Combination Immunotherapy Approaches Research Articles

M1A inhibition fuels oncolytic virus-elicited antitumor immunity via downregulating MYC/PD-L1 signaling

N1-methyladenosine (m1A) RNA methylation is critical for regulating mRNA translation; however, its role in the development, progression, and immunotherapy response of head and neck squamous cell carcinoma (HNSCC) remains largely unknown. Using Tgfbr1 and Pten conditional knockout (2cKO) mice, we found the neoplastic transformation of oral mucosa was accompanied by increased m1A modification levels. Analysis of m1A-associated genes identified TRMT61A as a key m1A writer linked to cancer progression and poor prognosis. Mechanistically, TRMT61A-mediated tRNA-m1A modification promotes MYC protein synthesis, upregulating programmed death-ligand 1 (PD-L1) expression. Moreover, m1A modification levels were also elevated in tumors treated with oncolytic herpes simplex virus (oHSV), contributing to reactive PD-L1 upregulation. Therapeutic m1A inhibition sustained oHSV-induced antitumor immunity and reduced tumor growth, representing a promising strategy to alleviate resistance. These findings indicate that m1A inhibition can prevent immune escape after oHSV therapy by reducing PD-L1 expression, providing a mutually reinforcing combination immunotherapy approach.

CAR-T Cell Therapy in Pancreatic and Biliary Tract Cancers: An Updated Review of Clinical Trials.

Pancreatic and biliary tract cancers are digestive system tumors with dismal prognosis and limited treatment options. The effectiveness of conventional surgical interventions, radiation therapy, and systemic therapy is restricted in these cases. Furthermore, clinical trials have shown that immunotherapy using immune checkpoint inhibitors has only demonstrated modest clinical results when applied to patients with pancreatobiliary tumors. This highlights the importance of implementing combination immunotherapy approaches or exploring alternative therapeutic strategies to improve treatment outcomes. We reviewed the relevant literature on chimeric antigen receptor (CAR)-T cell therapy for pancreatobiliary cancers from PubMed/Medline and ClinicalTrials.gov and retrieved the relevant data accordingly. Attention was additionally given to the examination of grey literature with the aim of obtaining additional details regarding ongoing clinical trials. We mainly focused on abstracts and presentations and e-posters and slides of recent important annual meetings (namely ESMO Immuno-Oncology Congress, ESMO Congress, ASCO Virtual Scientific Program, ASCO Gastrointestinal Cancers Symposium). CAR-T cell therapy has emerged as a promising and evolving treatment approach for pancreatic and biliary tract cancer. This form of adoptive cell therapy utilizes genetic engineering to modify the expression of specific antibodies on the surface of T cells enabling them to target specific cancer-associated antigens and to induce potent anti-tumor activity. The aim of this review is to provide an updated summary of the available evidence from clinical trials that have explored the application of CAR-T cell therapy in treating pancreatobiliary cancers. While the utilization of CAR-T cell therapy in pancreatobiliary cancers is still in its initial phases with only a limited amount of clinical data available, the field is advancing rapidly, incorporating novel technologies to mitigate potential toxicities and enhance antigen-directed tumor eradication.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Direct comparison of canine and human immune responses using transcriptomic and functional analyses

The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-γ than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-γ than human macrophages, as reflected by co-stimulatory molecule expression and TNF-α production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

Ibrutinib-based therapy reinvigorates CD8+ T cells compared to chemoimmunotherapy: immune monitoring from the E1912 trial

AbstractBruton tyrosine kinase inhibitors (BTKis) that target B-cell receptor signaling have led to a paradigm shift in chronic lymphocytic leukemia (CLL) treatment. BTKis have been shown to reduce abnormally high CLL-associated T-cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T-cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pretreatment and on-treatment (6 and 12 months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab with fludarabine, cyclophosphamide, and rituximab (FCR). Intriguingly, we report that despite reduced overall T-cell counts; higher numbers of T cells, including effector CD8+ subsets at baseline and at the 6-month time point, associated with no infections; and favorable progression-free survival in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T-cell killing function during ibrutinib-rituximab treatment, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T-cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T-cell cytotoxicity during ibrutinib-rituximab treatment, using the bispecific antibody glofitamab, supporting combination immunotherapy approaches.

Clinical Applications of Combined Immunotherapy Approaches in Gastrointestinal Cancer: A Case-Based Review.

Malignant neoplasms arising from the gastrointestinal (GI) tract are among the most common types of cancer with high mortality rates. Despite advances in treatment in a small subgroup harboring targetable mutations, the outcome remains poor, accounting for one in three cancer-related deaths observed globally. As a promising therapeutic option in various tumor types, immunotherapy with immune checkpoint inhibitors has also been evaluated in GI cancer, albeit with limited efficacy except for a small subgroup expressing microsatellite instability. In the quest for more effective treatment options, energetic efforts have been placed to evaluate the role of several immunotherapy approaches comprising of cancer vaccines, adoptive cell therapies and immune checkpoint inhibitors. In this review, we report our experience with a personalized dendritic cell cancer vaccine and cytokine-induced killer cell therapy in three patients with GI cancers and summarize current clinical data on combined immunotherapy strategies.

Comprehensive immunophenotyping of solid tumor-infiltrating immune cells reveals the expression characteristics of LAG-3 and its ligands.

Immune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies. Comprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy. LAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq. These results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade.

Phase II trial of XmAb20717 (vudalimab) in patients with advanced biliary tract cancers.

TPS4184 Background: For patients with advanced biliary tract cancers (BTCs), gemcitabine and cisplatin has been the standard-of-care first-line therapy, with a median overall survival (OS) of 11.7 months. More recently, TOPAZ-1 demonstrated a modest, but significant overall survival benefit (HR 0.80; 95% CI 0.66 – 0.97) to the addition of durvalumab to gemcitabine and cisplatin with a median OS of 12.8 months. However, for patients without targetable molecular variants, second-line and beyond options are limited, with FOLFOX demonstrating an overall response rate (ORR) of 5% and increase in median overall survival by 0.9 months compared to active symptom control. In patients with advanced BTCs, combination immunotherapy approaches with PD-1 or PD-L1 plus CTLA-4 inhibitors have been associated with improved ORRs of 10.8% - 23% compared to ORRs of 3 – 7% observed with single agent PD-1 or PD-L1 inhibitors. XmAb20717 (vudalimab) is a novel, bispecific antibody targeting PD-1 and CTLA-4 which demonstrated an ORR of 14.1% in a phase I dose expansion cohort of patients with advanced malignancies, including patients that had experienced disease progression on prior immune checkpoint inhibitors. Methods: We initiated a single-arm, phase II clinical trial with a Simon two-stage mini-max design to evaluate the efficacy of XmAb20717, in terms of ORR, in patients with advanced biliary tract cancers previously treated with gemcitabine-based chemotherapy. In the first stage, 13 patients evaluable for efficacy will be accrued. If no responses are observed in these 13 patients, the study will be stopped for futility. Otherwise, 14 additional patients evaluable for efficacy will be accrued to total 27 evaluable patients. If 4 or more responses are observed in these 27 patients, then the null hypothesis will be rejected. XmAb20717 (10 mg/kg) is administered intravenously on days 1 and 15 of a 28-day cycle. Important inclusion criteria include that patients with FGFR2 fusions, NTRK fusions, or IDH1 mutations must have received molecularly targeted therapy unless contraindicated or refused. Patients are not eligible if they have received prior immune checkpoint inhibitor therapy. Correlative studies will include longitudinal peripheral blood collection for circulating immune cell profiling. As of January, 2023, 8 patients have been enrolled with enrollment ongoing. Clinical trial information: NCT05297903 .

Tripartite antigen-agnostic combination immunotherapy cures established poorly immunogenic tumors

BackgroundSingle-agent immunotherapy has shown remarkable efficacy in selected cancer entities and individual patients. However, most patients fail to respond. This is likely due to diverse immunosuppressive mechanisms acting in a...

The BET Protein Inhibitor JQ1 Decreases Hypoxia and Improves the Therapeutic Benefit of Anti-PD-1 in a High-Risk Neuroblastoma Mouse Model.

Anti-programmed death 1 (PD-1) is a revolutionary treatment for many cancers. The response to anti-PD-1 relies on several properties of tumor and immune cells, including the expression of PD-L1 and PD-1. Despite the impressive clinical benefit achieved with anti-PD-1 in several cancers in adults, the use of this therapy for high-risk neuroblastoma remains modest. Here, we evaluated the therapeutic benefit of anti-PD-1 in combination with JQ1 in a highly relevant TH-MYCN neuroblastoma transgenic mouse model. JQ1 is a small molecule inhibitor of the extra-terminal domain (BET) family of bromodomain proteins, competitively binding to bromodomains. Using several neuroblastoma cell lines in vitro, we showed that JQ1 inhibited hypoxia-dependent induction of HIF-1α and decreased the expression of the well-known HIF-1α downstream target gene CA9. Using MRI relaxometry performed on TH-MYCN tumor-bearing mice, we showed that JQ1 decreases R2* in tumors, a parameter associated with intra-tumor hypoxia in pre-clinical settings. Decreasing hypoxia by JQ1 was associated with improved blood vessel quality and integrity, as revealed by CD31 and αSMA staining on tumor sections. By analyzing the immune landscape of TH-MYCN tumors in mice, we found that JQ1 had no major impact on infiltrating immune cells into the tumor microenvironment but significantly increased the percentage of CD8+ PD-1+, conventional CD4+ PD-1+, and Treg PD-1+ cells. While anti-PD-1 monotherapy did not affect TH-MYCN tumor growth, we showed that combinatorial therapy associating JQ1 significantly decreased the tumor volume and improved the therapeutic benefit of anti-PD-1. This study provided the pre-clinical proof of concept needed to establish a new combination immunotherapy approach that may create tremendous enthusiasm for treating high-risk childhood neuroblastoma.

Abstract CT236: INTERLINK-1: A phase 3, randomized, double-blind, placebo-controlled, multicenter, global study of monalizumab in combination with cetuximab in patients with recurrent or metastatic head and neck squamous cell carcinoma previously treated with an immune checkpoint inhibitor

Abstract Background: Head and neck (HN) cancer is the 7th most common cancer and 7th leading cause of cancer death worldwide with approximately 930,000 new cases and 470,000 deaths in 2020.1 HN squamous cell carcinoma (HNSCC) accounts for around 90% of HN cancers.2 Options for first-line treatment of patients (pts) with recurrent/metastatic (R/M) HNSCC include cetuximab (anti-epidermal growth factor receptor) in combination with (+) platinum-based chemotherapy (CT) and 5-fluorouracil (5- FU), or immune checkpoint inhibitor (ICI) therapy with single-agent pembrolizumab (anti-programmed cell death-1) or + CT and 5-FU.3-5 Pts who progress on/after CT may receive single-agent cetuximab, ICI pembrolizumab, or ICI nivolumab.3 However, treatment for pts who progress on/after ICI is not clearly defined and rechallenge is not recommended. Thus, there is a need to assess combination immunotherapy approaches. In a Phase 2 study, monalizumab (anti-NKG2A) + cetuximab combination therapy showed encouraging antitumor results in pts with R/M HNSCC who received prior CT and an ICI.6 INTERLINK-1 (NCT04590963) is a Phase 3 global study evaluating the efficacy and safety of monalizumab + cetuximab in pts with R/M HNSCC previously treated with CT and an ICI. Methods: Approximately 624 pts will be randomized 2:1 to monalizumab + cetuximab or placebo + cetuximab until disease progression or another discontinuation criterion is met. All pts will be followed for survival after discontinuation. Eligible pts will have measurable R/M HNSCC (per RECIST v1.1) of the oral cavity, oropharynx, hypopharynx or larynx, which is not amenable to curative therapy and has progressed on/after previous CT. They will have received 1-2 prior systemic regimens for R/M HNSCC, had prior treatment with a PD-(L)1 inhibitor, an ECOG performance status of 0 or 1, and must provide a fresh/recently acquired tumor sample. Key exclusion criteria include prior cetuximab therapy for R/M HNSCC, autoimmune/inflammatory disease or any concurrent anticancer treatment. The primary objective is overall survival (OS) in human papillomavirus-unrelated pts. Key secondary objectives include OS in all randomized pts, progression-free survival, objective response rate, duration of response, disease-related symptoms, functioning and quality of life, pharmacokinetics, safety and tolerability. Study enrollment is ongoing at 210 sites in 25 countries.

Coexpression of HHLA2 and PD-L1 on Tumor Cells Independently Predicts the Survival of Spinal Chordoma Patients.

BackgroundImmunotherapy only achieves efficacy in some cancer patients, and less is known about other immune checkpoint molecules in chordoma. Here, we aimed to determine the expression of PD-L1, HHLA2, B7H3, IDO-1 and Galectin-9 in spinal chordoma and evaluated their association with tumor infiltrating lymphocytes (TILs), clinicopathological characteristics and survival of patients.MethodsUsing multiplexed quantitative immunofluorescence (QIF), we simultaneously measured the levels of five different immune checkpoint molecules and major TIL subsets in 92 human spinal chordoma samples.ResultsTumor HHLA2 and PD-L1 were positive in 80.0% and 86.0% of cases, respectively. However, B7H3, IDO-1 and Galectin-9 positivity on tumor cells were only seen in 21.0% of cases, despite all showing predominantly stromal expression. Coexpression of these QIF markers in the tumor compartment was scarcely detected except for PD-L1 and HHLA2, which was observed in 69.6% of cases. While tumoral HHLA2 and stromal B7H3 expressions were associated with an aggressive tumor phenotype, suppressive immune response (specifically including elevated PD-1+ TILs level and decreased CD8+ TIL density) and poor prognosis, stromal levels of PD-L1 and Galectin-9 predicted the opposite outcomes. Importantly, HHLA2 and PD-L1 coexpression on tumor cells independently predicted both worse local recurrence-free survival and overall survival.ConclusionThese data provide a better understanding of the immunosuppressive mechanism in chordoma and may be useful for the development of combination or novel immunotherapy approaches aiming to improve therapeutic efficacy and survival.

Inactivated Cowpea Mosaic Virus in Combination with OX40 Agonist Primes Potent Antitumor Immunity in a Bilateral Melanoma Mouse Model.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

PI3K Isoform Immunotherapy for Solid Tumours.

Improving the anti-tumour T cell response as a consequence of immunotherapy can result in eradication of tumour burden, however, the majority of patients fail with current treatment regimens and so novel immunotherapies with greater efficacy and improved tolerability are needed. The phosphoinositide-3-kinase (PI3K) family members that are directly involved in cell signalling comprise PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, with the latter two isoforms expressed primarily by leukocytes. The survival and optimal function of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) is dependent on PI3Kδ, whereas tumour-associated macrophages (TAMs), use PI3Kγ. Blocking these signalling isoforms can boost development of effective anti-cancer immune responses and result in control of tumour burden. The dependence on different PI3K isoforms in immune cells makes targeting this pathway an attractive approach for tumour immunotherapy. Herein, we discuss how inhibiting specific PI3K isoforms in pro-tumoural Tregs, MDSCS and TAMs can unleash a powerful anti-tumour immune response, driven by CD8+ T cells, capable of controlling tumour burden and consider how the immune response to therapy needs careful investigation, to identify both the correlates of successful treatment and those that impede the generation of robust anti-tumour responses. Furthermore, we review how combination immunotherapy approaches with both PI3K inhibitors and subsequent immune checkpoint blockade can potentiate the efficacy of monotherapy. Finally, we discuss the recent advances in the use of PI3K isoform-specific inhibitors as an immunotherapy for solid tumours in clinical trials.

Abstract P053: Ablation of CCR1 relieves immunosuppression in pancreatic cancer

Abstract Pancreatic Cancer is one of the deadliest malignancies, with 5-year survival rate of 10%. The tumor microenvironment of pancreatic ductal adenocarcinoma (PDA) includes abundant fibroblasts and infiltrating immune cells, the latter largely immunosuppressive. Mono-immunotherapy or combination immunotherapy approaches has been ineffective in pancreatic cancer, pointing to the need for additional avenues to target in pancreatic cancer microenvironment. We previously showed that targeting regulatory T cell (Treg), a prevalent T cell population in pancreatic cancer, failed to relieve immunosuppression and led to accelerated tumor progression. We discovered that Treg depletion reprogrammed tumor associated fibroblasts and increased immunosuppressive myeloid cell recruitment, an effect that was partially mediated by CCLs/CCR1signaling. We found that tumor educated macrophages express the highest levels of Ccr1 compared to non-activated (M0), pro-inflammatory (M1), or anti-inflammatory (M2) bone marrow derived macrophage subsets. Thus, we sought to investigate the functional role of CCR1 in pancreatic cancer. By single cell RNA sequencing, we found CCR1 to be mainly expressed by tumor associated macrophages (TAMs) and neutrophils (or granulocytes) in both human and mouse PDA. We then orthotopically transplanted syngeneic mouse pancreatic cancer cells in CCR1 knockout hosts and observed reduced tumor growth which was rescued by CD8 T cell depletion. Histological analysis showed elevated Granzyme B expression in infiltrating T cells, as well as an increase in apoptotic cells in tumors implanted in Ccr1−/− mice. Through cytometry by time of flight (CyTOF) and co-immunofluorescence we also discovered that TAMs in tumors implanted in Ccr1−/− mice expressed less Arginase 1 and CD206 -both markers of immunosuppressive macrophages- compared to TAMs in wild type tumors. Thus, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell-mediated anti-tumor immunity. We are currently exploring combination approaches targeting CCR1 in pancreatic cancer. Citation Format: Yaqing Zhang, Kristee L. Brown, Wei Yan, Zeribe C. Nwosu, Eileen K. Carpenter, Katelyn L. Donahue, Ashley Velez-Delgado, Sion Yang, Marina Pasca di Magliano. Ablation of CCR1 relieves immunosuppression in pancreatic cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P053.

A randomized phase 2 trial of idiotype vaccination and adoptive autologous T-cell transfer in patients with multiple myeloma

AbstractWe hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)–keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

The Right Partner in Crime: Unlocking the Potential of the Anti-EGFR Antibody Cetuximab via Combination With Natural Killer Cell Chartering Immunotherapeutic Strategies.

Cetuximab has an established role in the treatment of patients with recurrent/metastatic colorectal cancer and head and neck squamous cell cancer (HNSCC). However, the long-term effectiveness of cetuximab has been limited by the development of acquired resistance, leading to tumor relapse. By contrast, immunotherapies can elicit long-term tumor regression, but the overall response rates are much more limited. In addition to epidermal growth factor (EGFR) inhibition, cetuximab can activate natural killer (NK) cells to induce antibody-dependent cellular cytotoxicity (ADCC). In view of the above, there is an unmet need for the majority of patients that are treated with both monotherapy cetuximab and immunotherapy. Accumulated evidence from (pre-)clinical studies suggests that targeted therapies can have synergistic antitumor effects through combination with immunotherapy. However, further optimizations, aimed towards illuminating the multifaceted interplay, are required to avoid toxicity and to achieve better therapeutic effectiveness. The current review summarizes existing (pre-)clinical evidence to provide a rationale supporting the use of combined cetuximab and immunotherapy approaches in patients with different types of cancer.

Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells.

Simple SummaryGlioblastoma (GBM) remains a particularly challenging cancer, with an aggressive phenotype and few promising treatment options. Future therapy will rely heavily on diagnosing and targeting aggressive GBM cellular phenotypes, both before and after drug treatment, as part of personalized therapy programs. Here, we use a genome-wide drug-induced gene expression (DIGEX) approach to define the cellular drug response phenotypes associated with two clinical drug candidates, the phosphodiesterase 10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib. We identify genes encoding specific drug targets, some of which we validate as effective antiproliferative agents and combination therapies in human GBM cell models, including HMGCoA reductase (HMGCR), salt-inducible kinase 1 (SIK1), bradykinin receptor subtype B2 (BDKRB2), and Janus kinase isoform 2 (JAK2). Individual, personalized treatments will be essential if we are to address and overcome the pharmacological plasticity that GBM exhibits, and DIGEX will play a central role in validating future drugs, diagnostics, and possibly vaccine candidates for this challenging cancer.We have used three established human glioblastoma (GBM) cell lines—U87MG, A172, and T98G—as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.

STINGing the Tumor Microenvironment to Promote Therapeutic Tertiary Lymphoid Structure Development

Tertiary lymphoid structures (TLS), also known as ectopic lymphoid structures (ELS) or tertiary lymphoid organs (TLO), represent a unique subset of lymphoid tissues noted for their architectural similarity to lymph nodes, but which conditionally form in peripheral tissues in a milieu of sustained inflammation. TLS serve as regional sites for induction and expansion of the host B and T cell repertoires via an operational paradigm involving mature dendritic cells (DC) and specialized endothelial cells (i.e. high endothelial venules; HEV) in a process directed by TLS-associated cytokines and chemokines. Recent clinical correlations have been reported for the presence of TLS within tumor biopsies with overall patient survival and responsiveness to interventional immunotherapy. Hence, therapeutic strategies to conditionally reinforce TLS formation within the tumor microenvironment (TME) via the targeting of DC, vascular endothelial cells (VEC) and local cytokine/chemokine profiles are actively being developed and tested in translational tumor models and early phase clinical trials. In this regard, a subset of agents that promote tumor vascular normalization (VN) have been observed to coordinately support the development of a pro-inflammatory TME, maturation of DC and VEC, local production of TLS-inducing cytokines and chemokines, and therapeutic TLS formation. This mini-review will focus on STING agonists, which were originally developed as anti-angiogenic agents, but which have recently been shown to be effective in promoting VN and TLS formation within the therapeutic TME. Future application of these drugs in combination immunotherapy approaches for greater therapeutic efficacy is further discussed.