findings define the landscape and constraints of tumor antigen cross-presentation in GBM and establish a framework for improved antigen selection in the development of next-generation GBM immunotherapies.

BackgroundPancreatic ductal adenocarcinoma (PDAC), a condition representing 90% of pancreatic cancers, shows one of the lowest 5-year survival rates across all cancer types. Current therapeutic approaches remain largely inefficient, in part due to the presence of a hostile tumor microenvironment (TME), impeding immune cells infiltration and function. Specifically, Natural Killer (NK) cells from PDAC patients exhibit impaired phenotype and cytotoxic functions. NK cell immunotherapy represents a safe and promising approach to restore NK cell cytotoxicity against PDAC. MethodsWe developed a dual strategy based on i) the re-activation of NK cells through Natural Killer activating multimeric immunotherapeutic complexes (NaMiX) composed of IL-15/IL-15R dimers coupled to anti-NKp46 single-chain variable fragments (scFvs) and ii) the crosslinking of activated NK cells to PDAC cells with a Trispecific Killer Engager (TriKE) targeting NKG2D, NKp30 and the tumor-associated antigen CEA. We evaluated the ability of these constructs to stimulate NK cell functions across BxPC-3 PDAC cell line and patient-derived organoid models and in humanized NSG mice bearing PDAC xenografts. ResultsNaMiX stimulated the activation and cytotoxic functions of NK cells towards pancreatic BxPC-3 cells in vitro while TriKE cross-linked NK cells to BxPC-3 cells. The cytotoxic effects of NaMiX were further enhanced when combined with the crosslinking abilities of TriKE for the killing of NK cell-mediated BxPC-3 spheroid and PDAC patient-derived organoids. In humanized mice bearing BxPC-3 xenografts, NaMiX induced cytotoxic lymphocyte expansion, and increased tumor infiltration of NK cells, while TriKE tended to slow tumor progression. ConclusionsThis proof-of-concept study reports for the first time that activating and engaging NK cells with immunoconjugates are a promising therapeutic avenue for PDAC treatment. Efforts should now focus on the optimization of NK cell therapeutic modalities to favor the infiltration of a high number of NK cells into the tumor.

Preconditioning regimens are essential for the success of adoptive cell therapies like CAR T-cells due to effects on the T-cell response, yet they are underexplored and generally absent from cancer vaccine clinical trials. To address this gap, we evaluated the impact of various preconditioning strategies on dendritic cell (DC) vaccine efficacy in a murine tumor model. Mice bearing syngeneic KP tumors expressing ovalbumin received preconditioning with low-dose radiation (LD RT; whole body or tumor only), cyclophosphamide, paclitaxel, LD RT plus cyclophosphamide, or no preconditioning, followed by administration of antigen-loaded DC1s. Tumor growth, survival, and antigen-specific CD8+ T-cell responses were assessed. LD RT preconditioning, whether whole body or tumor-directed, significantly enhanced vaccine-induced antitumor CD8+ T-cell responses and improved survival compared to DC vaccine alone and all other groups. Cyclophosphamide preconditioning reduced vaccine efficacy and negated the benefits of LD RT, while paclitaxel had no significant effect. Notably, whole-body LD RT induced the strongest antigen-specific T-cell response. These findings demonstrate that, similar to CAR T-cell therapy, preconditioning regimens can significantly influence cancer vaccine outcomes. Rational selection of preconditioning agents may either maximize or minimize the therapeutic potential of DC cancer vaccines, and should be considered carefully in clinical trials.

Humanized immune system (HIS) mouse models, generated by engrafting tumors and hematopoietic cells of human (Hu) origin into immunodeficient host mice, effectively recapitulate key aspects of the crosstalk between human immune cells and tumors. These models represent a valuable tool for the preclinical evaluation of immunotherapies. In this study, we provide a comprehensive comparison of two widely used HIS models: the Hu-CD34+ model, which engrafts Hu-hematopoietic cells derived from Hu-CD34+ hematopoietic stem cells (HSCs), and the Hu-PBMC model, which utilizes Hu-peripheral blood mononuclear cells (PBMCs). We assess the kinetics, quality and extent of immune cell engraftment, as well as the development of graft-versus-host disease (GVHD). Additionally, we investigate the impact of different immunodeficient host mouse strains on immune cell reconstitution in the Hu-CD34+ model. Both HIS models were engrafted with human tumors derived from either cell lines or patient-derived xenografts (PDX), revealing distinct immune-tumor interactions that influenced antitumor responses. Notably, tumor responses to T-cell-directed therapies, including anti-PD1 antibodies, IL-2-anti-IL-2 antibody complexes, and T-cell engagers, varied across these models. Our findings provide novel insights into the properties and limitations of HIS models, offering a critical resource for optimizing next-generation immuno-oncology strategies and guiding the design of future therapeutic interventions.

Resistance to immunotherapy is a cardinal feature of pancreatic ductal adenocarcinoma (PDAC). Inhibition of Small Ubiquitin-like MOdifier (SUMO), a post-translational modification with important immune regulatory functions, augments responsiveness to immunotherapy in non-PDAC models via pro-immunogenic effects on myeloid cells, cancer cells, and T-cells. Recently, it has been reported that SUMO inhibition has direct immunogenic effects on PDAC. Here, we report that the novel combination of SUMO inhibition with a small molecule, TAK-981, plus antibody-mediated CD40 agonism improves survival in an aggressive orthotopic mouse model of PDAC by enhancing anti-tumoral immunogenicity. This combination amplifies CD8+ T-cell tumor infiltration and induces significant changes among macrophages. TAK-981 also leads to enhanced cancer specific MHC-I expression both in vitro and in vivo by augmenting interferon signaling. We show that the improvement in survival is mediated by macrophages. Our findings show that SUMO inhibition complements CD40 agonism to enhance immune activity in PDAC via interferon signaling, improving survival in an aggressive pre-clinical model of PDAC and translating previous findings to a characteristically immunosuppressive and highly aggressive solid malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer, with limited therapeutic options and extremely high mortality rates. While immune checkpoint blockade (ICB) therapy is effective in many types of human cancers, responses in PDAC patients remain poor, partly due to the weak immunogenicity of PDAC tumors. We hypothesized that a whole-cell PDAC vaccine could improve anti-tumor responses if optimized to expose a more stimulatory repertoire of tumor antigens. To test this, we used murine Panc02 pancreatic cancer cells to screen several stress-inducing treatments (UV, hypoxia, heat shock, and hydrogen peroxide [H2O2]), among which low-dose oxidative stress (0.05% H2O2 for 2h) was identified as the optimal inducer of immunogenic cell death (including increased surface calreticulin, ERp57 exposure, HMGB1 release and MHC class I expression). We then prepared a whole-cell vaccine of fixed H2O2-treated Panc02 cells, which induced robust tumor-specific immunity in C57BL/6 mice bearing syngeneic Panc02 tumors. Vaccine-treated mice displayed a significant increase in tumor-reactive IFN{gamma}+ T cells, as well as extensive tumor infiltration by CD4 + and CD8 + T cells and NCR1+ NK cells. When used prophylactically, the vaccine significantly delayed tumor growth and extended survival, whereas therapeutic application markedly slowed tumor progression. Importantly, combining the whole-cell Panc02 vaccine with anti-PD-1 therapy induced complete tumor regression in a subset of animals. Together, these data demonstrate that controlled oxidative stress can convert autologous tumor cells into an effective whole-cell vaccine without the need for genetic modification or prior neoantigen identification, offering a scalable strategy for personalized immunotherapy in PDAC. STATEMENT OF SIGNIFICANCEThis study demonstrated that oxidative stress-induced immunogenic cell death reprograms pancreatic tumor cells to induce danger signaling and enhance antigen presentation, thereby promoting immune infiltration and sensitizing tumors to PD-1 blockade.

Pancreatic ductal adenocarcinoma (PDA) is a deadly malignancy with limited effective therapies. Adoptive cell therapy (ACT) is a promising treatment modality for patients with solid tumors but has been limited by the highly fibroinflammatory and immunosuppressive tumor microenvironment (TME). Transforming growth factor-{beta} (TGF{beta}) participates in the inordinately suppressive TME in PDA. Here, we test the impact of selective Tgfbr2 deletion using CRISPR/Cas9 or genetic approaches in mesothelin (Msln)-specific T cell receptor (TCR) engineered T cells during ACT of PDA. Abrogating TGF{beta} signaling augmented TCR-engineered T cell accumulation in autochthonous and orthotopic PDA models and promoted terminal effector T cells, although this largely required inclusion of a vaccine at the time of T cell transfer. While loss of Tgfbr2 impaired CD103 upregulation, it only modestly impaired donor T cell central, tissue resident, or Tcf1+Slamf6+ stem-like memory T cell formation. These attributes ultimately result in heightened functional capacity and delayed tumor growth. Unexpectedly, however, most tumor-infiltrating engineered T cells failed to differentiate into PD-1+Lag3+ exhausted T cells (TEX) regardless of TGF{beta}R2 expression and despite abundant Msln protein expression by PDA cells. Forcing Msln epitope processing in KPC tumor cells promoted donor T cell accumulation, acquisition of PD-1 and Lag3, increased IFN{gamma} production by TCR-engineered T cells refractory to TGF{beta} and bypassed the vaccine requirement for therapeutic efficacy. Thus, promoting increased antigen processing/presentation by tumor cells while abrogating Tgfbr2 in engineered T cells can sustain donor T cell function in the suppressive TME and enhance the therapeutic efficacy of ACT. Our study supports pursuit of strategies that modulate tumor intrinsic antigen processing while relieving T cell suppression to safely promote the antitumor activity of TCR-engineered T cells.

PurposeTo evaluate safety and immunogenicity of intramuscularly delivered personalized neoantigen synthetic long peptide (SLP) vaccines in patients with advanced solid tumors. Patients and MethodsIn this Phase I trial, 12 patients with advanced melanoma (n=9) or renal cell carcinoma (n=3) who could no longer access further standard treatments received intramuscular neoantigen SLP vaccines with poly-ICLC. Each vaccine contained [~]20 predicted neoantigen peptides. Adverse events were monitored throughout vaccination and follow-up. Immune profiling was performed at baseline and predefined post-vaccination time points. ResultsIntramuscular neoantigen vaccination was well tolerated, with only grade 1-2 local pain or fever and no immune-mediated toxicities. All participants developed de novo T-cell responses, detectable within one week. On average, 46% of peptides per patient were immunogenic, inducing both CD8 and CD4 neoantigen-specific responses. Patients previously treated with immune checkpoint inhibitors (ICIs) had higher baseline immunity but achieved comparable post-vaccination responses to ICI-naive patients. IFN-{gamma}-dominant CD8 and TNF--dominant CD4 responses were observed, along with increased effector memory differentiation. Two patients with higher CD8 TEMRA proportions were the longest survivors. Tumor biopsies revealed enhanced CD8 infiltration, and epitope spreading occurred in one of two evaluable cases. Analysis of 239 peptides showed greater immunogenicity for dual MHC I/II-binding, cysteine-containing, and in-frame indel- or low-VAF-derived mutations, while proline substitutions reduced responses. ConclusionsIntramuscular neoantigen SLP vaccination with poly-ICLC is safe and induces rapid, mutation-specific T-cell immunity with robust CD8 effector responses. These findings support intramuscular administration as a promising strategy for peptide-based cancer vaccines. Translational relevancePersonalized neoantigen vaccines offer a promising strategy to enhance tumor-specific immunity, but most prior studies using intradermal or subcutaneous delivery have shown limited induction of cytotoxic CD8 T cells. This study demonstrates that intramuscular administration of personalized neoantigen synthetic long peptide vaccines with poly-ICLC is safe, feasible, and capable of eliciting rapid, mutation-specific CD4 and CD8 T-cell responses in patients with advanced melanoma and renal cell carcinoma. Vaccine-induced immunity was dominated by IFN-{gamma}-producing cells and accompanied by a shift toward effector memory phenotypes. In selected cases, post-treatment tumor biopsies revealed increased CD8 infiltration. These findings support intramuscular delivery as a practical and effective platform for neoantigen-based cancer vaccine.

Colorectal cancer (CRC) is a highly prevalent and deadly disease that is largely refractory to immunotherapy. The only CRC subset that responds to these therapies is characterized by prevalent microsatellite instability (MSI), extensive CD8+ T cell infiltration and high expression of innate immune signaling pathways. Endogenous activation of the cGAS/STING pathway is essential for the CD8+ T cell antitumor response in MSI CRCs, suggesting that activating it in other CRCs could boost immunotherapy response rates. We show that cGAS/STING signaling can be enhanced by costimulation of the NLRP3 inflammasome and that dual stimulation increases CD8+ T cell-mediated antitumor immunity in both MSI and non-MSI CRCs. The ability of NLRP3 to boost cGAS/STING signaling was specific and did not occur with activation of other innate immune pathways such as AIM2 or TLRs. Cooperativity between cGAS/STING and NLRP3 proceeded via a positive feedback loop that was inflammasome-independent and required early crosstalk between the signaling mediators and regulation of their gene expression. Notably, increased cGAS/STING signaling enhanced CD8+ T cell activation when in conjunction with anti-PD1 immunotherapy, suggesting that signaling via NLRP3 could further boost this response and render otherwise resistant CRC susceptible to immunotherapy. SignificanceInnate immune signaling pathways cooperatively regulate CD8+ T cell-mediated antitumor immunity in both hot and cold tumors. In addition to serving as predictive biomarkers, these pathways can be therapeutically targeted to increase response rates to immunotherapy while minimizing undesirable adverse events.

Tertiary lymphoid structures (TLSs) are emerging as good predictive biomarkers of response to cancer immunotherapy. However, therapeutic strategies to induce these structures are currently limited. We evaluated the therapeutic benefit of efineptakin alfa (NT-I7), a long-acting form of IL-7, and its ability to induce TLSs in a murine lung tumor model. NT-I7 improved overall survival in tumor-bearing mice. It also increased the abundance of T, B, dendritic cells, and stem-like CD8 T cells and promoted the formation of immune aggregates in the tumor microenvironment (TME). Stem-like CD8 T cells were preferentially located in the immune aggregates. Spatial transcriptomic analyses of the TME further demonstrated that the immune aggregates induced by NT-I7 included TLSs with enrichment of Cd274 (PD-L1) transcripts and genes involved in antigen processing and presentation. Upregulation of Cd274 in the TLSs may provide opportunities for synergy between NT-I7 and PD-1-targeted immunotherapy. STATEMENT OF SIGNIFICANCEThis study demonstrates the ability of efineptakin alfa (NT-I7) to potentially augment the clinical efficacy of cancer immunotherapy by inducing tertiary lymphoid structures in the tumor microenvironment.

Concurrent KRAS LKB1 (STK11, KL) mutant Non-Small Cell Lung Cancers (NSCLC) is particularly difficult to treat and does not respond well to current immune checkpoint blockade (ICB) therapies. This is due to numerous mechanisms including low antigen presentation limiting T cell mediated killing. To activate anti-tumor immunity, we targeted tumor cell - natural killer (NK) cell interactions. We tested whether a novel antibody based therapeutic strategy that predominantly activates natural killer (NK) cells demonstrates efficacy in pre-clinical mouse models of KL NSCLC. NK cells rely on binding of ligands, such as Major Histocompatibility Complex (MHC) class I-related chain A or B (MICA/B), to the activating receptor NKG2D. Importantly MICA and MICB are widely expressed in elevated levels across NSCLC subtypes including KL lung cancers. Proteases with the tumor microenvironment (TME) can cleave these proteins rendering tumor cells less visible to NK cells. We therefore developed a MICA monoclonal antibody, AHA-1031, which utilizes two NK cell activating receptors. AHA1031 prevents ligand shedding without interfering with binding to NKG2D while targeting cancer cells to antibody mediated cell dependent cytotoxicity (ADCC). Our therapeutic novel antibody has significant monotherapy activity in KL cancer models including xenografts of human cell lines and patient derived xenografts. Activating NK cells through MICA/B stabilization and inducing ADCC offers an alternative and potent therapy option in KL tumors. MICA/B are shed across different tumors making this therapeutic strategy universally applicable.

Drugs that target the innate immune sensor STING are known to be effective in modulating the immune infiltrate of the tumour microenvironment. STING agonists have potential to enhance responses to checkpoint inhibitor therapy, however, their ability to influence and shape adaptive immune responses is poorly understood. Here, we investigated the impact of a range of synthetic STING agonists on antigen specific CD8+ T-cell responses to soluble antigen using the murine OT-1 adoptive transfer model with Ovalbumin as the antigen to monitor T cell responses. Our data demonstrate that synthetic STING agonists are able to stimulate antigen specific T-cell expansion in response to challenge in mice. This effect required expression of STING, an intact myeloid compartment and Type-I IFN and TNF signalling. Expanded T-cells post treatment differed from those induced by the established immune adjuvant, anti-CD40 antibody through lower induction of the immune checkpoint receptor PD-1. Furthermore, our data revealed a marked increase in the induction and persistence of CD8+ central memory cells after STING agonist and antigen challenge. Finally, we demonstrate that following rechallenge, STING agonism produced larger secondary responses that could be translated into enhanced tumour protection and survival. Therefore, synthetic STING agonists are capable of acting as potent immune adjuvants and can induce robust memory formation leading to better recall and tumour control. Critically, these benefits along with the lower expression of PD-1, have implications for their use as adjuvants for multiple immunotherapy and vaccine applications.

Immune checkpoint inhibition (ICI) has improved clinical outcomes for certain patients with cancer. However, only a minority of patients have durable responses with underlying causes of differential immune responses across individuals often being unknown. Lifestyle exposures impact immune function and may subsequently alter the response to ICI. Alcohol use is common among cancer patients with known detrimental effects on adaptive immune function. However, its impact on ICI efficacy remains unknown. To determine if alcohol impacts ICI therapies, we performed a retrospective assessment of outcomes for patients receiving anti-PD1 ICI across tumor types and employed preclinical mouse models of ICI for lung and bladder cancer. Alcohol use reduced ICI efficacy in human patients treated with anti-PD1 for lung and bladder cancer (HR [~]2.0) as well as in murine models ICI in lung (LN4K1) and bladder (MB49) cancer. Alcohol reduced tumoral T cell numbers, promoting less productive Th2 and Th17 CD4+ phenotypes intratumorally and regulatory phenotypes in the periphery. In both rodent and ex-vivo human T cells, alcohol disrupted T cell activation and effector functions. Thus, alcohol use negatively impacts ICI efficacy warranting alcohol cessation for this patient population.