Checkpoint Blockade Immunotherapy Research Articles

PiSTING: A Pocket-Independent Agonist Based on Multivalency-Driven STING Oligomerization.

The stimulator of interferon genes (STING) pathway is a potent therapeutic target for innate immunity. Despite the efforts to develop pocket-dependent small-molecule STING agonists that mimic the endogenous STING ligand, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), most of these agonists showed disappointing results in clinical trials owing to the limitations of the STING pocket. In this study, we developed novel pocket-independent STING-activating agonists (piSTINGs), which act through multivalency-driven oligomerization to activate STING. Additionally, a piSTING-adjuvanted vaccine elicited a significant antibody response and inhibited tumour growth in therapeutic models. Moreover, a piSTING-based vaccine combination with aPD-1 showed remarkable potential to enhance the effectiveness of immune checkpoint blockade (ICB) immunotherapy. In particular, piSTING can strengthen the impact of STING pathway in immunotherapy and accelerate the clinical translation of STING agonists.

PiSTING: A Pocket‐Independent Agonist Based on Multivalency‐Driven STING Oligomerization

The stimulator of interferon genes (STING) pathway is a potent therapeutic target for innate immunity. Despite the efforts to develop pocket‐dependent small‐molecule STING agonists that mimic the endogenous STING ligand, cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), most of these agonists showed disappointing results in clinical trials owing to the limitations of the STING pocket. In this study, we developed novel pocket‐independent STING‐activating agonists (piSTINGs), which act through multivalency‐driven oligomerization to activate STING. Additionally, a piSTING‐adjuvanted vaccine elicited a significant antibody response and inhibited tumour growth in therapeutic models. Moreover, a piSTING‐based vaccine combination with aPD‐1 showed remarkable potential to enhance the effectiveness of immune checkpoint blockade (ICB) immunotherapy. In particular, piSTING can strengthen the impact of STING pathway in immunotherapy and accelerate the clinical translation of STING agonists.

Tumor targeted porphyrin-based metal–organic framework for photodynamic and checkpoint blockade immunotherapy

Photodynamic therapy (PDT) has become a promising approach and non-invasive modality for cancer treatment, however the therapeutic effect of PDT is limited in tumor metastasis and local recurrence. Herein, a tumor targeted nanomedicine (designated as PCN@HA) is constructed for enhanced PDT against tumors. By modified with hyaluronic acid (HA), which could target the CD44 receptor that expressed on the cancer cells, the targeting ability of PCN@HA has been enhanced. Under light irradiation, PCN@HA can produce cytotoxic singlet oxygen (1O2) and kill cancer cells, then eliminate tumors. Furthermore, PCN@HA exhibits fluorescence (FL)/ photoacoustic (PA) effects for multimodal imaging-guided cancer treatment. And PCN@HA-mediated PDT also can induce immunogenic cell death (ICD) and stimulate adaptive immune responses by releasing of tumor antigens. By combining with anti-PD-L1 checkpoint blockade therapy, it can not only effectively suppress the growth of primary tumor, but also inhibit the metastatic tumor growth.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Tumor microenvironment reprogramming combined with immunogenic enhancement by nanoemulsions potentiates immunotherapy

The combination of immune checkpoint inhibitors and immunogenic cell death (ICD) inducers has become a promising strategy for the treatment of various cancers. However, its efficacy remains unmet because of the dense stroma and defective vasculatures in the tumor microenvironment (TME) that restricts the intratumoral infiltration of cytotoxic T lymphocytes (CTLs). Herein, cancer-associated fibroblasts (CAFs)-targeted nanoemulsions are tailored to combine the ICD induction and the TME reprogramming to sensitize checkpoint blockade immunotherapy. Melittin, as an ICD inducer and an antifibrotic agent, is efficiently encapsulated into the nanoemulsion accompanied by a nitric oxide donor to improve its bioavailability and tumor targeting. The nanoemulsions exhibited dual functionality by directly inducing direct cancer cell death and enhancing the tumoral immunogenicity, while also synergistically reprogramming the TME through reversing the activated CAFs, decreasing collagen deposition and restoring tumor vessels. Consequently, these nanemulsions successfully facilitated the CTLs infiltration and suppressing the recruitment of immunosuppressive cells. A combination of AE-MGNPs and anti-CTLA-4 antibody greatly elicited a striking level of antitumor T-cell response to suppress tumor growth in CAFs-rich colorectal tumor models. Our work emphasized the integration of the ICD induction with simultaneous modulation of the TME to enhance the sensitivity of patients to checkpoint blockade immunotherapy.

Abstract 164: Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages

Abstract Response to checkpoint blockade immunotherapy (CBI) is not universal, partly due to a wide variation in baseline immune cell infiltration in the tumor microenvironment (TME). One mechanism contributing to this variability is tumor cell-intrinsic activation of the β-catenin signaling pathway, which has previously been shown to result in a non-T cell-inflamed TME. Constitutive β-catenin signaling resulted in reduced Batf3-lineage dendritic cell (cDC1) recruitment, leading to reduced T cell activation and infiltration and loss of anti-CTLA-4+anti-PD-L1 therapeutic efficacy. Efforts to target the Wnt/β-catenin pathway to enhance immune responses in β-catenin-active tumors have seen limited success. A genetic experiment eliminating β-catenin expression after establishment of a non-T cell-inflamed TME might illustrate the maximal biologic effect that could be expected with blockade of the pathway. We developed a genetically engineered mouse (GEM) model that allows for dynamic regulation of β-catenin on and off. This model employs tyrosinase-driven tamoxifen-regulated Cre-recombinase to activate the BRAF V600E oncogene and delete the tumor suppressor gene PTEN, along with a transactivator for doxycycline-regulated expression of a non-degradable β-catenin. Topical application of 4-hydroxytamoxifen resulted in melanoma formation, and administration of doxycycline resulted in robust nuclear expression of melanocyte-specific β-catenin compared to non-treated controls. This was accompanied by a significant reduction in CD3+ T cell infiltration within the TME, confirming the link between active β-catenin signaling and T cell exclusion. Discontinuing doxycycline treatment led to complete reduction of nuclear β-catenin expression in the tumor cells within seven days and a substantial return of CD3+ T cells into the TME. However, despite the re-infiltration of CD3+ T cells, the tumors previously expressing β-catenin (ex-β-catenin tumors) did not regain therapeutic responsiveness to anti-CTLA-4+anti-PD-L1. Single cell RNA sequencing of the ex-β-catenin tumors indicated the presence of an immunosuppressive-like macrophage population, characterized by the expression of Ccl8, Gas6 and Cd163. This finding suggests that the prior presence of β-catenin may induce long-lasting changes in the TME that suppress the immune response even after β-catenin levels are reduced. These data corroborate previous findings on the role of β-catenin in modulating the immunological landscape of melanoma and extend it by demonstrating the long-term persistence of immune evasion by suppressive myeloid cells even after β-catenin signaling is eliminated. These insights have implications for clinical strategies aiming to block Wnt/β-catenin signaling, which may additionally require myeloid cell modulation to restore immunotherapy efficacy. Citation Format: Alexandra Cabanov, Ruxandra Tonea, Jason Shapiro, Anna Martinez, Yuanyuan Zha, Thomas F. Gajewski. Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 164.

Abstract 4154: PET imaging to characterize the tumor microenvironment in a breast cancer model of obesity

Abstract Introduction: Obesity is known to reduce efficacy for cancer treatment patients and is thought to modulate the tumor microenvironment and glucose metabolism. In this study, we aim to quantify obesity-induced differences in the breast cancer tumor microenvironment with positron emission tomography (PET) imaging of hypoxia, glucose metabolism, and granzyme B. These experiments are testing the hypothesis that tumors in obese mice have increased glucose metabolism and hypoxia, measured by [18F]F-fluorodeoxyglucose (FDG) and [18F]F-fluoromisonidazole (FMISO) uptake. We also hypothesize that immunotherapy (IMT) increases immune activation, as measured by [68Ga]Ga-Granzyme B peptide (GZP), and that obese mice will respond less to immunotherapy than lean mice. Methods: C57/Bl6 mice were placed on a high-fat diet (HFD) with 60% kcal fat (n=18) or low-fat diet (LFD) composed of 10% kcal fat (n=18) to induce obesity. Blood serum was collected for analysis of cholesterol levels prior to tumor implantation. Body weight and food intake were measured weekly. 14 weeks after initiating each diet, 5x105 E0771 cells were implanted into the 3rd mammary fat pad and allowed to grow to 50-150mm3. Tumor-bearing mice were imaged with [18F]F-FDG and [18F]F-FMISO PET to measure glucose metabolism and hypoxia, respectively prior to treatment. Following imaging, mice began treatment with either saline (n=6/diet) or checkpoint blockade IMT with 200 ug anti-PD-1 and 100 ug anti-CTLA-4 (n=12/diet). After 3 doses of IMT, mice were imaged with [68Ga]Ga-GZP PET to measure immune activation. Tumor volume measurements and treatment administration occurred every 3 days. Metrics of standardized uptake value (SUV) including mean, max, and peak were quantified from imaging data. A one-way independent ANOVA or independent t-test was used to compare differences between groups. Results: HFD-fed mice had significantly increased body weight (p<0.0001) and serum cholesterol (p=0.005) compared to LFD-fed mice. SUVmean for tumor hypoxia (p=0.003) and glucose metabolism (p=0.02) were significantly increased at baseline in HFD-fed mice compared to LFD-fed mice. IMT increases immune activation for both LFD-fed (p=0.009) and HFD-fed mice (p=0.05). IMT treatment significantly reduces tumor burden for LFD mice (p<0.0001), but not HFD mice (p=0.096). Conclusions: Obesity plays a role in the reduction of the effectiveness of immunotherapy. At baseline, tumors of obese mice have increased tumor glucose metabolism and hypoxia. Although immunotherapy increases immune activation, this is not sufficient to reduce tumor burden or improve overall survival. Understanding obesity-induced differences in tumor biology may provide imaging-directed treatment decision making. Citation Format: Shannon E. Lynch, Corinne Crawford, Addison Hunt, Luke Sligh, Benjamin M. Larimer, Suzanne E. Lapi, Anna G. Sorace. PET imaging to characterize the tumor microenvironment in a breast cancer model of obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4154.

Abstract 5100: Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade

Abstract Background: Why some patients fail or have short lived response to immune checkpoint blockade (ICB) immunotherapy remains largely unknown. While baseline molecular assessments have provided clues to prognostic factors, insights into resistance drivers remains elusive. This is partially due to the difficulty in getting access to post progression samples from patients that were either primary resistant or developed acquired resistance after an initial response to ICB. Thus, the tumor-intrinsic and -extrinsic features that are selected for during progression and potentially drive primary and acquired resistance to immunotherapy remain underexplored. Methods: To compare clinical features and immunogenomic drivers of acquired and primary resistance to ICB across major cancers, we analysed and annotated de-identified patient records in the Tempus real-world database. We built an immuno-oncology cohort consisting of >2500 multimodal (DNA, RNA and clinical outcome data) pre-treatment baseline with >1500 post-treatment tumour biopsy samples from mainly NSCLC, TNBC, HNC and Bladder cancer patients. We used bulk RNA-seq data to estimate activation of the hallmark oncogenic pathways and immune cell composition and used panel DNA-seq data (>500 genes) to quantify mutation selection at the gene and pathway levels using dndscv. Results: Compared to acquired, primary resistant patients tended to have a higher observation of liver lesions at progression. Post-ICB, acquired resistant NSCLC and HNC patients showed a significantly inflamed tumour microenvironment (TME) characterised by higher estimation of infiltration of T cells and myeloid cells and higher activation of interferon gamma (IFNg) signalling as compared to primary resistant patients. In addition, in post-ICB acquired resistance in NSCLC we observed selection for mutations in genes involved in known immunomodulatory pathways, including loss-of-function mutations in B2M. Consistently, acquired resistance patients showed stronger selection for mutations in Hedgehog and Notch pathways as compared to primary resistance patients across NSCLC, HNC and TNBC post-ICB. Conclusions: Acquired and primary ICB resistant patients have distinct clinical and molecular features at progression. Their tumours’ TME is fundamentally different with acquired resistance TMEs being infiltrated with immune cells albeit escaped post progression. In addition, ICB selects mutations that potentially activate pathways such as Notch and Hedgehog. This multi-modal Real-World Data with post therapy biopsies has given insights for patient selection strategies and provides rational into combination treatment options for acquired resistant patients. Citation Format: Mohamed Reda Keddar, Sebastian Carrasco Pro, Kathleen Burke, Ana Camelo Stewart, Mark Cobbold, Ross Stewart, Sajan Khosla, Ben Sidders, Scott Hammond, Douglas C. Palmer, Jonathan Dry, Martin L. Miller. Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5100.

Abstract 3961: Impact of CD8 resident memory T cells on antitumor immunity and response to cancer immunotherapy

Abstract CD8+ resident memory T (TRM) cells expressing the CD103 integrin accumulate in human lung tumors and are associated with a favorable prognosis. We have previously demonstrated that a high density of CD103+CD8+ T cells in human lung tumors is a biomarker of response to immune checkpoint blockade (ICB) immunotherapy. However, a large fraction of human tumors is only weakly infiltrated by CD8+ TRM cells, and the reason of this TRM desert is not explained. Therefore, defining the molecular signals that give rise and maintain TRM cells within the tumor microenvironment (TME) and the mechanisms that potentiate antitumor TRM functions are important challenges. Data obtained in pre-clinical mouse colorectal cancer and melanoma models demonstrate that knocking-out CD103 impairs antitumor T-cell immunity and response to ICB and therapeutic peptide vaccine. Moreover, immunization of melanoma-bearing mice with the cancer vaccine results in a dramatic decrease in the percentage of CD103+ TRM cells among CD8+ tumor-infiltrating T lymphocytes (TIL). Algorithm analyses applied to spectral cytometry data of TIL support the conclusion that upon activation with the peptide vaccine a TRM subset gives rise to tumor-specific effector T cells. The mechanisms associated with the decrease in CD103 expression on CD8+ T cells and lead to a TRM differentiation shift will be presented as well as the key role of TGF-b in CD8+CD103+ TRM formation and persistence in the TME. Our data demonstrate that tumor CD8+CD103+ TRM are capable of mounting a potent antitumor immunity and suggest that a fine balance between diverse signals in the tumor ecosystem is required to induce their differentiation and thereby optimize response to cancer immunotherapy. Citation Format: Stephanie Corgnac, Isabelle DAMEI, Fathia Mami-Chouaib. Impact of CD8 resident memory T cells on antitumor immunity and response to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3961.

Abstract 1957: OPM-116, a highly potent and specific PI3Kγ inhibitor to enhance antitumor immunity

Abstract Tumor-associated macrophages are an integral part of the tumor microenvironment. They play a crucial role in cancer initiation and progression by orchestrating tumor proliferation, angiogenesis, extracellular matrix remodeling, metastasis, immunosuppression, and resistance to checkpoint blockade immunotherapy and chemotherapy (Gao J, 2022). On the other hand, when properly stimulated, macrophages can effectively interact with the innate and adaptive immune systems arms, promoting cancer cell phagocytosis and tumor destruction by T cell-mediated cytotoxicity (Mantovani A, 2022). PI3Kγ has a unique pattern of expression and biological function, and it has been investigated for a long time as a therapeutic target for different disorders, including cancer. Inhibition of PI3Kγ reprograms M2 macrophages into M1 cells in the tumor microenvironment which translates into the expansion of activated T cells by relieving macrophage suppression. Recent clinical data indicates the compelling role of PI3Ky in combination with PDL1 in anti-tumor activity, irrespective of PDL1 status (Hamilton E, 2021). PI3Ky selectivity has been difficult to achieve due to the high sequence homology across the class 1 PI3K isoforms (Drew SL, 2020). Macrocyclization of small molecules with a linear structure strongly reduces their conformational flexibility and alters a variety of their physicochemical and biological characteristics. The unexpected selectivity these compounds can exhibit, even amongst closely similar target molecules, is explained by the conformationally limited structure (Ma J, 2022). Here, we describe the discovery of a potent and selective, ATP-competitive inhibitor of PI3Kγ. OPM-116, macrocyclic compound display >300-fold selectivity for PI3Kγ over the other class I isoforms. Its selectivity in biochemical and cellular assays as well as in human PBMC assays is presented. In vitro, OPM-116 can inhibit the IL-4-induced polarization of M2 macrophages. Pharmacodynamic analysis performed in animals that received oral compound administration confirmed the absolute selectivity and good PK profiles of the drug. OPM-116 is a highly selective, PI3Kγ inhibitor that displays high potency and specificity and represents a promising step toward the identification of a clinical candidate. Drew S.L., et al. Discovery of potent and selective PI3Kγ inhibitors. J. Med. Chem. 2020. Ma J., et al. Development of small macrocyclic kinase inhibitors. Futur. Med. Chem. 2022. Gao J., et al. Shaping polarization of tumor-associated macrophages in cancer immunotherapy. Front. Immunol. 2022. Mantovani A, et al. Macrophages as tools and targets in cancer therapy. Nat Rev Drug Discovery. 2022. Hamilton E, et al. Mario-3 phase II study safety run-in evaluating a novel triplet combination of eganelisib, atezolizumab, and nab-paclitaxel as first-line therapy for locally advanced or metastatic TNBC. AACR, Cancer Res 2021. Citation Format: Kenji Shoji, Petra Blom, Maria Eugenia Riveiro, Jan Hoflack. OPM-116, a highly potent and specific PI3Kγ inhibitor to enhance antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1957.

Abstract 3186: An Erythrocyte-αPD-1 conjugate suppresses cancers resistant to checkpoint blockade immunotherapy: A first-in-human study

Abstract Background: Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, yet resistance to this therapy can lead to immune escape and disease progression. Here, we introduce WTX-212, an erythrocyte-antibody conjugate covalently linking anti-PD-1 antibody to erythrocyte membranes. Unlike conventional antibodies, WTX-212 naturally accumulates in the spleen and remodels the splenic immune landscape in tumor-bearing mice. Pre-clinical studies have demonstrated that T cells activated by WTX-212 reduce the splenic reservoir of immunosuppressive myeloid cells, including those within tumors. This results in a systemic anti-tumor immune response and suppression of tumor growth in xenograft tumor models resistant to anti-PD-1 immunotherapies. A first-in-human (FIH) clinical trial is now investigating its potential in human cancer patients. Methods: The FIH trial (NCT05707325) is aimed to investigate the safety, pharmacokinetics, mechanisms and preliminary efficacy of WTX-212 in cancer patients with advanced malignancies. All of patients had previously received anti-PDs therapies and developed resistance to these treatments. By November 15th, 2023, 7 patients across two cohorts had received WTX-212 monotherapy (1 × 1011-2 × 1011 cells, Q3W IV). The tumor lesions were assessed using RECIST v1.1 criteria. Blood and tumor samples were collected for correlative analysis. Results: 7 patients with various solid tumors, having undergone a median of 3.1 prior lines of therapy (ranging from 1-6), received WTX-212 monotherapy. No dose-limiting toxicities or treatment-related adverse events greater than Grade 3 were reported. WTX-212 was detectable in peripheral blood at the end of cycle in a dose dependent manner. Disease control, including partial remission (PR) and stable disease (SD), was observed in 5/7 patients (DCR, 71%). One patient with esophageal cancer achieved a PR with a significant 44% reduction in target lesion size after two cycles of treatment. Two patients, one with esophageal cancer (3 lines prior therapies) and another with HPV-negative cervical cancer (4 lines prior therapies), maintained disease control for over 30 weeks after WTX-212 treatment. Consistent with preclinical findings, WTX-212 systemically induced anti-tumor responses, leading to a reduction of myeloid-derived suppressor cells (MDSCs) particularly the PMN-MDSC subset in 6/7 patients (reductions ranging from 24%-82% compared to baseline), and a median 1.5-fold increase of T cells in 7/7 patients in the peripheral blood after the 1st cycle of treatment. Conclusion: The initial promising data support further investigation and exploration of the combination therapy strategies to expand the therapeutic potential of WTX-212 in cancer patients. Erythrocyte-drug conjugation may serve as a unique strategy for targeting the peripheral immune system to treat cancers. Citation Format: Liu Yang, Xiaoqian Nie, Zheling Chen, Yuehua Liu, Jiawei Zheng, Xiaofei Gao. An Erythrocyte-αPD-1 conjugate suppresses cancers resistant to checkpoint blockade immunotherapy: A first-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3186.

Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer.

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

Machine learning-based extrachromosomal DNA identification in large-scale cohorts reveals its clinical implications in cancer.

The clinical implications of extrachromosomal DNA (ecDNA) in cancer therapy remain largely elusive. Here, we present a comprehensive analysis of ecDNA amplification spectra and their association with clinical and molecular features in multiple cohorts comprising over 13,000 pan-cancer patients. Using our developed computational framework, GCAP, and validating it with multifaceted approaches, we reveal a consistent pan-cancer pattern of mutual exclusivity between ecDNA amplification and microsatellite instability (MSI). In addition, we establish the role of ecDNA amplification as a risk factor and refine genomic subtypes in a cohort from 1015 colorectal cancer patients. Importantly, our investigation incorporates data from four clinical trials focused on anti-PD-1 immunotherapy, demonstrating the pivotal role of ecDNA amplification as a biomarker for guiding checkpoint blockade immunotherapy in gastrointestinal cancer. This finding represents clinical evidence linking ecDNA amplification to the effectiveness of immunotherapeutic interventions. Overall, our study provides a proof-of-concept of identifying ecDNA amplification from cancer whole-exome sequencing (WES) data, highlighting the potential of ecDNA amplification as a valuable biomarker for facilitating personalized cancer treatment.

Immune cell infiltrates in peritoneal metastases from colorectal cancer.

The presence of peritoneal metastases (PMs) in patients with colorectal cancer (CRC) confers a poor prognosis and only a minority of patients will benefit from the available treatment options. In primary CRC tumors, it is well established that a high infiltration of CD8+ effector T cells correlates to a favorable patient outcome. In contrast, the immune response induced in PMs from CRC and how it relates to patient survival is still unknown. In this study, we characterized the immune infiltrates and the distribution of immune checkpoint receptors on T cells from PMs from CRC, in order to evaluate the potential benefit of checkpoint blockade immunotherapy for this patient group. Surgically resected PM tissue from CRC patients (n=22) and synchronous primary tumors (n=8) were processed fresh to single cell suspensions using enzymatic digestion. Surface markers and cytokine production were analyzed using flow cytometry. T cells dominated the leukocyte infiltrate in the PM specimens analyzed, followed by monocytes and B cells. Comparing two different PMs from the same patient usually showed a similar distribution of immune cells in both samples. The T cell infiltrate was characterized by an activated phenotype and markers of exhaustion were enriched compared with matched circulating T cells, in particular the checkpoint receptors PD-1 and TIGIT. In functional assays most cytotoxic and helper T cells produced INF-γ and TNF following polyclonal stimulation, while few produced IL-17, indicating a dominance of Th1-type responses in the microenvironment of PMs. Immune cells were present in all PMs from CRC examined. Although infiltrating T cells express markers of exhaustion, they produce Th1-type cytokines when stimulated. These results indicate the possibility to augment tumor-specific immune responses within PMs using checkpoint blockade inhibitors.

89Zr]-CD8 ImmunoPET imaging of glioblastoma multiforme response to combination oncolytic viral and checkpoint inhibitor immunotherapy reveals CD8 infiltration differential changes in preclinical models.

Rationale: Novel immune-activating therapeutics for the treatment of glioblastoma multiforme (GBM) have shown potential for tumor regression and increased survival over standard therapies. However, immunotherapy efficacy remains inconsistent with response assessment being complicated by early treatment-induced apparent radiological tumor progression and slow downstream effects. This inability to determine early immunotherapeutic benefit results in a drastically decreased window for alternative, and potentially more effective, treatment options. The objective of this study is to evaluate the effects of combination immunotherapy on early CD8+ cell infiltration and its association with long term response in orthotopic syngeneic glioblastoma models. Methods: Luciferase positive GBM orthotopic mouse models (GSC005-luc) were imaged via [89Zr]-CD8 positron emission tomography (PET) one week following treatment with saline, anti-PD1, M002 oncolytic herpes simplex virus (oHSV) or combination immunotherapy. Subsequently, brains were excised, imaged via [89Zr]-CD8 ImmunoPET and evaluated though autoradiography and histology for H&E and CD8 immunohistochemistry. Longitudinal immunotherapeutic effects were evaluated through [89Zr]-CD8 PET imaging one- and three-weeks following treatment, with changes in tumor volume monitored on a three-day basis via bioluminescence imaging (BLI). Response classification was then performed based on long-term BLI signal changes. Statistical analysis was performed between groups using one-way ANOVA and two-sided unpaired T-test, with p < 0.05 considered significant. Correlations between imaging and biological validation were assessed via Pearson's correlation test. Results: [89Zr]-CD8 PET standardized uptake value (SUV) quantification was correlated with ex vivo SUV quantification (r = 0.61, p < 0.01), autoradiography (r = 0.46, p < 0.01), and IHC tumor CD8+ cell density (r = 0.55, p < 0.01). Classification of therapeutic responders, via bioluminescence signal, revealed a more homogeneous CD8+ immune cell distribution in responders (p < 0.05) one-week following immunotherapy. Conclusions: Assessment of early CD8+ cell infiltration and distribution in the tumor microenvironment provides potential imaging metrics for the characterization of oHSV and checkpoint blockade immunotherapy response in GBM. The combination therapies showed enhanced efficacy compared to single agent immunotherapies. Further development of immune-focused imaging methods can provide clinically relevant metrics associated with immune cell localization that can inform immunotherapeutic efficacy and subsequent treatment response in GBM patients.

Cancer vaccines: from an immunology perspective.

The concept of a therapeutic cancer vaccine to activate anti-tumour immunity pre-dates innovations in checkpoint blockade immunotherapies. However, vaccination strategies have yet to show the hoped-for successes in patients, and unanswered questions regarding the underlying immunological mechanisms behind cancer vaccines have hampered translation to clinical practice. Recent advances in our understanding of the potential of tumour mutational burden and neo-antigen-reactive T cells for response to immunotherapy have re-ignited enthusiasm for cancer vaccination strategies, coupled with the development of novel mRNA-based vaccines following successes in prevention of COVID-19. Here we summarise current developments in cancer vaccines and discuss how advances in our comprehension of the cellular interplay in immunotherapy-responsive tumours may inform better design of therapeutic cancer vaccines, with a focus on the role of dendritic cells as the orchestrators of anti-tumour immunity. The increasing number of clinical trials and research being funnelled into cancer vaccines has demonstrated the 'proof-of-principle', supporting the hypothesis that therapeutic vaccines have potential as an immuno-oncology agent. For efficacious and safe cancer vaccines to be developed, better understanding of the underpinning immunological mechanisms is paramount.

TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

Heterogeneity of tertiary lymphoid structures in cancer.

The success of immunotherapy approaches, such as immune checkpoint blockade and cellular immunotherapy with genetically modified lymphocytes, has firmly embedded the immune system in the roadmap for combating cancer. Unfortunately, the majority of cancer patients do not yet benefit from these therapeutic approaches, even when the prognostic relevance of the immune response in their tumor entity has been demonstrated. Therefore, there is a justified need to explore new strategies for inducing anti-tumor immunity. The recent connection between the formation of ectopic lymphoid aggregates at tumor sites and patient prognosis, along with an effective anti-tumor response, suggests that manipulating the occurrence of these tertiary lymphoid structures (TLS) may play a critical role in activating the immune system against a growing tumor. However, mechanisms governing TLS formation and a clear understanding of their substantial heterogeneity are still lacking. Here, we briefly summarize the current state of knowledge regarding the mechanisms driving TLS development, outline the impact of TLS heterogeneity on clinical outcomes in cancer patients, and discuss appropriate systems for modeling TLS heterogeneity that may help identify new strategies for inducing protective TLS formation in cancer patients.

Poly(ethyl ethylene phosphate): Overcoming the "Polyethylene Glycol Dilemma" for Cancer Immunotherapy and mRNA Vaccination.

Polyethylene glycol conjugation (PEGylation) is the most successful strategy to promote the stability, pharmacokinetics, and efficacy of therapeutics; however, anti-PEG antibodies induced by repeated treatments raise serious concerns about the future of PEGylated therapeutics. In order to solve the "PEG dilemma", polymers with excellent water solubility and biocompatibility are urgently desired to attenuate the generation of anti-PEG antibodies. Here, poly(ethyl ethylene phosphate) (PEEP) with excellent degradability and stealth effects is used as an alternative to PEG to overcome the "PEG dilemma". PEEPylated liposomes exhibit lower immunogenicity and generate negligible anti-PEEP antibodies (IgM and IgG) after repeated treatments. In vivo studies confirm that PEEPylated liposomes loaded with oxaliplatin (PEEPlipo@OxPt) show better pharmacokinetics compared to PEGlipo@OxPt, and they exhibit potent antitumor performances, which can be further promoted with checkpoint blockade immunotherapy. In addition, PEEPylated lipid nanoparticle is also used to develop an mRNA vaccine with the ability to evoke a potent antigen-specific T cell response and achieve excellent antitumor efficacy. PEEP shows promising potentials in the development of next-generation nanomedicines and vaccines with higher safety and efficacy.

Abstract IA009: Antibody-lectin chimeras for glyco-immune checkpoint blockade

Abstract Despite the curative potential of checkpoint blockade immunotherapy, a majority of patients remain unresponsive to existing treatments. Glyco-immune checkpoints – interactions of cell-surface glycans with lectin, or glycan binding, immunoreceptors – have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a tumor-targeting arm as well as a lectin “decoy receptor” domain that directly binds tumor glycans and blocks their ability to engage lectin receptors on immune cells. AbLecs enhanced cancer cell killing in vitro via macrophage phagocytosis and NK cell and granulocyte cytotoxicity compared to their parent monoclonal antibodies. AbLec treatment reduced tumor burden in vivo compared to treatment with the parent antibody in a humanized model of metastatic HER2+ cancer. Furthermore, AbLecs synergized with blockade of the “don’t eat me” signal CD47 for enhanced tumor killing. AbLecs can be readily designed to target numerous tumor-associated antigens and glyco-immune checkpoint ligands, and therefore represent a new modality of cancer immunotherapy. Citation Format: Jessica Stark. Antibody-lectin chimeras for glyco-immune checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA009.