IMMU-13. COMBINED MODULATION OF THE TGF-Β PATHWAY AND GITR IMMUNE CHECKPOINT SIGNALING PROMOTES ANTI-TUMOR IMMUNITY IN SYNGENEIC GLIOMA MODELS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi94-vi94
Author(s):  
Daniela Lorizio ◽  
Michael Weller ◽  
Manuela Silginer ◽  
Alan Epstein ◽  
Patrick Roth
Keyword(s):  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Transforming Growth Factor ◽  
Immune Cell ◽  
Single Agent ◽  
Underlying Mechanism ◽  
T Effector Cells ◽  
Promising Therapeutic Approach

Abstract The profound local immunosuppressive microenvironment is one hallmark of glioblastoma, which results in resistance to most immunotherapeutic strategies that have been explored so far. Reverting this condition in order to reinvigorate anti-glioma immunity might be a promising therapeutic approach. Transforming growth factor (TGF)-β signaling is deregulated in different cancer types and contributes to the malignant phenotype of glioma cells. Glioma-derived TGF-β is also a major immunosuppressive factor in the tumor microenvironment. Furthermore, intratumoral regulatory T (Treg) cells and activated T effector cells express high levels of the co-stimulatory immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR). Agonistic anti-GITR antibodies have been explored in preclinical tumor models and are under investigation in clinical trials for the treatment of solid tumors. We evaluated the effect of TGF-β and GITR targeting on anti-tumor immune responses in syngeneic mouse glioma models. In co-culture settings, GITR modulation with a GITR ligand (GITRL)-Fc fusion protein, given alone or in combination with a pharmacological TGF-β receptor inhibitor, led to increased T cell activation. Furthermore, the combined targeting of the two pathways resulted in significantly higher immune cell-mediated tumor cell killing than either treatment alone. In vivo, TGF-β inhibition and GITR signaling modulation resulted in a higher fraction of long-term surviving glioma-bearing mice than single-agent treatment. Surviving mice were resistant to tumor re-challenge, suggesting adaptive immunity as an underlying mechanism. These data support the assumption that combined immunotherapeutic strategies may represent a promising approach for the treatment of glioma.

EP.WE.805From bench to bedside; A rationale for Immunotherapy in the adjuvant setting for Oesophageal cancer

2021 ◽  
Vol 108 (Supplement_7) ◽  
Author(s):  
Noel Donlon ◽  
Maria Davern ◽  
Andrew Sheppard ◽  
John Reynolds ◽  
Joanne Lysaght
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Single Agent ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Post Surgery ◽  
Post Operative Period

Abstract Background Immunotherapy is being intensively investigated for its utilisation in the curative setting as a single agent and in the multimodal setting, however, the most appropriate time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumour immunity perioperatively to provide a rationale for adjuvant immunotherapy. Methods Systemic immunity was immunophenotyped pre and post-oesophagectomy on days 0, 1, 3, 7 and week 6 by flow cytometry (n = 14). The frequency of circulating lymphocytes, T cells, cytotoxic and helper T lymphocytes was profiled longitudinally including the proportion of T cell subsets in circulation. This study also profiled immune checkpoint expression on circulating T cells including: PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, PD-L1 and PD-L2. Markers of immunogenicity (calreticulin, HMGB1 and MIC-A/B) were also assessed. Results The frequency of circulating CD27 + T cells increases sequentially in the immediate post-operative period peaking on day 7 in OAC patients. (p < 0.01) There is a sequential decrease in the percentage of effector memory and central memory T cells in circulation and an increase in the percentage of naïve T cells in peripheral circulation of OAC patients in the immediate post-operative period. The expression of CTLA-4 on the surface of circulating CD4 + T cells decreases 6 weeks post-operatively in OAC patients. Conclusions We observed increased T cell activation and immune checkpoints immediately post-surgery with returns to baseline by week 6. These results suggest that immune checkpoint inhibitors such as anti-PD-1 may be beneficial immediately post-surgery to maintain T cell activation and prevent exhaustion of this increased population of activated T cells observed immediately post-surgery.

scholarly journals Acidosis-Induced TGF-β2 Production Promotes Lipid Droplet Formation in Dendritic Cells and Alters Their Potential to Support Anti-Mesothelioma T Cell Response

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1284
Author(s):  
Natalia Trempolec ◽  
Charline Degavre ◽  
Bastien Doix ◽  
Davide Brusa ◽  
Cyril Corbet ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Lipid Droplet ◽  
T Cell Activation ◽  
Cell Response ◽  
Cell Activation ◽  
Transforming Growth Factor ◽  
T Cell Response ◽  
Mesenchymal Transition

For poorly immunogenic tumors such as mesothelioma there is an imperious need to understand why antigen-presenting cells such as dendritic cells (DCs) are not prone to supporting the anticancer T cell response. The tumor microenvironment (TME) is thought to be a major contributor to this DC dysfunction. We have reported that the acidic TME component promotes lipid droplet (LD) formation together with epithelial-to-mesenchymal transition in cancer cells through autocrine transforming growth factor-β2 (TGF-β2) signaling. Since TGF-β is also a master regulator of immune tolerance, we have here examined whether acidosis can impede immunostimulatory DC activity. We have found that exposure of mesothelioma cells to acidosis promotes TGF-β2 secretion, which in turn leads to LD accumulation and profound metabolic rewiring in DCs. We have further documented how DCs exposed to the mesothelioma acidic milieu make the anticancer vaccine less efficient in vivo, with a reduced extent of both DC migratory potential and T cell activation. Interestingly, inhibition of TGF-β2 signaling and diacylglycerol O-acyltransferase (DGAT), the last enzyme involved in triglyceride synthesis, led to a significant restoration of DC activity and anticancer immune response. In conclusion, our study has identified that acidic mesothelioma milieu drives DC dysfunction and altered T cell response through pharmacologically reversible TGF-β2-dependent mechanisms.

scholarly journals IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Marilin Koch ◽  
Mykola Zdioruk ◽  
M Oskar Nowicki ◽  
Estuardo Aguilar ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Symptomatic Treatment ◽  
Tumor Cell Death ◽  
The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

A phase III comparative study of nivolumab (anti-PD-1; BMS-936558; ONO-4538) versus docetaxel in patients with previously treated advanced or metastatic squamous cell non-small cell lung cancer (NSCLC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. TPS8122-TPS8122 ◽  
Author(s):  
Hossein Borghaei ◽  
Thomas James Lynch ◽  
Naiyer A. Rizvi ◽  
Laura Quan Man Chow ◽  
Robert Reilly ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Squamous Cell ◽  
Immune Checkpoint ◽  
Clinical Benefit ◽  
Cell Activation ◽  
Tumor Regression ◽  
Phase 1 ◽  
Single Agent ◽  
Phase Iii ◽  
Symptom Improvement

TPS8122 Background: NSCLC is the leading cause of cancer death worldwide, exceeding breast, colon and prostate cancer combined. Most patients (pts) are diagnosed with advanced/recurrent disease. NSCLC therapies have incrementally improved overall survival (OS), but benefit has reached a plateau (median OS for late stage pts is just 1 yr). Squamous cell carcinoma (SCC) represents one quarter of NSCLC cases and has limited treatment options. Pemetrexed and bevacizumab are not approved in SCC, and molecularly targeted therapies have limited application. Single-agent chemotherapy is standard of care following progression with platinum-based doublet chemotherapy (Pt-doublet), resulting in median OS of approximately 7 months. Immune checkpoint blockade in NSCLC may promote tumor regression by reversing tumor-induced immunosuppression and restoring the antitumor immune response. Programmed death-1 (PD-1), an immune checkpoint receptor that negatively regulates T-cell activation, is upregulated in tumor infiltrating lymphocytes; expression of its ligand PD-L1 has been associated with poor prognosis in NSCLC. Nivolumab, a PD-1 receptor blocking antibody, showed encouraging antitumor activity in SCC pts in a phase 1 study, with objective responses (ORs) in 4 pts (27%) and stable disease ≥24 weeks in 1 pt (7%) (3 mg/kg; n=15) (Gettinger S et al; ESMO 2012 [Abstr 1237PD]). We describe an ongoing open-label phase 3 study to compare the clinical benefit of nivolumab vs docetaxel in advanced/metastatic SCC pts following failure of Pt-doublet. Methods: A total of 264 pts will be randomized 1:1 to nivolumab monotherapy 3 mg/kg IV every 2 weeks or docetaxel 75 mg/m2 every 3 weeks until disease progression or unacceptable toxicity. Pts will be stratified by prior paclitaxel use and geographic region. Tumor response will be assessed using modified RECIST 1.1. The co-primary objectives are OR rate and OS. Secondary objectives include progression free survival, clinical benefit in PD ligand 1 (PD-L1+) and PD-L1– subgroups (archived tissue required for entry), durability of and time to OR, and pt proportion with symptom improvement. Clinical trial information: NCT01642004.

scholarly journals Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

2021 ◽  
Vol 9 ◽  
Author(s):  
Juan Yang ◽  
Xianzhi Yang ◽  
Wenfeng Pan ◽  
Mingshuo Wang ◽  
Yuxiong Lu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Progression ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Synergistic Effects ◽  
Immune Checkpoint Blockade ◽  
Stat Pathway

Immune checkpoint blockade (ICB) therapies such as PD-1 antibodies have produced significant clinical responses in treating a variety of human malignancies, yet only a subset of cancer patients benefit from such therapy. To improve the ICB efficacy, combinations with additional therapeutics were under intensive investigation. Recently, special dietary compositions that can lower the cancer risk or inhibit cancer progression have drawn significant attention, although few were reported to show synergistic effects with ICB therapies. Interestingly, Fucoidan is naturally derived from edible brown algae and exhibits antitumor and immunomodulatory activities. Here we discover that fucoidan-supplemented diet significantly improves the antitumor activities of PD-1 antibodies in vivo. Specifically, fucoidan as a dietary ingredient strongly inhibits tumor growth when co-administrated with PD-1 antibodies, which effects can be further strengthened when fucoidan is applied before PD-1 treatments. Immune analysis revealed that fucoidan consistently promotes the activation of tumor-infiltrating CD8+ T cells, which support the evident synergies with ICB therapies. RNAseq analysis suggested that the JAK-STAT pathway is critical for fucoidan to enhance the effector function of CD8+ T cells, which could be otherwise attenuated by disruption of the T-cell receptor (TCR)/CD3 complex on the cell surface. Mechanistically, fucoidan interacts with this complex and augments TCR-mediated signaling that cooperate with the JAK-STAT pathway to stimulate T cell activation. Taken together, we demonstrated that fucoidan is a promising dietary supplement combined with ICB therapies to treat malignancies, and dissected an underappreciated mechanism for fucoidan-elicited immunomodulatory effects in cancer.

159 METFORMIN DOWNREGULATES PD-L1 EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA BY INHIBITING IL-6 SIGNALING PATHWAY

2021 ◽  
Vol 34 (Supplement_1) ◽  
Author(s):  
Feng Wang ◽  
Dao Xin ◽  
Xiangrui Meng ◽  
Qingxia Fan
Keyword(s):  
T Cell ◽  
Signaling Pathway ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Antitumor Effects ◽  
Killing Assay

Abstract   ESCC is an aggressive cancer with poor prognosis in China. Metformin with low cost and toxicity have proved to have anti-cancer effects in many kinds of cancers, while its role in combination of immune checkpoint blockade and mechanism in ESCC has seldom been studied. This study was designed to evaluate the potential role of combinatorial anti-tumor effect with metformin and immunomodulatory monoclonal antibodies (mAbs) targeting programmed cell death protein-1 (PD-1) in vitro and in vivo. Methods Reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemistry (IHC) assays were used to study the effects of metformin on the expression levels of PD-L1. In addition, T cell activation and killing assay were performed in the co-culture system of ESCC cell and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6 to evaluate the function of inhibiting PD-L1 expression. In vivo assay, we used NPIdKOTM mice model, which was re-constructed replaced immune system by transplanting PBMCs through intravenously injecting. Mice were treated with metformin alone as well as in combination with anti-PD-1 mAbs. Results PD-L1 expression were downregulated by metformin via JAK2 and STAT3 signaling pathway. The results showed that metformin promoted CD3/CD28 induced ERK phosphorylation at a dose dependent measure. And IL-2 was increased in the supernatant of KYSE-450/PBMC coculture system after metformin treatment. Both of which are indicators of T cell activation. T cell mediated tumor killing assay showed that metformin inducing PD-L1 downregulation could protect T cell function, and it could be reversed by IL-6. It showed that metformin enhanced its efficacy in TE-7 cells-harboring mice in the combination treatment group, reflected by tumor sizes assay, and in vivo imaging system. Conclusion Various approaches are under way to expand the benefits and improve the efficacy of these immune checkpoint inhibitors. Another approach is combining immunotherapy with existing anticancer therapies. In this regard, we have evaluated the mechanism of metformin inhibiting PD-L1 expression in ESCC. This finding indicated that metformin significantly improved the antitumor effects by anti-PD-1 blockade without detectable toxicity and suggested that metformin has strong potential to be used in combination with immunotherapy.

scholarly journals 886 Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A928-A928
Author(s):  
Steve Sazinsky ◽  
Phuong Nguyen ◽  
Mohammad Zafari ◽  
Ryan Phennicie ◽  
Joe Wahle ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immune Cell ◽  
Ex Vivo ◽  
Syngeneic Mouse ◽  
Pathogen Clearance

BackgroundVSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism.1–3 VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion.3–6 Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response.MethodsThe ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce T cell activation was assessed in vitro and ex vivo, by measuring production of inflammatory mediators. In vitro assays were performed primarily with M-CSF plus IL-10 driven monocyte-derived M2c macrophages from healthy donors. Ex vivo assays were performed with fresh, patient-derived tumor samples in culture. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies and characterized for changes in tumor volume and immune cell populations.ResultsIn in vitro and ex vivo assays anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Targeting VSIG4 upregulates pro-inflammatory cytokines in M2c macrophages, as well as upregulates pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines in M2c macrophages co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation. To assess targeting VSIG4 in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). In vivo, tumor growth inhibition is observed in syngeneic mouse models dosed with anti-mouse-VSIG4 alone and in combination with anti-PD-1.ConclusionsTaken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms.Referencesvan Lookeren Campagne M, Verschoor A. Pathogen clearance and immune adherence “revisited”: immuno-regulatory roles for CRIg. Semin Immunol 2018;37:4–11.Xu S, Sun Z, Li L, Liu J, He J, Song D, Shan G, Liu H, Wu X. Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus. Immunol Lett 2010;128(1):46–50.Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, Fei L, Ni B, Ruan Z. VSIG4 expression on macrophages facilitates lung cancer development. Lab Invest 2014;94(7):706–715.Roh J, Jeon Y, Lee A, Lee S, Kim Y, Sung C, Park C, Hong J, Yoon D, Suh C, Huh J, Choi I, Park C. The immune checkpoint molecule V-set Ig domain-containing 4 is an independent prognostic factor for multiple myeloma. Oncotarget 2017;8(35):58122–58132.Xu T, Jiang Y, Yan Y, Wang H, Lu C, Xu H, Li W, Fu D, Lu Y, Chen J. VSIG4 is highly expressed and correlated with poor prognosis of high-grade glioma patients. Am J Transl Res 2015;7(6):1172–1180.Byun J, Jeong D, Choi I, Lee D, Kang M, Jung K, Jeon Y, Kim Y, Jung E, Lee K, Sung M, Kim K. The significance of VSIG4 expression in ovarian cancer. Int J Gynecol Cancer 2017;27(5):872–878.Ethics ApprovalAll legal and ethical requirements were met with regards to the humane treatment of animals described in the study. The animal study was conducted in compliance with CRL IACUC under IACUC No. I033.

scholarly journals Targeted Desialylation Overcomes Glyco-Immune Checkpoints and Potentiates the Anticancer Immune Response in Vivo

2019 ◽  
Author(s):  
Melissa Gray ◽  
Michal A. Stanczak ◽  
Han Xiao ◽  
Johan F. A. Pijnenborg ◽  
Stacy A. Malaker ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Immune Cell ◽  
Immune Therapy ◽  
Treatment Options ◽  
Immune Checkpoints ◽  
Alternative Pathways ◽  
Her2 Breast Cancer

<div><div><div><p>Currently approved immune checkpoint inhibitor (ICI) therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, the majority of patients across cancer types still fail to respond. Addressing alternative pathways that mediate immune suppression could enhance ICI efficacy. One such mechanism is the increase in sialic acid-containing proteins and lipids (sialoglycans) in malignancy, which recently has been shown to inhibit immune cell activation through multiple mechanisms including Siglec receptor binding, and therefore represents a targetable glyco-immune checkpoint. Here, we report the design of a trastuzumab- sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells in vivo. In a syngeneic orthotopic HER2+ breast cancer model, targeted desialylation delayed tumor growth and enhanced immune cell infiltration and activation, leading to prolonged survival of mice with trastuzumab-resistant breast cancer. Thus, antibody-sialidase conjugates represent a promising modality for cancer immune therapy.</p></div></div></div>

CTLA4 and CD47 combinational therapy to extend survival in melanoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e21025-e21025
Author(s):  
Anthony L. Schwartz ◽  
Pulak Nath ◽  
Elizabeth Lessey-Morillon ◽  
Lisa Ridnour ◽  
Michael Allgaeuer ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Cell Activation ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Granzyme B ◽  
Suppressor Cells ◽  
Cell Infiltration

e21025 Background: Irradiation (IR) combined with chemotherapy is the post-surgical standard of care treatment for melanoma, but metastasis still results in high mortality rates. Immune checkpoint inhibitors such as cytotoxic T-lymphocyte antigen-4 (CTLA4) have proven effective for immunotherapy of melanoma. CTLA-4 is up-regulated post-T cell activation and blockade enhances tumor responses in immunocompetent rodents and humans. Trials suggest that combinations of immune checkpoint inhibitors are more efficacious than single agents, but tumors remain resistant. We are investigating CD47 blockade for the treatment of cancer. CD47 is frequently elevated in cancers and serves as an inhibitory receptor for thrombospondin-1 on immune cells in the tumor stroma. CD47 blockade on CD8 T or tumor cells significantly enhances immune-targeted tumor cell killing post-IR compared to IR alone. Here we explore the potential for antisense CD47 and anti-CTLA4 therapy alone or in combination with IR using a syngeneic mouse melanoma model. Methods: C57BL/6 mice were inoculated with 1x106B16F10 melanoma cells in the hind limb and treated with 10 Gy IR combined with CTLA4 blocking antibody, CD47 translational blocking morpholino, or the combination of CTLA4/CD47 therapies. Granzyme B along with CD4/CD8 T cell infiltration were examined in tumors. Histology was evaluated for CD3 and necrosis. Results: The combination of CD47/CTLA4 with IR significantly increased survival by 25% compared to IR/CTLA4 alone at 50 days. Granzyme B expression was significantly increased in IR mice with CTLA4/CD47 combination, which correlated with infiltration of CD8+ T cells and a concomitant decrease in Gr1+CD11b suppressor cells compared to controls. In non-IR tumors, histology revealed minimal necrosis, while all IR groups showed increased necrosis. Tumor IR in combination with CTLA4 or CD47 increased immune cell infiltration. However, the combination of IR with CTLA4/CD47 showed widespread necrosis. All groups treated with the CD47 exhibited focal hemorrhage, which was more extensive when combined with CTLA4. Conclusions: Results herein suggest IR combined CTLA4/CD47 checkpoint blockade provides a survival benefit by activating a beneficial adaptive immune response.

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