Defective Antigen Presentation Research Articles

Abstract PO1-19-08: Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the cDC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal doxorubicin, CDX-1140 (CD40 agonist monoclonal antibody), and CDX-301 (recombinant Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal doxorubicin only) for one cycle prior to receiving triplet therapy with tissue biopsies done before and after the lead-in treatment. CDX-301 will be given for only two cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase II dose based on treatment-related adverse events and dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line treatment for metastatic disease (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999) and is currently open at University of Texas Southwestern Medical Center, Texas Oncology, University of Chicago, University of Texas San Antonio, Sarah Cannon Research Institute, and Johns Hopkins. Citation Format: Sangeetha Reddy, Joyce O'Shaughnessy, Navid Sadeghi, Samira Syed, Cesar Santa-Maria, Virginia Kaklamani, Nan Chen, Denise Yardley, Yisheng Fang, Isaac Chan, Nisha Unni, Sarah Kashanian, Namrata Peswani, Shahbano Shakeel, Meredith Carter, Kelly Kyle, Farjana Fattah, Chul Ahn, Ina Patel, Joshua Gruber, Dawn Klemow, Glenda Delgado, Nicole Sinclair, Michael Yellin, Heather McArthur, Rita Nanda, Suzanne Conzen, Carlos Arteaga. Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-19-08.

IGSF8 is an innate immune checkpoint and cancer immunotherapy target

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival invitro or invivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells invitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and Tcell signaling invivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Abstract 5314: Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells

Abstract Background: There is a need for improved immunotherapy strategies beyond current FDA approved treatments for triple negative breast cancer. Defective antigen presentation has been shown to play a contributory role in deficient anti-tumor immunity in breast cancers. Flt3 ligand (Flt3L) is a growth factor that increases differentiation into DC1 dendritic cells. CD40 agonist (CD40a) activates all 3 classes of antigen presenting cells - DCs, B cells, and macrophages. Synergy with chemotherapy and CD40a has been seen in other cancers but not explored in breast cancers. Methods: C57BL/6 mice (aged 6-8 weeks) were injected with 150,000 E0771 breast cancer cells within the mammary fat pad. Tumors were allowed to grow to a volume of ~50 mm3 before randomization into treatment groups. Mice were treated with triplet therapy of pegylated liposomal doxorubicin (PLD) via tail vein followed by Flt3L intraperitoneal (IP) daily for 5 days and CD40a IP 1 week later as well as monotherapy and doublet therapy controls. Tumor growth and survival were monitored throughout the experiment. Tumors were harvested for single cell RNA sequencing (scRNAseq) and immunohistochemistry. To further study subset specific effects on efficacy of the immunotherapy, CD8 and CD4 T-cells were systemically depleted with anti-CD8 and anti-CD4 antibodies administered IP every 4 days starting 2 days before PLD treatment. Results: Triplet therapy led to improved tumor control compared to no treatment, monotherapy, and doublet controls (p<0.0001). Increased tumor infiltration with CD8 T cells was observed with triplet therapy via immunohistochemistry. scRNAseq showed increased cDC1 subset with Flt3L treatment. Contrastingly, CD40a decreased DC subsets likely due to activation and emigration out of the tumor, remodeled macrophage populations, and decreased T regulatory cells. CD8 depletion led to faster tumor growth whereas CD4 depletion reduced tumor growth. Triplet therapy maintained efficacy over PLD with both CD8 and CD4 depletion but showed reduced magnitude of benefit with CD8 T cell depletion suggesting a partial CD8 mediated process. CD4 T cell depletion did not hinder triplet therapy efficacy compared to PLD but depletion further enhanced tumor control in combination with triplet therapy, with 20% (2/10) of animals achieving complete regression. Conclusion: Triplet therapy with PLD, Flt3L, and CD40a improved tumor control and survival in syngeneic E0771 breast cancer mouse model. Added benefit of immunotherapy to PLD is partially CD8 T cell mediated. CD4 depletion further enhances tumor control in combination with triplet therapy. Experiments to explore the interactive effect of triplet therapy with other pertinent immune subsets are ongoing. A clinical trial with this combination in metastatic triple negative breast cancer patients is open and recruiting (NCT05029999). Citation Format: Preteesh L. Mylabathula, Shruthi Nooka, Anas Alkhani, James Zhu, Ashley Vu, Riyasat Ali, Varshini Ramasamy Balasubramanian, Isaac Chan, Sangeetha M. Reddy. Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells [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 5314.

Abstract 3914: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target

Abstract Background: Loss of MHC-I antigen presentation is often associated with T-cell excluded tumors, and represents a common mechanism of both primary and acquired resistance to immune checkpoint blockade (ICB) treatment. MHC-I normally acts as a marker of “self” and cells that lack MHC-I are recognized and killed by innate immunity. In this study, we investigated the mechanism of innate immune evasion in tumors with MHC-I antigen presentation defects. Methods: Integrating functional genomics, big data, and artificial intelligence, we discovered that IGSF8 expressed on cancer cells is an evolutionarily conserved innate immune checkpoint. IGSF8 is normally expressed in neuronal tissues and is not essential in vitro or in vivo. However, knockout of IGSF8 in B16-F10 melanoma cell line decreased tumor growth in vivo. For clinical relevance of IGSF8 in tumor immunity, we analyzed genomics, transcriptomics, and clinical data from The Cancer Genome Atlas (TCGA). We developed an antibody (GV20-0251) against IGSF8 and tested its function to induce immune cell killing of cancer cells in vitro and inhibit tumor growth in vivo. Results: IGSF8 has receptors on both natural killer (NK) cells and dendritic cells (DC) to strongly suppress NK cytotoxicity and antigen presentation. In the TCGA tumor profiles, IGSF8 has highest mRNA expression in melanoma and is significantly overexpressed in many cancer types. IGSF8 also shows frequent copy number amplifications. In addition, IGSF8 expression is associated with low antigen presentation, low immune infiltration, and worse overall survival in patients with low MHC class I expression. Immunohistochemistry staining of various cancers showed that IGSF8 expression was primarily observed in malignant cells with low MHC-I. We developed a cross-species reactive antibody against IGSF8 (GV20-0251) which blocks IGSF8 interaction with NK and DC. Flow cytometry of the tumor infiltrating CD45+ cells revealed that IGSF8 inhibition significantly increased NK and dendritic cell infiltration. GV20-0251 enhances NK killing of cancer cells in vitro and increases antigen presentation, NK-mediated cytotoxicity, and T cell signaling in vivo. In multiple syngeneic tumor models (B16-F10, CT26, LLC, 4T1 and EMT6), anti-IGSF8 shows single-agent efficacy and is synergistic with anti-PD1 in controlling tumor growth. Conclusions: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target. A phase 1 study is ongoing to explore the IGSF8 inhibitor GV20-0251 in patients with advanced or metastatic solid tumors (NCT05669430). Citation Format: X. Shirley Liu, Yulong Li, Xiangyang Wu, Hailing Liu, Huizhu Liu, Caibin Sheng, Qingyang Ding, Yixuan Tang, Chao Liu, Bin Xie, Xi Xiao, Quan Yu, Rongbin Zheng, Xihao Hu, Tengfei Xiao. IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target [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 3914.

CXCL9/10-engineered dendritic cells promote T cell activation and enhance immune checkpoint blockade for lung cancer

Immune checkpoint blockade (ICB) with PD-1/PD-L1 inhibition has revolutionized the treatment of non-small cell lung cancer (NSCLC). Durable responses, however, are observed only in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment (TME) can lead to deficient Tcell recruitment and ICB resistance. We evaluate intratumoral (IT) vaccination with CXCL9- and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a strategy to overcome resistance. IT CXCL9/10-DC leads to enhanced Tcell infiltration and activation in the TME and tumor inhibition in murine NSCLC models. The antitumor efficacy of IT CXCL9/10-DC is dependent on CD4+ and CD8+ Tcells, as well as CXCR3-dependent Tcell trafficking from the lymph node. IT CXCL9/10-DC, in combination with ICB, overcomes resistance and establishes systemic tumor-specific immunity in murine models. These studies provide a mechanistic understanding of CXCL9/10-DC-mediated host immune activation and support clinical translation of IT CXCL9/10-DC to augment ICB efficacy in NSCLC.

A Comparison of Spatial and Phenotypic Immune Profiles of Pancreatic Ductal Adenocarcinoma and Its Precursor Lesions.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of 12.5%. PDAC predominantly arises from non-cystic pancreatic intraepithelial neoplasia (PanIN) and cystic intraductal papillary mucinous neoplasm (IPMN). We used multiplex immunofluorescence and computational imaging technology to characterize, map, and compare the immune microenvironments (IMEs) of PDAC and its precursor lesions. We demonstrate that the IME of IPMN was abundantly infiltrated with CD8+ T cells and PD-L1-positive antigen-presenting cells (APCs), whereas the IME of PanIN contained fewer CD8+ T cells and fewer PD-L1-positive APCs but elevated numbers of immunosuppressive regulatory T cells (Tregs). Thus, immunosuppression in IPMN and PanIN seems to be mediated by different mechanisms. While immunosuppression in IPMN is facilitated by PD-L1 expression on APCs, Tregs seem to play a key role in PanIN. Our findings suggest potential immunotherapeutic interventions for high-risk precursor lesions, namely, targeting PD-1/PD-L1 in IPMN and CTLA-4-positive Tregs in PanIN to restore immunosurveillance and prevent progression to cancer. Tregs accumulate with malignant transformation, as observed in PDAC, and to a lesser extent in IPMN-associated PDAC (IAPA). High numbers of Tregs in the microenvironment of PDAC went along with a markedly decreased interaction between CD8+ T cells and cancerous epithelial cells (ECs), highlighting the importance of Tregs as key players in immunosuppression in PDAC. We found evidence that a defect in antigen presentation, further aggravated by PD-L1 expression on APC, may contribute to immunosuppression in IAPA, suggesting a role for PD-L1/PD-1 immune checkpoint inhibitors in the treatment of IAPA.

Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation.

Co-inhibitory and checkpoint molecules suppress T cell function in the tumor microenvironment, thereby rendering T cells dysfunctional. Although immune checkpoint blockade is a successful treatment option for multiple human cancers, severe autoimmune-like adverse effects can limit its application. Here, we show that the gene encoding peptidoglycan recognition protein 1 (PGLYRP1) is highly coexpressed with genes encoding co-inhibitory molecules, indicating that it might be a promising target for cancer immunotherapy. Genetic deletion of Pglyrp1 in mice led to decreased tumor growth and an increased activation/effector phenotype in CD8+ T cells, suggesting an inhibitory function of PGLYRP1 in CD8+ T cells. Surprisingly, genetic deletion of Pglyrp1 protected against the development of experimental autoimmune encephalomyelitis, a model of autoimmune disease in the central nervous system. PGLYRP1-deficient myeloid cells had a defect in antigen presentation and T cell activation, indicating that PGLYRP1 might function as a proinflammatory molecule in myeloid cells during autoimmunity. These results highlight PGLYRP1 as a promising target for immunotherapy that, when targeted, elicits a potent antitumor immune response while protecting against some forms of tissue inflammation and autoimmunity.

Biomaterial-Mediated Genetic Reprogramming of Merkel Cell Carcinoma and Melanoma Leads to Targeted Cancer Cell Killing In Vitro and In Vivo.

Tumor immunotherapy is a promising anticancer strategy; however, tumor cells may employ resistance mechanisms, including downregulation of major histocompatibility complex (MHC) molecules to avoid immune recognition. Here, we investigate reprogramming nanoparticles (NPs) that deliver immunostimulatory genes to enhance immunotherapy and address defective antigen presentation in skin cancer in vitro and in vivo. We use a modular poly(beta-amino ester) (PBAE)-based NP to deliver DNA encoding 4-1BBL, IL-12, and IFNγ to reprogram human Merkel cell carcinoma (MCC) cells in vitro and mouse melanoma tumors in vivo to drive adaptive antitumor immune responses. Optimized NP formulations delivering 4-1BBL/IL-12 or 4-1BBL/IL-12/IFNγ DNA successfully transfect MCC and melanoma cells in vitro and in vivo, respectively, resulting in IFNγ-driven upregulation of MHC class I and II molecules on cancer cells. These NPs reprogram the tumor immune microenvironment (TIME) and elicit strong T-cell-driven immune responses, leading to cancer cell killing and T-cell proliferation in vitro and slowing tumor growth and improving survival rates in vivo. Based on expected changes to the tumor immune microenvironment, particularly the importance of IFNγ to the immune response and driving both T-cell function and exhaustion, next-generation NPs codelivering IFNγ were designed. These offered mixed benefits, exchanging improved polyfunctionality for increased T-cell exhaustion and demonstrating higher systemic toxicity in vivo. Further profiling of the immune response with these NPs provides insight into T-cell exhaustion and polyfunctionality induced by different formulations, providing a greater understanding of this immunotherapeutic strategy.

Dendritic Cell Vaccination in Non-Small Cell Lung Cancer: Remodeling the Tumor Immune Microenvironment.

Non-small-cell lung cancer (NSCLC) remains one of the leading causes of death worldwide. While NSCLCs possess antigens that can potentially elicit T cell responses, defective tumor antigen presentation and T cell activation hinder host anti-tumor immune responses. The NSCLC tumor microenvironment (TME) is composed of cellular and soluble mediators that can promote or combat tumor growth. The composition of the TME plays a critical role in promoting tumorigenesis and dictating anti-tumor immune responses to immunotherapy. Dendritic cells (DCs) are critical immune cells that activate anti-tumor T cell responses and sustain effector responses. DC vaccination is a promising cellular immunotherapy that has the potential to facilitate anti-tumor immune responses and transform the composition of the NSCLC TME via tumor antigen presentation and cell-cell communication. Here, we will review the features of the NSCLC TME with an emphasis on the immune cell phenotypes that directly interact with DCs. Additionally, we will summarize the major preclinical and clinical approaches for DC vaccine generation and examine how effective DC vaccination can transform the NSCLC TME toward a state of sustained anti-tumor immune signaling.

Single-cell RNA sequencing reveals the transcriptomic characteristics of peripheral blood mononuclear cells in hepatitis B vaccine non-responders.

The emergence of a vaccine against hepatitis B has proven to be an important milestone in the prevention of this disease; however, 5%-10% of vaccinated individuals do not generate an immune response to the vaccine, and its molecular mechanism has not been clarified. In this study, single-cell RNA sequencing was performed on peripheral blood mononuclear cells (PBMCs) from three volunteers with a high immune response (HR) and three with no immune response (NR) to the hepatitis B vaccine. We found that the antigen-presenting activity scores of various antigen-presenting cells, the mitogen-activated protein kinase (MAPK) pathway activity scores of naive B cells, and the cell activity scores of three types of effector T cells were significantly decreased, whereas the cytotoxicity scores of CD3highCD16lowKLRG1high natural killer T (NKT) cells were significantly increased in the NR group compared with those in the HR group. Additionally, the expression levels of some classical molecules associated with distinct signaling pathways-including HLA-B, HLA-DRB5, BLNK, BLK, IL4R, SCIMP, JUN, CEBPB, NDFIP1, and TXNIP-were significantly reduced in corresponding subsets of PBMCs from the NR group relative to those of the HR group. Furthermore, the expression of several cytotoxicity-related effector molecules, such as GNLY, NKG7, GZMB, GZMM, KLRC1, KLRD1, PRF1, CST7, and CTSW, was significantly higher in CD3highCD16lowKLRG1high NKT cells derived from non-responders. Our study provides a molecular basis for the lack of response to the hepatitis B vaccine, including defective antigen presentation, decreased T cell activity, and reduced IL-4 secretion, as well as novel insight into the role of NKT cells in the immune response to the hepatitis B vaccine.

Defects in lysosomal function and lipid metabolism in human microglia harboring a TREM2 loss of function mutation

TREM2 is an innate immune receptor expressed by microglia in the adult brain. Genetic variation in the TREM2 gene has been implicated in risk for Alzheimer’s disease and frontotemporal dementia, while homozygous TREM2 mutations cause a rare leukodystrophy, Nasu-Hakola disease (NHD). Despite extensive investigation, the role of TREM2 in NHD pathogenesis remains poorly understood. Here, we investigate the mechanisms by which a homozygous stop-gain TREM2 mutation (p.Q33X) contributes to NHD. Induced pluripotent stem cell (iPSC)-derived microglia (iMGLs) were generated from two NHD families: three homozygous TREM2 p.Q33X mutation carriers (termed NHD), two heterozygous mutation carriers, one related non-carrier, and two unrelated non-carriers. Transcriptomic and biochemical analyses revealed that iMGLs from NHD patients exhibited lysosomal dysfunction, downregulation of cholesterol genes, and reduced lipid droplets compared to controls. Also, NHD iMGLs displayed defective activation and HLA antigen presentation. This defective activation and lipid droplet content were restored by enhancing lysosomal biogenesis through mTOR-dependent and independent pathways. Alteration in lysosomal gene expression, such as decreased expression of genes implicated in lysosomal acidification (ATP6AP2) and chaperone mediated autophagy (LAMP2), together with reduction in lipid droplets were also observed in post-mortem brain tissues from NHD patients, thus closely recapitulating in vivo the phenotype observed in iMGLs in vitro. Our study provides the first cellular and molecular evidence that the TREM2 p.Q33X mutation in microglia leads to defects in lysosomal function and that compounds targeting lysosomal biogenesis restore a number of NHD microglial defects. A better understanding of how microglial lipid metabolism and lysosomal machinery are altered in NHD and how these defects impact microglia activation may provide new insights into mechanisms underlying NHD and other neurodegenerative diseases.

Abstract OT2-17-01: Phase 1 trial of anthracycline chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the DC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and also repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal-doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal-doxorubicin, CDX-1140 (CD40 agonist), and CDX-301 (Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal-doxorubicin only) for 1 cycle prior to receiving triplet therapy with fresh tissue biopsies before and after the lead-in treatment. CDX-301 will be discontinued after 2 cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase 2 dose based on treatment-related adverse events including dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal-doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line metastatic treatment setting (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999). Citation Format: Sangeetha Reddy, Meredith Carter, Isaac Chan, Nisha Unni, Namrata Peswani, Dawn Klemow, Samira Syed, Shahbano Shakeel, Farjana Fattah, Chul Ahn, Yisheng Fang, Heather McArthur, Nicole Sinclair, Michael Yellin, Denise Yardley, Nan Chen, Joyce O’Shaughnessy, Rita Nanda, Suzanne D. Conzen, Carlos Arteaga. Phase 1 trial of anthracycline chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-17-01.

Abstract P2-20-03: Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model

Abstract Background: Intrinsic resistance to immunotherapy observed in breast cancer is attributed to low neoantigen levels, defective antigen presentation, low mutational burden, reduced programmed death ligand (PD-L1) expression and the presence of immunosuppressive signals like transforming growth factor-beta (TGFβ) in the tumor microenvironment (TME). These collectively attenuate the effector functions of T cells and natural killer cells (NK cells). NK cells are an important component of innate immunity. While generally TGFβ dampens the NK cell response in the breast cancer TME, chronic stimulation in vivo with TGFβ during the expansion process of donor NK cells produces TGFβ imprinted NK cells which exhibit high cytotoxicity and resistance to suppression by TGFβ due to down regulation of SMAD3 and hypersecretion of interferon gamma and tumor necrosis factor alpha. NK cell activation also promotes antibody dependent cellular cytotoxicity (ADCC) and augments monoclonal antibody directed killing. GD2 is a disialoganglioside and a tumor-associated antigen with limited expression in healthy tissues and overexpression in a variety of cancers, including breast cancer. Up to 60% of high grade breast cancers have GD2 expression with higher expression linked to worse overall survival. Given GD2 expression in aggressive BC subtypes and the role of NK cells in ADCC and antigen presentation, there is a strong rationale for evaluating an anti-GD2 antibody along with NK cells in the immunosuppressive breast TME. Based upon established preclinical data, we hypothesized that combining (1) an IgG1 anti-GD2 monoclonal antibody (anti-GD2), (2) TGFβ imprinted, IL-21 expanded allogenic universal donor NK cells (TGFβiNK) and (3) the standard chemotherapy agent gemcitabine (gem) which has immunomodulatory effects including improved NK cell function, will improve tumor control and survival in a TNBC mouse model. Methods: First, we evaluated if anti-GD2 treatment would increase ADCC against MD-MBA-231 tumor cells. To do this, tumor cells with or without anti-GD2 were cocultured with TGFβiNK + anti-GD2 and evaluated by real time cell analysis (RTCA). Next we evaluated the effect of gem on both tumor and immune cells. Finally to evaluate the effect of our novel combination therapy, female NSG mice were injected with 1 × 10e6 MDA-MB-231 cells into the mammary fat on day 0 and tumor burden was followed for 83 days. On day 21, mice were divided into 4 groups: control, gem only (standard of care), TGFβiNK+anti-GD2 (NK-GD2), and gem+TGFβiNK+anti-GD2 (G-NK-GD2) and tumor growth was measured weekly. Mice were observed for toxicity and bioluminescence evaluation for NK cells were performed prior to each cycle. Results: RTCA data confirmed anti-GD2 treatment increased ADCC mediated cell lysis by TGFβiNK. Furthermore, at clinically relevant doses, gem did not alter GD2 expression by flow cytometry on MDA-MB-231 tumor cells but induced MICA expression which suggests potential improved NK cell activity. In the mouse model, monitoring of mouse weights showed greater weight stability in the G-NK-GD2 group, suggesting a tolerable regimen. Serial measurement of tumors showed a significant reduction in tumor growth in NK-GD2 and G-NK-GD2 arms when compared to Gem or control. Additionally, mice who received G-NK-GD2 or NK-GD2 had significantly improved survival. Histological evaluation and immune assessment of the mice are ongoing. Conclusion: Our data showed no significant toxicities in the murine model and improved efficacy of G-NK-GD2 over the standard of care gemcitabine. Our preclinical data supports moving forward with a phase 1b/2 Bench-To-Bedside clinical trial, that will evaluate this combination in patients with aggressive subtypes of metastatic breast cancer where there is a great clinical need for effective treatments. Citation Format: Marcelo Pereira, Prashant Trikha, Sumithira Vasu, Zihai Li, Daniel Stover, Dean Lee, Margaret Gatti-Mays. Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-03.

A Novel Tongue Squamous Cell Carcinoma Cell Line Escapes from Immune Recognition due to Genetic Alterations in HLA Class I Complex.

Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8+ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)-a component of all HLA class I complex allotypes-was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins' translation and inhibited the activation and killing effect of CD8+ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8+ T cells.

733MO γδT cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

DNA mismatch repair deficient (MMR-d) cancers present an abundance of neoantigens that likely underlies their exceptional responsiveness to immune checkpoint blockade (ICB). Early evidence has indicated that MMR-d cancers that evade CD8+ T cells through loss of Human Leukocyte Antigen (HLA) class I-mediated antigen presentation still respond to ICB, suggesting compensatory responsiveness of other immune effector cells in this context.

The immune phenotypes and different immune escape mechanisms in colorectal cancer.

The tumor microenvironment (TME) plays a crucial role in tumor progression and metastasis. However, the immune phenotypes of colorectal cancer (CRC) and the underlying immune escape mechanism have not been studied sufficiently. A total of 1802 and 619 CRC samples from the microarray and TCGA cohorts were enrolled, respectively. The ssGSEA algorithm and unsupervised clustering were used for TME cell infiltration speculation and immune phenotype recognition in the above cohorts. A total of 447 samples from Zhongshan Hospital were collected for validation. Immunohistochemistry was performed in this cohort to quantify TME cell infiltration. The single-cell RNA-seq (scRNA-seq) data of 252,940 cells from 60 CRC samples was analyzed for further mechanistic exploration. CRC samples can be classified into three distinct immune phenotypes. Subtype 1, the immune-active subtype, was characterized by high infiltration of activated adaptive immune cells. Subtype 2, the immune-desert subtype, featured high tumor purity and low infiltration of immune and stromal cells. Subtype 3, the stroma-rich subtype, had high infiltration of stromal cells. The stroma-rich subtype conferred a significantly worse prognosis. The three subtypes had different immune escape mechanisms. The immune-active subtype has the highest immune checkpoint expression level. In comparison, the immune-desert subtype had the lowest immunogenicity and defective antigen presentation. The stroma-rich subtype lacked activated immune cells. In conclusion, distinct immune phenotypes and immune escape mechanisms may provide inspiration and direction for further research on CRC immunotherapy.

Geospatial Immune Heterogeneity Reflects the Diverse Tumor–Immune Interactions in Intrahepatic Cholangiocarcinoma

This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies. This article is highlighted in the In This Issue feature, p. 2221.

Multimodule characterization of immune subgroups in intrahepatic cholangiocarcinoma reveals distinct therapeutic vulnerabilities

BackgroundImmune microenvironment is well recognized as a critical regulator across cancer types, despite its complex roles in different disease conditions. Intrahepatic cholangiocarcinoma (iCCA) is characterized by a tumor-reactive milieu, emphasizing...

Abstract 4197: Treatment with chemotherapy, CD40 agonist, and Flt3 ligand triplet combination enhances antigen presentation and leads to cures in triple negative breast cancer

Abstract Introduction: Triple negative breast cancers (TNBC) have shown limited responses to immune checkpoint blockade (ICB). Breast cancer is associated with defects in antigen presentation which may contribute to resistance to ICB. Flt3 ligand (Flt3L) is a growth factor that increases differentiation of DC1 dendritic cells, critical mediators of antigen presentation. CD40 agonist activates all 3 classes of antigen presenting cells - dendritic cells, B cells, and macrophages. Synergy has been demonstrated between Flt3L and CD40 agonist as well as between CD40 agonist and chemotherapy in other cancers, however the combination of all three has not been studied in breast cancer. Methods: 6-8 week old Balb/c mice were injected with 4T1 triple negative breast cancer cells. When tumors were 50 mm3, mice were first treated with intraperitoneal pegylated liposomal doxorubicin (PLD) once and subcutaneous Flt3L daily for 5 days in different sequencing schedules (PLD first followed by Flt3L immediately or 4 days later or Flt3L first followed by PLD 7 days or 10 days later). Based on optimal scheduling of PLD and Flt3L, mice were treated with PLD alone, Flt3L alone, CD40 agonist alone, PLD + Flt3L, PLD + CD40 agonist, PLD + PD-1 blockade, or PLD + CD40 agonist + Flt3L. In addition to serial tumor measurements, mice were sacrificed and tumors and lymphoid organs harvested for flow cytometry analyses. Results: Treatment with PLD at least 4 days before Flt3L led to an optimal increase in intra-tumoral DC1 cells (p=0.0002), increase in polyfunctional CD8 T cell response (p<0.0001), reduced PD-L1 expression on DC1 cells (p<0.0001), and reduced PD-1 expression on CD8 T cells (p<0.0001) compared to other sequencing schedules. Therefore, this regimen was advanced for further study. Treatment with novel triplet combination of PLD, Flt3L, and CD40 agonist resulted in improved tumor control and survival compared to PLD alone (p<0.0001 and p<0.0001, respectively), Flt3L alone (p<0.0001 and p<0.0001), CD40 agonist alone (p <0.0001 and p<0.0001), PLD + Flt3L (p<0.0001 and p<0.0001), PLD + CD40 agonist (p<0.0001 and p<0.0001), and PLD + PD-1 blockade (p<0.0001 and p<0.0001). 22% of mice were tumor-free in the triplet therapy group only and were resistant to tumor re-challenge. Treatment with triplet therapy was associated with an increase in antigen specific CD8 T cells. Additional mechanistic studies will also be presented. Conclusions: Novel triplet combination with PLD, CD40 agonist, and Flt3L leads to enhanced tumor control in the 4T1 TNBC mouse model. A clinical trial with this combination in metastatic TNBC patients is expected to begin recruitment soon (NCT05029999). Citation Format: Vijay Ramani, Shruthi Nooka, Yu-An Zhang, Serena Gibbs, Hai-Cheng Huang, Melanie Hullings, Suzanne Conzen, Carlos L. Arteaga, Isaac Chan, Sangeetha M. Reddy. Treatment with chemotherapy, CD40 agonist, and Flt3 ligand triplet combination enhances antigen presentation and leads to cures in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4197.

Abstract 4201: Immune priming with low intensity focused ultrasound and radiation therapy in murine breast cancer

Abstract Introduction: Our long-term goal is to use focal therapy, such as radiation therapy (RT) and ultrasound, to induce stress and engineer the tumor microenvironment (TME) to drive a systemic anti-tumoral immunity, while achieving primary tumor control. Current RT-induced systemic anti-tumoral immunity is dampened by defective tumor antigen presentation resulting in immune escape, an immunosuppressive TME, and the presence of anergic T cells in the immune privileged tumor draining lymph nodes. We have previously developed a proprietary non-ablative low intensity focused ultrasound (LOFU) therapy that induces unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in LOFU-treated cells and reverses T cell anergy in draining lymph nodes in a mouse model of metastatic melanoma. To further extend our findings to other solid tumors, we hypothesized that a combination of acoustic stress and immune priming by non-ablative LOFU, followed by ablative RT will promote the release of heat shock proteins and other damage-associated molecular pattern (DAMP) factors along with tumor-associated antigens for launching a systemic anti-tumor response in a murine breast cancer (TSA). Methods: TSA cells and palpable orthotopic mammary tumors in BALB/c thymic and athymic nude mice were treated with varying parameters of multi-frequency LOFU and RT (8-10 Gy/fraction), over 1-3 consecutive days. Cell surface immunomodulation of TSA cells and tumor infiltrating immune cell populations were analyzed with flow cytometry and tumor volumes were recorded to calculate growth delay along with survival. Results: LOFU-treated TSA cells exhibited an increase in cell surface chaperones, BiP/GRP78, calreticulin, HSP70 and HSP90 at 24 hours post treatment. Furthermore, there was increased secretion of HMGB1, an immunomodulatory DAMP activator for dendritic cells. LOFU+RT-treated tumors had higher numbers of GP70 tetramer-positive CD8+ T cells by day 10 with higher levels of Ki67+ and GzB+ markers. In a dual tumor abscopal model, we observed significantly lower average tumor volumes of both the primary treated, and secondary untreated tumors, 35 days post treatment. There was a trend towards better tumor control and survival in mice with tumors treated with LOFU+RT, when compared to RT alone. Parallel studies in athymic immunodeficient mice failed to regress tumors treated with LOFU+RT. Conclusions: LOFU + RT poses to be a promising combination therapy not only for elimination of primary tumor tissue but also the induction of a tumor-specific immune response. Further optimization of LOFU parameters is underway to maximize the immunogenic effects of LOFU+RT for combination therapy with immunotherapy. Citation Format: Michelle M. Schumacher, Claudia Gutierrez Chavez, Saurabh Singh, Chandra Karunakaran, Sanjay Pandey, Mayank Kumar, Rodney Macedo Gonzales, Chandan Guha. Immune priming with low intensity focused ultrasound and radiation therapy in murine breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4201.