Human Checkpoint Research Articles

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37)and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) invitro and invivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

BMIM]BF4 Mediated Multi-component Synthesis of Phenyl-4-(1Hpyrazol- 5-yl)-1H-pyrazolo[3,4-b]quinolin-5(4H)-ones and Evaluation of their Anti-cancer Activity

Abstract: Pyrazolo bridged quinolones scaffolds are an important building block in many of the medicinally active new chemical entities. In the current work, synthesis of phenyl-4-(1H-pyrazol-5-yl) conjugated with 1H-pyrazolo[3,4-b]quinolin-5(4H)-ones derivatives has been achieved by one-pot threecomponent reaction of 3-methyl-1-phenyl-1H-pyrazol-5-amine 1, 5,5-dimethylcyclohexane-1,3-diones 2, and 1H-pyrazole-5-carbaldehydes 3 using [BMIM]BF4 as green reaction medium with good yields. Furthermore, the synthesized compounds were evaluated for cytotoxic potential towards HeLa and DU145 cells. Of the eight derivatives, compound 4d exhibited good cytotoxicity with an IC50 value of 8.5 and 8.9 μM against DU145 and HeLa cells, respectively. The molecular docking studies revealed that compound 4d displayed good binding energy (-8.4 kcal/mol) against human checkpoint kinase 1.

Abstract 14233: Checkpoint Kinase 1 Combines PKM2 C-domain to Stimulate Endogenous Cardiomyocyte Renewal and Cardiac Repair by Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury

Background: The regenerative capacity of adult mammalian hearts is limited. We previously reported CHK1 in adult mice could promote cardiac regeneration after MI. However, the specific mechanism of CHK1 needs more comprehensive and in-depth interpretation. Methods: I/R injury was induced in pigs, recombinant human checkpoint kinase 1 (rhCHK1) protein encapsulated in hydrogel was injected into the infarct border zone. The therapeutic efficacy of rhCHK1 was evaluated by echocardiography, cMRI, hematological indices and immunofluorescence. Multi-omics analysis were performed to explore the potential mechanisms of rhCHK1. Further, protein interaction prediction and CO-IP experiments were conducted to verify the specific interaction pattern and domains between CHK1 and PKM2. Hipsc-CMs and porcine were used for confirming whether rhCHK1 promotes cardiac regeneration through combining with the PKM2 C-domain. Results: RhCHK1 protein stimulated CMs proliferation and reduced cardiac inflammation at 3 dpi, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 dpi. Mechanistically, multiomics analysis showed that glycolysis and mTOR pathways were significantly enriched in this process. Further CO-IP experiments and protein docking prediction showed that CHK1 directly bound to and activated the S37 and Y105 sites of PKM2 to promote metabolic reprogramming. We further constructed plasmids that knock out different CHK1 and PKM2 domains, and transfected them into H293T cells for CO-IP experiments. It was found that the 391-476 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Further hipsc-CMs and pig experiments both demonstrated that CHK1 stimulates CMs renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming and mTORC1 pathway. Conclusion: This study demonstrates that rhCHK1 could combine with PKM2 391-476 amino acid domain to mediate metabolic reprogramming and mTORC1 pathways to promote CM proliferation and myocardial repair. This study is the first to clarify the specific functional mechanism of CHK1 in pigs. Our results underscore the potential of the local applied rhCHK1 as a safe and simple feasible treatment option for repairing heart.

Abstract 5194: Humanized PD-1 knock-in mice as a model system for combination therapies with human specific PD-1 therapeutics

Abstract Checkpoint inhibitor treatment has become a common therapy for various cancer types. As an escape mechanism, tumor cells express PD-L1 on their surface, as a ligand for PD-1 with players of the immune system (such as T cells), aimed at preventing the immune system from exerting its anti-tumour activities. Antibodies blocking the PD-L1/PD-1 interaction have emerged as front-line treatments for various oncological indications. Several anti-PD-L1 and anti-PD-1 therapeutics are already approved and in clinical use. For preclinical testing of such human specific antibodies, syngeneic tumour models are of limited use due to the limited cross-specificity of such antibodies, giving rise to the development of humanized mouse models in which combination therapies testing new drugs with already clinically approved human checkpoint inhibitors addressing human specific targets can be evaluated. The anti-tumor efficacies of pembrolizumab (Keytruda) a human specific anti-hPD-1 checkpoint inhibitor and the mouse specific anti-mPD-1 counterpart (clone RPMI1-14) were tested in C57BL/6 wildtype and humanized hPD-1 C57BL/6 mice engrafted with syngeneic MC38-CEA colorectal carcinoma cells. The MC38-CEA tumor cells were implanted into the mammary fat pad (subQperiorTM), a procedure that prevents tumor ulceration and leads to a more homogenous tumor growth compared to subcutaneous implantation. Treatment with pembrolizumab extended the survival of tumor-bearing human PD-1 C57BL/6 mice significantly, whereas no effect in C57BL/6 wildtype mice was observed, in line with the reported specificity of Keytruda towards the human version of PD-1. Further supporting this finding, the application of the anti-mPD-1 antibody led to a prolonged survival of C57BL/6 mice with no life extension of hPD-1 C57BL/6 mice. The validation of the humanized subQperior™ platform was further complemented by conducting flow cytometry analysis of intra-tumoral immune populations present in tumor tissues at 7 days post-treatment. Two staining panels were executed: a well-established and validated 17 marker staining allowing to quantify the frequency of all major immune cell populations and a staining panel addressing the activity of T and NK cells after ex vivo stimulation with PMA/Ionomycin. An increased activity of CD8+ T cells and NK cells was observed as major players in the response to treatment with these anti-PD-1 antibodies. In summary, the humanized subQperior™ platform using hPD-1 C57BL/6 mice is a suitable tool to evaluate novel cancer therapies in combination with human specific anti-PD-1 therapeutics and tumor ulceration was completely prevented by subQperior implantation of the tumor cells. Citation Format: Philipp Metzger, Fabiane Sônega, Hervé Berthomme, Cynthia Obodozie, Holger Weber. Humanized PD-1 knock-in mice as a model system for combination therapies with human specific PD-1 therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5194.

Abstract 12329: Checkpoint Kinase 1 Protein Therapy Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair in a Porcine Model of Myocardial Ischemia/Reperfusion Injury

Objective: To investigate the impact of recombinant human checkpoint kinase 1 (rhCHK1) on cardiomyocyte renewal and myocardial repair after myocardial ischemia/reperfusion (I/R) injury in swine. Methods: Quantitative phosphoproteomics profiling was performed on infarct border zone of neonatal myocardium, and kinase-substrate network analysis using iGPS algorithm revealed CHK1 kinase with enriched substrates and upregulated phosphorylation levels. Swine myocardial ischemia/reperfusion injury was induced by ligating the left anterior descending coronary artery for 60 minutes, and followed by reperfusion. Immediately after reperfusion, rhCHK1 protein (1mg/kg) dissolved in hydrogel or equivalent hydrogel was injected into the infarcted border zone. The therapeutic efficacy of rhCHK1 post I/R injury was evaluated by ECG, echocardiography, MRI, hematological indices and immunofluorescence. Results: Intramyocardial injection of rhCHK1-hydrogel efficiently delivered rhCHK1 to the cardiomyocytes surrounding the infarcted area of pig hearts without affecting other organs. A single administration of rhCHK1 protein (1mg/kg) prominently stimulated cardiomyocytes proliferation, reduced cardiac inflammation and improved vascularity 3 days after I/R. At 28-days post rhCHK1-hydrogel therapy, improved cardiac function, reduced infarct size and cardiac scar area, and attenuated ventricular remodeling were evidenced in I/R swine. Mechanistically, rhCHK1 stimulated CMs renewal and cardiac repair by activating mTOR/CTHRC1/P70S6K signaling pathways. Conclusion: Our study demonstrates that myocardial injection of rhCHK1 could promote cardiomyocyte proliferation, inhibit local myocardial inflammation and promote angiogenesis to facilitate myocardial repair and cardiac function after I/R injury in adult pigs through activating mTOR/CTHRC1/P70S6K signaling pathways.

Modulation of urelumab glycosylation separates immune stimulatory activity from organ toxicity.

Checkpoint control and immunomodulatory antibodies have become important tools for modulating tumor or self-reactive immune responses. A major issue preventing to make full use of the potential of these immunomodulatory antibodies are the severe side-effects, ranging from systemic cytokine release syndrome to organ-specific toxicities. The IgG Fc-portion has been demonstrated to contribute to both, the desired as well as the undesired antibody activities of checkpoint control and immunomodulatory antibodies via binding to cellular Fcγ-receptors (FcγR). Thus, choosing IgG subclasses, such as human IgG4, with a low ability to interact with FcγRs has been identified as a potential strategy to limit FcγR or complement pathway dependent side-effects. However, even immunomodulatory antibodies on the human IgG4 background may interact with cellular FcγRs and show dose limiting toxicities. By using a humanized mouse model allowing to study the immunomodulatory activity of human checkpoint control antibodies in vivo, we demonstrate that deglycosylation of the CD137-specific IgG4 antibody urelumab results in an amelioration of liver toxicity, while maintaining T cell stimulatory activity. In addition, our results emphasize that antibody dosing impacts the separation of side-effects of urelumab from its therapeutic activity via IgG deglycosylation. Thus, glycoengineering of human IgG4 antibodies may be a possible approach to limit collateral damage by immunomodulatory antibodies and allow for a greater therapeutic window of opportunity.

Abstract 5080: Age- and pregnancy-related impact on soluble immune checkpoint protein profiles in mice as analyzed by a novel multiplex immunoassay

Abstract Immune checkpoint proteins are key regulators of the immune system and drug targets of cancer immunotherapy. There is extensive literature on the use of various types of mouse models for biomarker discovery and drug testing. Only a few studies have examined the association between soluble immune checkpoint proteins and the immune status. Following the development of multiplex immunoassay panels for profiling human co-inhibitory and co-stimulatory immune checkpoint proteins, here we report the development of a novel magnetic bead-based Luminex multiplex assay for the quantitative detection of 28 mouse immune checkpoint proteins and immune regulators in 25 μL of sample (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). Immune function declines with age, while the incidence of cancer rises. In this study, we examined age-related serum samples by quantitative profiling soluble checkpoint proteins from young (age of 8-month-old, n=8) and old-aged mice (48-month-old, n=8) of C57BL/6 strain. A significant age effect was observed on the levels of soluble checkpoint proteins in mouse serum samples tested. Comparing with the young mice, decreased levels of circulating HVEM, Granzyme B, LAG-3, PD-L1, CD73, B7-H2, CD25, and CD27; and increased levels of circulating TIM-3, PD-1, PD-L2, CD40, Galectin-3, IL-10, BCA-1, and TLR-2 were observed in aged C57BL/6 mice (Mann Whitney test, P value <0.05). The immune system also changes during pregnancy. We used the similar multiplex array approach to profile the soluble checkpoint proteins in non-pregnant (n=16) and pregnant CD-1 mice (n=9). Significant decreased levels of soluble LAG-3, B7-H2, B7-H3, CD25, CD27, and Galectin-1; and significant increase of soluble CD137, Galectin-3, and CD73 expression levels were detected in the serum samples of pregnant mice, as compared to the samples from the non-pregnant mice (Mann Whitney test, P value <0.05). Altogether, using bead-based quantitative multiplex analysis, our results demonstrate that the changes on circulating immune checkpoint protein profiles occur in the immunologic adaptations with age and during pregnancy in mice. Citation Format: Wen-Rong Lie, Christine Kornmeier, Ling Zeng, James Hoberg. Age- and pregnancy-related impact on soluble immune checkpoint protein profiles in mice as analyzed by a novel multiplex immunoassay [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 5080.

Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

Abstract 1621: Comprehensive profiling of immune regulatory molecules in baseline sera from advanced stage NSCLC patients receiving anti-PD-1 and anti-PD-L1 immunotherapy: A prognostic potential

Abstract Introduction: Immune checkpoint inhibitor (ICI) therapy has shown remarkable benefits in treating non-small-cell lung cancer (NSCLC). However, progress beyond PD-L1 expression has not yielded biomarkers that can better select responders to ICI. This study comprehensively evaluated the prognostic value of soluble immune checkpoints and immune regulators in previously treated lung cancer patients receiving anti PD-L1/PD1 single agents. Method: We profiled 47 immune checkpoint molecules and regulatory molecules in baseline serum from 135 metastatic NSCLC patients receiving atezolizumab (n=40), pembrolizumab (n=32), durvalumab (n=10), or nivolumab (n=53) after failing frontline therapy. Immune checkpoints and regulators were evaluated via the MILLIPLEX® Human Immuno-Oncology Checkpoint Protein Panels 1 and 2 (MilliporeSigma) using manufacturer-defined protocols. All statistical relationships were determined using the Log-Rank test in relation to overall survival (OS) and progression-free survival (PFS). Results: The study cohort consisted of 50% males, 72% caucasians, and histologically divided into 72% adenocarcinoma and 25% squamous cell. The median follow-up time was ten months from the time of ICI therapy commencement. The median PFS was 4.1 months, and overall survival was 13.1 months for the entire cohort. Twenty eight molecules were found to be significantly associated (p <0.05) with progression free survival (PFS), out of which the baseline serum levels for circulating CD27, GITR, PD-1, CTLA-4, CD80, CD86, CD40L, B7-H2/ICOSL, E-Cadherin, FGL1/Hepassocin, Galectin-1, and Perforin showed the highest significance (all p-values <0.01). Similarly, twenty-six of the profiled molecules were found to have significant association (p <0.05) with overall survival (OS), with baseline serum levels of PD-1, CTLA-4, CD80, CD86, CD40L, B7-H2/ICOSL, E-Cadherin, FGL1/Hepassocin, Galectin-1, and Perforin being the most highly associated with OS (all p-values <0.01). Conclusion: We demonstrated the promising value of circulating immune checkpoints and immune regulatory molecules in precision immuno-oncology related to advanced-stage NSCLC. This work requires validation but, with further development, may have implications for single agent anti PD-L1/PD-1 treatment selection in the front-line setting. Citation Format: Imad Tarhoni, Hita Moudgalya, Sanjib Basu, Wen_Rong Lie, Danielle Pepin, Mary Jo Fidler, Marta Batus, Philip Bonomi, Jeffrey Borgia. Comprehensive profiling of immune regulatory molecules in baseline sera from advanced stage NSCLC patients receiving anti-PD-1 and anti-PD-L1 immunotherapy: A prognostic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1621.

Abstract 2847: Multiplex bead array approach for quantitative profiling of 95 cancer-immunity biomarkers in breast cancer

Abstract Immune regulators, including immune checkpoint proteins and cytokines/chemokines, have an emerging role as both disease/treatment biomarkers and targeted agents in cancer therapies. To contribute to the understanding of the roles of these immune regulators in cancer, we have developed 3 new Luminex-based multiplex immunoassay panels, a 48-plex human cytokine/chemokine panel, a 17-plex human immune checkpoint protein panel, and a 30-plex human immune checkpoint protein panel, to simultaneously quantitate the expression levels of 95 key immune regulator proteins in biofluids or cell/tissue homogenates. Here we report the quantitative profiles of these 95 immune regulators in 4 types of breast cancer samples: cancer versus healthy control serum samples, the lysates from breast cancer tumor biopsies versus the adjacent normal tissues, the conditioned media and lysates from the established breast cancer cell lines, and breast cancer cell-derived exosomes. Analysis of the circulating immune protein signatures generated from this multiplex approach reveals an elevated level of IL-6, IL-27, M-CSF, MDC, MIG, sCD27, sTIM3, sCD40, Galectin-3, Galectin-1, FGL-1, BAFF and low level of EGF and sCD40L in breast cancer serum samples compared to the healthy serum controls. The expression profiling of tumor and adjacent normal tissues from 3 patients with metastatic breast cancer revealed differential expression of multiple protein markers including IL-6, IL-8, MIG, IL-1ra, IL-18, IP-10, MCP-1, MIP-1b, VEGF, BTLA, HVEM, CTLA-4, CD40, TLR-2, Siglec-9, CD25, Granzyme B, APRIL, BAFF, Nectin-2, Nectin-4, E-cadherin, and IDO-1 in the matched lysates. We performed similar analysis using cultured breast tumor cells. In these samples we identified markers with significantly altered expression in conditioned media, cell lysates, and tumor cell-derived exosomes from MCF10CA1d tumor cells versus MCF-10A non-tumorigenic mammary epithelial cells as well as triple negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells. Altogether, our results suggest Luminex-based profiling allows for sensitive and versatile multiplexed analysis of stimulating and inhibitory immune mediators in circulation, tissues, and cell lines which will assist in the discovery of biomarkers and therapeutic targets for cancer interventions. Citation Format: Wen-Rong Lie, Alexander Davies, Danielle Pepin, Michael Godeny. Multiplex bead array approach for quantitative profiling of 95 cancer-immunity biomarkers in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2847.

Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells

PurposeChemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses.MethodsPancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry.ResultsThese agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine.DiscussionThese data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy.

Circadian variation in nivolumab efficacy in patients with advanced non-small cell lung cancer.

e21585 Background: Despite efficacy of human checkpoint inhibitors, only 20 to 30% of treated patients present long term benefits. Circadian variations of the delivery of nivolumab to patients with advanced non-small-cell lung cancer (NSCLC) are proposed here as a factor potentially influencing the efficacy. Methods: A total of 1318 courses of nivolumab were administered in 57 consecutive patients with advanced NSCLC from September 2015 to December 2019 in Medical Oncology Unit of Montfermeil hospital. Percentage of total dose per patient delivered before and after 12:00 pm was computed and corelated to objective response rate (ORR), progression free survival (PFS) and overall survival (OS). Results: Twenty eight patients (49%) received ≥ 50% of their treatment before 12:00 pm (morning group) and 29 patients (51%) after 12:00 pm (afternoon group). ORR was 53.6% in morning group and 31.0% in afternoon group; p = 0.083. Both PFS and OS were significantly longer in morning group as compared to afternoon group. Median PFS was 18.6 months [95%CL, 8.6 – 28.7] and 3.5 months [2.9 – 4.1] in morning and afternoon group respectively; p < 0.001. After a maximum follow-up of 48 months, median OS was not reached in morning group whereas it was 14.4 months [0.0 – 29.5] in afternoon group; p = 0.004. Conclusions: The significant difference in the efficacy of nivolumab between morning and afternoon group is indirect evidence of the influence of circadian rhythm on the metabolism of this checkpoint inhibitor. The application of this finding in clinical practice should allow an increase of nivolumab efficacy in patients with advanced NSCLC.

Abstract B81: Efficacy of nivolumab, pembrolizumab, and atezolizumab against MC38 colon cancer expressing human PD-1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes

Abstract The human checkpoint genes PD-1 and PD-L1 have been the target of extensive research for the treatment of human malignancies. Checkpoint inhibitor therapy has shown great promise in the clinic. Animal models that can use the human clinical antibodies will be beneficial for evaluating new therapies. A novel transgenic mouse model was developed that expresses the human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We evaluated the efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. The tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 μg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. We have shown that both PD-1 and PD-L1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. Citation Format: Murray Stackhouse, Ted Green, Jay Liu, Charlotte Hammond, LaJuana Durbin, Anna Chen, Mike Koratich. Efficacy of nivolumab, pembrolizumab, and atezolizumab against MC38 colon cancer expressing human PD-1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B81.

Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct

Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct

Bioinformatics Model of Serum Biomarkers to Prognosticate the Response to Programmed Death-1/Ligand-1 Targeted Immunotherapy in Metastatic Non–Small Cell Lung Cancer

Abstract Introduction Immune-checkpoint inhibitors revolutionized the therapeutic paradigm for metastatic non–small cell lung cancer (NSCLC). The average response, however, still hovers at 20%, demonstrating the urgent need for biomarkers predictive of response. High-throughput laboratory technology promises to serve as an insightful and robust tool to recognize and select patterns of biomarkers in serum. We applied machine learning on serum immune-checkpoint biomarkers for prognostication of response to immunotherapy in advanced NSCLC. Method Pretreatment sera from 106 advanced NSCLC cases who failed frontline chemotherapy were evaluated for 16 soluble immune-checkpoint molecules using the Human Immuno-Oncology Checkpoint Protein Panel (MilliporeSigma). This panel constituted BTLA, CD27, CD28, TIM-3, HVEM, CD40, GITR, GITRL, LAG-3, TLR-2, PD-1, PD-L1, CTLA-4, CD80, CD86, and ICOS. Primary data points were collected and calculated via a Luminex FLEXMAP 3D system (xPONENT v4.0.3 Luminex Corp). The minimum follow-up after treatment was 12 months. Response patterns were categorized based on their overall survival (OS) as long-term responders (>12 months) or short-term responders (<12 months). Values were analyzed with the clinical outcomes using “Survminer” and “survival” R packages to determine the log-rank-based cutoff values associated with overall survival. Finally, machine learning methods were implemented using “caret” and “rpart” R packages to fit a classification model to predict the response pattern. The model was trained and tested on random fractions of the cohort. Results BTLA4, HVEM, CD40, GITRL, LAG-3, PD-1, CD80, and CD86 serum levels significantly correlated with OS (all P values ≤.02 and HR of 0.27, 0.5, 4.59, 0.17, 0.12, 0.48, 3.64, and 0.37, respectively). The algorithm composing PD-1, LAG-3, CD86, and CTLA4 predicted the response pattern with PPV of 81%, specificity of 87%, and accuracy of 75%. Conclusion The serum immune-checkpoint predictive model might assist in the tissue and gene-based profiling of immune-checkpoints to predict the benefit from immunotherapy.

Divergent Synthesis and Evaluation of the in vitro Cytotoxicity Profiles of 3,4-Ethylenedioxythiophenyl-2-propen-1-one Analogues.

A new series of 3,4-ethylenedioxythiophene (EDOT)-appended propenones were prepared by condensation reaction and their in vitro cytotoxicity effects were evaluated against five human cancer cell lines. Preliminary structure-activity relationships of EDOT-incorporated 2-propenone derivatives were also established. The EDOT-appended enones demonstrated significant cytotoxicity against human cancer cell lines. The most active analogue, (E)-3-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3 p, GI50 =110 nm), severely inhibited the clonogenic potential of cancer cells, and induced cell-cycle arrest in the G2/M phase and caused an accumulation of HCT116 colon cancer cells with >4 N DNA content. Also, 3 p exhibited weak inhibition of the enzymatic activity of human topoisomerase I. Molecular docking studies indicated preferential binding of the compounds to the ATP-binding pocket of the human checkpoint 2 kinase (Chk2) catalytic domain, thus, identifying a novel diaryl 2-propenone chemotype for the development of potent inhibitors of Chk2.

Abstract 4087: Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes

Abstract Human checkpoint genes PD-1 and PD-L1 are the target of extensive research for treating human cancers. Checkpoint inhibitor therapy shows great promise in the clinic. Combining checkpoint inhibitors with standard chemotherapeutic agents is an important for these inhibitors. Animal models that use human clinical antibodies and chemotherapeutic agents will be beneficial for evaluating new therapies. A novel transgenic mouse model is described that expresses human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We initially evaluated early treatment efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. Tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 µg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To extend the utility of the model we implanted mice and allowed the tumors to grow to 63 to 126 mg before treatment initiation (Day 10). Similar results were observed in established tumors as for early treatment with the PD-1 inhibitors nivolumab and pembrolizuman. In contrast there was little activity from the PD-L1 inhibitor atezolizumab in established tumors. To prepare for combination studies, we also tested cisplatin, irinotecan, gemcitabine, decitabine, rucaparib, and thiarabine near the MTD for each agent with various treatment schedules against the established tumors. The doses for cisplatin, irinotecan and decitabine resulted in excess animal deaths and require reduced doses for combination therapies. Gemcitabine, decitabine, and thiarabine (in house development drug) were too active with complete regressions observed; the doses for combination treatment will be reduced. Due to the effectiveness of the PD-1 checkpoint inhibitors the number of doses will be reduced from four to two treatments in combination studies; no change for atezolizumab. We have shown that PD-1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice in early treatment and in established tumors while the PD-L1 inhibitor was only effective in early treatment. We also have identified doses and schedules of five chemotherapeutic agents to use in future combination studies in this model. Citation Format: Murray A. Stackhouse, Ted Green, Jay Liu, Charlotte Hammond, LaJuana Durbin, Ana Chen, Jerry Zhou, Mike Koratich. Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4087.

Abstract 2375: Developing double knock-in models of human immune checkpoint targets (dKI HuGEMM) for efficacy assessment of combinatorial therapeutic antibodies

Abstract Immune checkpoint inhibitors (ICIs), i.e. PD1, PDL1 and CTLA4 therapeutic antibodies, have led to long-term survival in many late stage solid tumor patients. Despite their revolutionary clinical impact, the overall response rate is still low. One major roadblock is that compensatory immune inhibitory pathways are turned on to protect tumor cells from being attacked by T cells. Various combinatory ICI treatments have now been investigated in clinical trials to tackle this challenge. We have previously reported the development of immune checkpoint target humanized mice (HuGEMM) through CRISPR-Cas9 knock-in. We have generated PD1 and CTLA4 HuGEMM mice and used them for efficacy assessment of corresponding therapeutic antibodies. We have also generated human PDL1 expressing MC38 cells and PDL1 HuGEMM, and demonstrated robust efficacy of several PDL1 therapeutic antibodies. Here we report the establishment of double knock-in (dKI) mice of ICI targets, i.e., PD1/PDL1 and PD1/CTLA4 dKI HuGEMM, and use them as tools to test immunotherapies with PD1/PDL1 and PD1/CTLA4 combos. Similar to clinical benefit of PD1 and CTLA4 combo shown in the melanoma and NSCLC patients, we found combined treatment of Nivolumab and Ipilimumab antibody leads to 103% TGI in the corresponding PD1/CTLA4 dKI-HuGEMM, with complete tumor remission in 80% of treated mice. These mice remained disease free throughout a tumor re-challenge study for over 40 days. We have also developed PD1/PDL1 dKI HuGEMM and tested combined treatment of Atezolizumab and Nivolumab. Interestingly, we observed improved efficacy in the combo treatment group despite the two ICIs are thought to target the same PD1/PDL1 axis. Monotherapies may not fully release the PD1/PDL1 blockade due to complicity of multiple compatible ligands & receptors, including PDL2 and B7-1. We have also tested PD1/OX40 combo treatment with dKI HuGEMM, which is reported separately. Taken together, Our dKI HuGEMM models offer robust tests on ICI combinations, as well as combos of ICIs with other anti-tumor therapeutic modalities. Citation Format: Lei Zheng, Daniel Xianfei He, Ruilin Sun, Annie Xiaoyu An, Jian Fei, Henry Qixiang Li, Davy Xuesong Ouyang. Developing double knock-in models of human immune checkpoint targets (dKI HuGEMM) for efficacy assessment of combinatorial therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2375.

Abstract 404: Prognostic value of soluble and cell surface immune-checkpoint molecules in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD-1/-L1 immunotherapy

Abstract Current methods to stratify immunotherapy candidates, including PD-L1 expression and tumor mutational burden (TMB) profiling, have limitations that hamper their clinical value. This study explores the prognostic potential of soluble and cell-surface immune-checkpoint (IC) molecules as a means to improve treatment selection for NSCLC patients being considered for PD-1/-L1 blockade. Pretreatment sera from 111 cases of previously-treated advanced NSCLC receiving PD-1/L-1 targeting checkpoint inhibitors (nivolumab, atezolizumab, or pembrolizumab) were evaluated for 16 soluble IC molecules and immune regulators via the MILLIPLEX® MAP Human Immuno-Oncology Checkpoint Protein Panel (MilliporeSigma) using manufacturer-defined protocols. PBMCs from a subset of this cohort (n=28) were profiled on a LSRFortessa™ for cell-surface IC molecules. T-cell subsets (CD4,CD8) were analyzed for CD27\CD28, iCOS, PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, GITR and PD-L1. NK cells (CD56\CD16) were analyzed for CD96, CD94, CD86, CD80, PD-1, CTLA-4, TIM-3, TIGIT, LAG-3 and PD-L1. Classical (CD14++\CD16-), intermediate (CD14++\CD16+) and non-classical (CD14dim\-\CD16++) monocyte subsets were analyzed for these identical markers. All statistical relationships were determined using the Log-Rank test in relation to overall survival (OS) and progression-free survival (PFS). Pearson correlations were calculated between the circulating and PBMCs levels of IC molecules. Sixteen soluble IC molecules were detectable in patient serum, with twelve of these being prognostic for OS, PFS, or both (see Table). In the 28 patients with PBMCs, a set of cell-surface molecules showed prognostic value, but had no apparent correlation to soluble molecules. These findings suggest that both soluble and cell-surface IC molecules may have promise for identifying advanced NSCLC patients that may benefit from anti-PD-1/-L1 immunotherapy. PFSOSp-valueHRp-valueHRSoluble moleculesBMI0.0030.790.0540.75Lymphocyte0.0040.700.0020.60NLR0.0051.69<0.0012.12BTLA0.0140.790.010.90CD270.0020.680.0170.80TIM30.0370.880.0280.93HVEM0.0040.620.0040.53GITR0.0010.003<0.0010.00LAG30.0400.820.030.72GITRL0.0140.100.0070.03PD10.0020.490.0030.34CTLA4<0.0010.180.0020.27CD800.0010.680.0020.67CD86<0.0010.63<0.0010.54iCOS0.0140.840.0470.90PBMCs - cell surface moleculesCD30.0520.660.110.65CD3-CD800.370.990.0051.45CD4-iCOS0.050.740.491.35CD8-CTLA40.020.660.21.34CD8-GITR0.0230.490.30.70CD8-TIGT0.020.630.370.78CD8-LAG30.0160.620.031.18CL-CD860.360.900.050.56CL-CD640.141.330.0151.89NC-CD640.0261.520.0021.74NC-PDL20.0382.840.351.52NC-TIM30.0032.070.041.19 Citation Format: Imad Tarhoni, Mary Jo Fidler, Ibtihaj Fughhi, Connor Wakefield, Revathi Kollipara, Maneet Multani, Marta Batus, Sanjib Basu, Wen-Rong Lie, Donna Russell, Jeffrey Martinson, Alan L. Landay, Timothy M. Kuzel, Philip Bonomi, Jeffrey A. Borgia. Prognostic value of soluble and cell surface immune-checkpoint molecules in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD-1/-L1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 404.

Abstract 1504: Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1

Abstract The human checkpoint targets PD-1 and PDL-1 continue to demonstrate great promise in the clinic. With much of the current focus turning toward combination regiments, an animal model using the human clinical antibodies will be beneficial for evaluating new combination strategies. Here we continue characterization of a C57BL/6 transgenic mouse model expressing both the human PD-1 and PD-L1 combined with a modified murine MC38 colon tumor cell line expressing human PD-L1. Both PD-1 and PD-L1 expression of the transgenic mice were verified in ex vivo stimulated splenocytes by flow cytometry analysis. Expression of PD-L1 on the genetically modified MC38 cells was also demonstrated by flow cytometry. For in vivo efficacy evaluation, tumor-implanted mice were treated with the clinical agents nivolumab and pembrolizumab at 100 µg and atezolizumab at 1 mg on Days 3, 7, 10, and 14 post implant. Treatment with nivolumab and pembrolizumab initiated tumor regression by Day 17. Complete tumor regressions were seen at Day 28 in nivolumab, 62.5% complete regression, and pembrolizumab, 71.4% complete regression. Growth inhibition was 83.9%, 69.7%, and 95.0% on Day 28 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To compare the human anti-PD-1 and PD-L1 clinical agent specificity, the non-transgenic parent C57BL/6 mouse strain was implanted with the unmodified MC38 colon tumor cells. Treatment with pembrolizumab and atezolizumab was conducted as with the transgenic animals. Through 28 days no growth inhibition, tumor size 105.9% of control, was seen in the pembrolizumab treated group, demonstrating lack of cross reactivity of the human therapeutic in the standard mouse model. Atezolizumab did demonstrate a 56.9% growth inhibition compared to controls and is consistent with the known cross reactivity of atezolizumab between human and mouse. We have shown a genetically modified MC38 colon tumor expressing human PD-L1 in transgenic mice expressing both human PD-1 and PD-L1 to be a suitable model for checkpoint inhibitor evaluation of the human form of the particular checkpoint therapeutic. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. Citation Format: Michael Koratich, Ted Green, Jie Liu, Charlotte Hammond, LaJuana Durbin, Jerry Zhou, Anna Chen, Murray Stackhouse. Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1504.