Effect of STING agonist on tumor immune microenvironment of non-inflamed lung cancer and efficacy of immune checkpoint blockade.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 178-178
Author(s):  
Hongjae Chon
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Lung Carcinoma ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Immune Checkpoints ◽  
Anti Cancer

178 Background: Cancer immunotherapy targeting immune checkpoints are now emerging as a promising therapeutic strategy in various tumors. However, the treatment of T cell non-inflamed tumor which lacks intratumoral T cell infiltrates are still major clinical hurdle. Therefore, drugs that target signaling pathways to increase T cell infiltration in non-inflamed tumor microenvironment (TME) should be investigated. In this study, we aimed to explore the therapeutic potential of STING agonist in murine model of non-small cell lung cancer to overcome immunotherapy resistance. Methods: C57BL/6 mice, which are 6 to 8 weeks of age, were used for the experiment. Mice were injected with Lewis lung carcinoma cells on the right flank. STING agonist (cGAMP) was injected intratumorally. CD8+ and CD31+ cells were detected using immunofluorescence (IF) staining. Gene expressions of tumor microenvironment were analyzed by NanoString RNA sequencing. Flow cytometry (FACS) was performed to detect CD8+, CD4+, Treg and myeloid cell population. Tumor growths were evaluated in combination with anti-PD1 and STING agonist treatment. Results: Local injection of STING agonist effectively delayed tumor growth of LLC. STING agonist increased intratumoral CD8+ T cells and vascular disruption. Expressions of inhibitory checkpoint molecules (PD-1, PD-L1), cytokines (IFN), CD8+ and CD4+ T cells were increased, which showed that anti-cancer immune responses were augmented. Combination treatment of anti-PD-1 antibody and STING agonist synergistically decreased tumor growth. Conclusions: In this study, STING agonist was shown to delay tumor growth and remodel tumor microenvironment in non-inflamed lung carcinoma model. Combination therapy of STING agonist and immune checkpoint inhibitors (ICI) targeting PD-1 synergistically suppressed the growth of lung cancer which is resistant to ICI monotherapy. Collectively, our findings demonstrated that localized STING therapy effectively sensitizes non-inflamed lung cancer to systemic ICI treatment and induces a maximal anti-cancer immune response.

scholarly journals A Systematic Review of the Tumor-Infiltrating CD8+ T-Cells/PD-L1 Axis in High-Grade Glial Tumors: Toward Personalized Immuno-Oncology

2021 ◽  
Vol 12 ◽  
Author(s):  
Mahdi Abdoli Shadbad ◽  
Zahra Asadzadeh ◽  
Negar Hosseinkhani ◽  
Afshin Derakhshani ◽  
Nazila Alizadeh ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Checkpoint ◽  
Response Rate ◽  
Checkpoint Inhibitors ◽  
Immune Checkpoints ◽  
Cancer Therapies ◽  
High Grade ◽  
Glial Tumors ◽  
Anti Cancer ◽  
The Impact

Based on preclinical findings, programmed death-ligand 1 (PD-L1) can substantially attenuate CD8+ T-cell-mediated anti-tumoral immune responses. However, clinical studies have reported controversial results regarding the significance of the tumor-infiltrating CD8+ T-cells/PD-L1 axis on the clinical picture and the response rate of patients with high-grade glial tumors to anti-cancer therapies. Herein, we conducted a systematic review according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statements to clarify the clinical significance of the tumor-infiltrating CD8+ T-cells/PD-L1 axis and elucidate the impact of this axis on the response rate of affected patients to anti-cancer therapies. Indeed, a better understanding of the impact of this axis on the response rate of affected patients to anti-cancer therapies can provide valuable insights to address the futile response rate of immune checkpoint inhibitors in patients with high-grade glial tumors. For this purpose, we systematically searched Scopus, Web of Science, Embase, and PubMed to obtain peer-reviewed studies published before 1 January 2021. We have observed that PD-L1 overexpression can be associated with the inferior prognosis of glioblastoma patients who have not been exposed to chemo-radiotherapy. Besides, exposure to anti-cancer therapies, e.g., chemo-radiotherapy, can up-regulate inhibitory immune checkpoint molecules in tumor-infiltrating CD8+ T-cells. Therefore, unlike unexposed patients, increased tumor-infiltrating CD8+ T-cells in anti-cancer therapy-exposed tumoral tissues can be associated with the inferior prognosis of affected patients. Because various inhibitory immune checkpoints can regulate anti-tumoral immune responses, the single-cell sequencing of the cells residing in the tumor microenvironment can provide valuable insights into the expression patterns of inhibitory immune checkpoints in the tumor micromovement. Thus, administrating immune checkpoint inhibitors based on the data from the single-cell sequencing of these cells can increase patients’ response rates, decrease the risk of immune-related adverse events development, prevent immune-resistance development, and reduce the risk of tumor recurrence.

scholarly journals Alpha Thalassemia/Intellectual Disability X-Linked Deficiency Sensitizes Non-Small Cell Lung Cancer to Immune Checkpoint Inhibitors

2020 ◽  
Vol 10 ◽  
Author(s):  
Tao Hou ◽  
Shun Jiang ◽  
Yapeng Wang ◽  
Yangchun Xie ◽  
Haixia Zhang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Small Cell Lung Cancer ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Tumor Volume ◽  
Checkpoint Inhibitors ◽  
Small Cell ◽  
Small Cell Lung

BackgroundThe immune checkpoint inhibitors (ICIs) have achieved great success in the treatment of non-small cell lung cancer (NSCLC) patients. However, the response rate is low. The molecular mechanism involved in the effectiveness of ICIs remains to be elucidated.MethodsATRX mutation incidence among human cancers was analyzed from TCGA database. Atrx-deficient Lewis lung cancer cell line (LLC-sgAtrx) was established via AAV-CRISPR. Subcutaneous and metastasis models were established by subcutaneous and intravenous injection of LLC-sgAtrx and LLC-sgNTC cells into female C57BL/6 mice. The mice were treated with anti-PD1, anti-CLTA4 or Rat IgG2a. Tumor volume was determined by Vernier calipers and the IVIS imaging system. The proportions of CD3+ T cells, CD45+ immune cells, and the expression of pMHC I and PDL1 were determined by flow cytometry. The T cell cytotoxicity was determined by co-culture experiment.ResultsTCGA data showed that Atrx is a tumor suppressor mutated at high frequency among various human cancers. The tumor volume of mice bearing LLC-sgAtrx was significantly shrinked and the median survival of mice was significantly longer after anti-PD1 and anti-CTLA4 treatment. Flowcytometry results showed that Atrx deficiency increase the penetration of CD3+ T cell into the tumor microenvironment and enhanced antigen presentation after IFNγ stimulation. Additionally, the tumor cells with Atrx deficiency were more easily to be damaged by T cells under IFNγ stimulation.ConclusionThe present study demonstrated that Atrx deficiency sensitize lung cancer cells to ICIs by multiple mechanisms. And ATRX may serve as a promising biomarker for ICIs which helps patient stratification and decision making.

Abstract 350: Immune Profiling and Causal Antigen Discovery in Mouse and Human Models of Immune Checkpoint Inhibitor-induced Myocarditis

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Han Zhu ◽  
Daniel Lee ◽  
Waliany Sarah ◽  
Francisco X Galdos ◽  
Jessica D’Addabbo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Myocardial Damage ◽  
T Cell Subsets ◽  
Immune Checkpoints ◽  
Fulminant Myocarditis ◽  
T Cell Infiltration ◽  
Life Threatening

Introduction: Immune checkpoint inhibitors (ICIs) are novel drugs that activate T cell-mediated anti-tumor response by blocking immune checkpoints such as PD-1 or CTLA-4, leading to improved cancer patient survival. Despite these benefits, ICIs can result in autoimmune side effects including fulminant myocarditis and heart failure. While ICI-induced myocarditis is characterized by myocardial T cell infiltration, the causal mechanisms remain unknown. We hypothesize that ICI-induced myocarditis is caused by cardiac-specific auto-antigens triggering clonal expansion of myocardial CD8+ T-cells, leading to T-cell mediated myocardial damage. Methods/Results: We have explored the ICI-induced inflammatory response in a mouse model of myocarditis induced by PD-1 knockout and in patients with ICI-induced myocarditis. PD-1 deficient-mice on a lupus-like autoimmune background (i.e. MRL/Pcd1-/- mice) develop spontaneous fatal myocarditis in 70% of animals by 5 weeks of age, with massive cardiac infiltration of CD8>CD4+ T-cells. Likewise, patients with ICI-induced myocarditis have CD8>CD4+ T-cell infiltrate in the heart. We have performed time-of-flight mass cytometry (CyTOF) to immunophenotype the T-cell subsets in the blood/myocardium of MRL/Pcd1-/- mice and in ICI-myocarditis patients. We have also conducted single cell sequencing of T-cell receptors (TCRs) from the blood +/- myocardial-derived T-cell samples of the mice and patients. Our preliminary results in ICI-myocarditis patients confirmed the previously reported CD8+ T-cell expansion in the blood and myocardium of myocarditis patients compared with healthy control. We are currently identifying candidate cardiac auto-antigen(s) responsible for this disease by performing Grouping Lymphocyte Interactions by Paratope Hotspots (GLIPH). Conclusion: Myocarditis is a serious and life-threatening complication of ICI treatment. By understanding the unique immune response present during ICI-induced myocarditis and the responsible cardiac auto-antigen(s) involved, we will pave the way for the development of adjuvant therapies that target these antigens and mitigate their deleterious effects.

Effect of NKTR-214 on the number and activity of CD8+ tumor infiltrating lymphocytes in patients with advanced renal cell carcinoma.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 454-454 ◽  
Author(s):  
Michael E. Hurwitz ◽  
Adi Diab ◽  
Chantale Bernatchez ◽  
Cara L. Haymaker ◽  
Harriet M. Kluger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Tumor Infiltrating Lymphocytes ◽  
Poor Response ◽  
Post Treatment ◽  
Open Label

454 Background: Patients with low baseline CD8+ T-cells within the tumor microenvironment (TILs) have a poor response to immune checkpoint inhibitors. Agents designed to specifically activate and expand CD8+ T cells may improve clinical outcomes in patients with low TILs. NKTR-214 is a CD-122-biased agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβɣ) and preferentially activate and expand NK and effector CD8+ T cells over CD4+ T regulatory cells. Methods: A dose escalation, open-label, trial was initiated to assess the safety of NKTR-214 and explore immune changes in the blood and tumor microenvironment in patients with advanced solid tumors. NKTR-214 was administered IV in an outpatient setting with initial dosing at 0.003 mg/kg. Pre and post treatment blood and tumor samples were analyzed for immune phenotyping, gene expression, T cell receptor diversity, and changes in the tumor microenvironment by immunohistochemistry. Results: Among 25 patients dosed, 15 had RCC ([email protected]/kg, [email protected]/kg, and [email protected]/kg). Treatment with NKTR-214 was well tolerated and the MTD was not reached. One patient experienced DLTs (Gr3 syncope and hypotension) at 0.012 mg/kg. There were no immune-related AEs. Of 12 patients evaluable for response, 75% had SD at their first on treatment scan. Of 5 patients, who were immune checkpoint naïve with ≥ 1 prior TKI treatments, 3 experienced tumor shrinkage, 1 with PR per RECIST 1.1 (unconfirmed). Interrogation of the tumor microenvironment revealed many significant immunological changes post treatment, including increase in total and proliferating NK, CD8+, and CD4+ T cells. There was good correlation between increase in activated CD4+ and CD8+ T cells in peripheral blood with an increase in T cell infiltrates within the tumor tissue. Conclusions: NKTR-214 increased immune infiltration in the tumor and anti-tumor activity in patients who previously progressed on TKIs, with a favorable safety profile. The ability to alter the immune environment and increase PD-1 expression on effectors T cells may improve the effectiveness of anti-PD-1 blockade. A trial combining NKTR-214 and nivolumab is enrolling. Clinical trial information: 02869295.

scholarly journals Therapy of lymphoma by immune checkpoint inhibitors: the role of T cells, NK cells and cytokine-induced tumor senescence

2021 ◽  
Vol 9 (1) ◽  
pp. e001660
Author(s):  
Fatima Ahmetlic ◽  
Josia Fauser ◽  
Tanja Riedel ◽  
Vera Bauer ◽  
Carolin Flessner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Nk Cells ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Immune Checkpoint Blockade ◽  
Ifn Γ

BackgroundAlthough antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors.MethodsGrowth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry.ResultsOn immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF.ConclusionsTumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence.

scholarly journals Targeting the Tumor Microenvironment for Improving Therapeutic Effectiveness in Cancer Immunotherapy: Focusing on Immune Checkpoint Inhibitors and Combination Therapies

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1188
Author(s):  
I-Tsu Chyuan ◽  
Ching-Liang Chu ◽  
Ping-Ning Hsu
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Cancer Therapy ◽  
Cancer Immunotherapy ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Cell Activity ◽  
Immune Checkpoint Blockade ◽  
Immune Checkpoints

Immune checkpoints play critical roles in the regulation of T-cell effector function, and the effectiveness of their inhibitors in cancer therapy has been established. Immune checkpoint inhibitors (ICIs) constitute a paradigm shift in cancer therapy in general and cancer immunotherapy in particular. Immunotherapy has been indicated to reinvigorate antitumor T-cell activity and dynamically modulate anticancer immune responses. However, despite the promising results in the use of immunotherapy in some cancers, numerous patients do not respond to ICIs without the existence of a clear predictive biomarker. Overall, immunotherapy involves a certain degree of uncertainty and complexity. Research on the exploration of cellular and molecular factors within the tumor microenvironment (TME) aims to identify possible mechanisms of immunotherapy resistance, as well as to develop novel combination strategies involving the specific targeting of the TME for cancer immunotherapy. The combination of this approach with other types of treatment, including immune checkpoint blockade therapy involving multiple agents, most of the responses and effects in cancer therapy could be significantly enhanced, but the appropriate combinations have yet to be established. Moreover, the in-depth exploration of complexity within the TME allows for the exploration of pathways of immune dysfunction. It may also aid in the identification of new therapeutic targets. This paper reviews recent advances in the improvement of therapeutic efficacy on the immune context of the TME and highlights its contribution to cancer immunotherapy.

scholarly journals CAR-T “the living drugs”, immune checkpoint inhibitors, and precision medicine: a new era of cancer therapy

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Delong Liu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Stage Iv ◽  
Oncolytic Viruses ◽  
Immune Checkpoints ◽  
Car T Cell ◽  
Car T

Abstract New advances in the design and manufacture of monoclonal antibodies, bispecific T cell engagers, and antibody-drug conjugates make the antibody-directed agents more powerful with less toxicities. Small molecule inhibitors are routinely used now as oral targeted agents for multiple cancers. The discoveries of PD1 and PD-L1 as negative immune checkpoints for T cells have led to the revolution of modern cancer immunotherapy. Multiple agents targeting PD1, PD-L1, or CTLA-4 are widely applied as immune checkpoint inhibitors (ICIs) which alleviate the suppression of immune regulatory machineries and lead to immunoablation of once highly refractory cancers such as stage IV lung cancer. Tisagenlecleucel and axicabtagene ciloleucel are the two approved CD19-targeted chimeric antigen receptor (CAR) T cell products. Several CAR-T cell platforms targeting B cell maturation antigen (BCMA) are under active clinical trials for refractory and/or relapsed multiple myeloma. Still more targets such as CLL-1, EGFR, NKG2D and mesothelin are being directed in CAR-T cell trials for leukemia and solid tumors. Increasing numbers of novel agents are being studied to target cancer-intrinsic oncogenic pathways as well as immune checkpoints. One such an example is targeting CD47 on macrophages which represents a “do-not-eat-me” immune checkpoint. Fueling the current excitement of cancer medicine includes also TCR- T cells, TCR-like antibodies, cancer vaccines and oncolytic viruses.

Immune checkpoint and checkpoint inhibitors in hepatocellular carcinoma

2018 ◽  
Vol 1 (1) ◽  
pp. 28-32
Author(s):  
Piyawat Komolmit
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell Receptor ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Cell Receptor ◽  
Immune Checkpoints ◽  
Histocompatibility Complex

การรักษามะเร็งด้วยแนวความคิดของการกระตุ้นให้ภูมิต้านทานของร่างกายไปทำลายเซลล์มะเร็งนั้น ปัจจุบันได้รับการพิสูจน์ชัดว่าวิธีการนี้สามารถหยุดยั้งการแพร่กระจายของเซลล์มะเร็ง โดยไม่ก่อให้เกิดภาวะแทรกซ้อนทางปฏิกิริยาภูมิต้านทานต่ออวัยวะส่วนอื่นที่รุนแรง สามารถนำมาใช้ทางคลินิกได้ ยุคของการรักษามะเร็งกำลังเปลี่ยนจากยุคของยาเคมีบำบัดเข้าสู่การรักษาด้วยภูมิต้านทาน หรือ immunotherapy ยากลุ่ม Immune checkpoint inhibitors โดยเฉพาะ PD-1 กับ CTLA-4 inhibitors จะเข้ามามีบทบาทในการรักษามะเร็งตับในระยะเวลาอันใกล้ จำเป็นแพทย์จะต้องมีความรู้ความเข้าใจในพื้นฐานของ immune checkpoints และยาที่ไปยับยั้งโมเลกุลเหล่านี้ Figure 1 เมื่อ T cells รับรู้แอนทิเจนผ่านทาง TCR/MHC จะมีปฏิกิริยาระหว่าง co-receptors หรือ immune checkpoints กับ ligands บน APCs หรือ เซลล์มะเร็ง ทั้งแบบกระตุ้น (co-stimulation) หรือยับยั้ง (co-inhibition) TCR = T cell receptor, MHC = major histocompatibility complex

scholarly journals Inhibition of the PI3K/mTOR Pathway in Breast Cancer to Enhance Response to Immune Checkpoint Inhibitors in Breast Cancer

2021 ◽  
Vol 22 (10) ◽  
pp. 5207
Author(s):  
Chi Yan ◽  
Jinming Yang ◽  
Nabil Saleh ◽  
Sheau-Chiann Chen ◽  
Gregory D. Ayers ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Mtor Pathway ◽  
Complete Regression ◽  
Mtor Inhibition

Objectives: Inhibition of the PI3K/mTOR pathway suppresses breast cancer (BC) growth, enhances anti-tumor immune responses, and works synergistically with immune checkpoint inhibitors (ICI). The objective here was to identify a subclass of PI3K inhibitors that, when combined with paclitaxel, is effective in enhancing response to ICI. Methods: C57BL/6 mice were orthotopically implanted with syngeneic luminal/triple-negative-like PyMT cells exhibiting high endogenous PI3K activity. Tumor growth in response to treatment with anti-PD-1 + anti-CTLA-4 (ICI), paclitaxel (PTX), and either the PI3Kα-specific inhibitor alpelisib, the pan-PI3K inhibitor copanlisib, or the broad spectrum PI3K/mTOR inhibitor gedatolisib was evaluated in reference to monotherapy or combinations of these therapies. Effects of these therapeutics on intratumoral immune populations were determined by multicolor FACS. Results: Treatment with alpelisib + PTX inhibited PyMT tumor growth and increased tumor-infiltrating granulocytes but did not significantly affect the number of tumor-infiltrating CD8+ T cells and did not synergize with ICI. Copanlisib + PTX + ICI significantly inhibited PyMT growth and increased activation of intratumoral CD8+ T cells as compared to ICI alone, yet did not inhibit tumor growth more than ICI alone. In contrast, gedatolisib + ICI resulted in significantly greater inhibition of tumor growth compared to ICI alone and induced durable dendritic-cell, CD8+ T-cell, and NK-cell responses. Adding PTX to this regimen yielded complete regression in 60% of tumors. Conclusion: PI3K/mTOR inhibition plus PTX heightens response to ICI and may provide a viable therapeutic approach for treatment of metastatic BC.

scholarly journals 281 Measuring immune checkpoint inhibitor efficacy using primary patient-derived 3D spheroids

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A305-A305
Author(s):  
Kathryn Appleton ◽  
Katy Lassahn ◽  
Ashley Elrod ◽  
Tessa DesRochers
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Clinical Success ◽  
Single Cells ◽  
Granzyme B ◽  
Immune Checkpoints ◽  
Dependent Manner ◽  
Luminex Technology

BackgroundCancerous cells can utilize immune checkpoints to escape T-cell-mediated cytotoxicity. Agents that target PD-1, PD-L1 and CTLA4 are collectively deemed immune checkpoint inhibitors (ICIs), and many have been approved for treatment of non-small cell lung cancer (NSCLC) and melanoma. Unfortunately, many patients do not respond to these therapies and often experience disease progression. Immunohistochemistry assays to predict response to ICIs have been inconsistent in their readouts and often patients with low expression levels respond to ICIs. Understanding the determinants of ICI response in individual patients is critical for improving the clinical success of this drug class. Using patient-derived spheroids from NSCLC and melanoma primary tissue, we developed a multi-plexed assay for detecting ICI efficacy.MethodsNine NSCLC and 11 melanoma primary tumor samples were dissociated to single cells, classified for immune checkpoint expression and cell content by flow cytometry, and seeded for spheroid formation. Spheroids were treated with pembrolizumab, nivolumab, atezolizumab, ipilimumab or durvalumab across a range of concentrations and monitored for cytotoxicity at 24-hours and viability at 72-hours by multiplexing CellTox™ Green Cytotoxicity Assay and CellTiter-Glo® 3D Cell Viability Assay. IFNγ and granzyme B secretion was assessed using Luminex technology. ICI response was evaluated by determining the concentration-response relationship for all three read-outs.ResultsIncreased IFNγ and granzyme B were detected for every ICI in one or more patient samples. ICI-induced IFNγ secretion inversely correlated with PD-1+ immune cells. Durvalumab was significantly more cytotoxic for both NSCLC and melanoma spheroids compared to the other ICIs and significantly reduced spheroid viability with mean spheroid survival decreasing to 19.5% for NSCLC and 58.2% for melanoma. We evaluated if there was an association between durvalumab response and cell composition and found that percent spheroid survival significantly correlated with CD8+ T-cells for both NSCLC (r=-0.7920, p=0.0191) and melanoma (r=-0.6918, p=0.0390). Furthermore, CD8+ T-cells correlated with durvalumab-induced granzyme B secretion for NSCLC (r=-0.7645, p=0.0271) and melanoma (r=-0.7419, p=0.0221).ConclusionsIn this study we show ICI-specific increases in immune-related analytes in a concentration-dependent manner for NSCLC and melanoma patient-derived spheroids. We detected spheroid cytotoxicity following short term ICI treatment which closely mirrored decreased spheroid viability at a later timepoint. Finally, we can decipher response mechanisms as exemplified by durvalumab-induced granzyme B secretion correlating with the presence of CD8+ T-cells which results in reduced spheroid viability for both tested cancer indications.

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