scholarly journals 233 Human PD-L2 triggers a unique T cell inhibitory program through PD-1 engagement distinct from that of PD-L1

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A248-A248
Author(s):  
Anupallavi Srinivasamani ◽  
Michael Curran ◽  
Qinying Liu ◽  
Shwetha Hegde ◽  
Chao-Hsien Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
Cancer Immunotherapy ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
List Type ◽  
Inhibitory Signal ◽  
Kinetics Of ◽  
L2 Cells

BackgroundPD-1/PD-L1 blockade is responsible for the majority of the success of cancer immunotherapy.1 However, only 14% of patients eligible to receive checkpoint blockade achieve objected clinical responses.2 3 The reason for the failure of PD-L1 blockade may be attributed to the recently appreciated widespread expression of PD-L2 across human cancers and its immunosuppresive stromal cells.4 PD-L2 expression was shown to be as or more predictive of response to PD-1 blockade than PD-L14. PD-L2 traditionally was dismissed as functionally redundant to PD-L1 varying only in pattern of expression. We hypothesize that PD-L2 engages PD-1 to generate a distinct inhibitory signal from that of PD-L1, and antibody mediated blockade and depletion of PD-L2+ cells may promote anti-tumor immunity that is superior to PD-L1 blockade alone.MethodsCell based bioluminescent assay demonstrated the nature of regulation mediated by human PD-L2 through the PD-1 co-receptor. RNA-sequencing identified key differences in the signaling pathways generated in Jurkat T cells by PD-1 binding to PD-L1 or PD-L2. Multidimensional flow cytometry determined the differential effects of PD-L1 and PD-L2 on human T cell proliferation and effector function. Western blot elucidated the temporal kinetics of inhibition mediated by PD-L1 and PD-L2. Survival studies in murine syngeneic lymphoma model evaluated the efficacy of antibody mediated blockade and depletion of PD-L2+ cells.ResultsWe validated that human PD-L2, unlike murine PD-L2, generates a purely co-inhibitory signal in human T cells, albeit with a reduced inhibitory potential relative to PD-L1. We discovered significant differences in downstream T cell signaling pathways generated by PD-L1 versus PD-L2 through PD-1 engagement. Human PD-L1 and PD-L2 differentially modulated T cell effector function and proliferation with PD-L2 preferentially arresting T cells in S-phase of cell cycle. PD-L1 and PD-L2 also differed in the temporal kinetics of dephosphorylation of the membrane proximal proteins in the TCR-CD3 signaling complex. We observed that combination blockade of PD-L1 and PD-L2 improves on blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our data in a syngeneic murine model of EL4 showed that effector-function capable PD-L2 blocking antibodies are therapeutically superior to PD-L1 or PD-L2 blockade alone.ConclusionsWe are the first to report on T cell immunoregulatory functions of PD-L2 which are distinct from those of PD-L1, and demonstrate that the more tumor-selective expression pattern of PD-L2 relative to PD-L1 provides a therapeutic advantage to effector-function capable PD-L2 antibodies.AcknowledgementsAS was supported by the CPRIT Research Training Grant(RP170067)ReferencesRibas A, Wolchok JD (2018). Cancer immunotherapy using checkpoint blockade. Science 359:1350–1355.Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Tomassini JE, Loboda A, Kaufman D (2018). Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science 362.Haslam A, Prasad V (2019). Estimation of the percentage of US patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs. JAMA Netw Open 2:e192535.Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T (2017). PD-L2 expression in human tumors: relevance to anti-PD-1 therapy in cancer. Clin Cancer Res 23:3158–3167.

scholarly journals 234 Distinct efficacy and immunological responses to αPD-1, αPD-L1 and αPD-L2 immunotherapy in aged versus young hosts

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A249-A250
Author(s):  
Yilun Deng ◽  
Harshita Gupta ◽  
Myrna Garcia ◽  
Aravind Kancharla ◽  
Ryan Reyes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Blockade ◽  
List Type ◽  
Aged Mice ◽  
Melanoma Model ◽  
Ifn Γ

BackgroundAging is the biggest risk factor for cancer, yet there are limited pre-clinical/clinical data regarding aging effects on immune checkpoint (IC) inhibition (ICI) outcomes. αPD-1 can potentially block PD-L1 and PD-L2 while αPD-L1 can block PD-1 and CD80. Melanoma response to αPD-1/αPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.1 Lack of host IL-17 can lead to increased IFN-γ production.2 3MethodsWe tested αPD-1 (200 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18–33 months) and young (3–8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) or TPN61R melanoma (NRAS mutation melanoma model)4 (αPD-L2 only) (SQ). Tumors were analyzed by flow. We tested αPD-L2 (20 μg/ml) effects by co-culturing young or aged T cells ± young or aged myeloid cells.ResultsWe reported that αPD-1 treats young and aged with B16 whereas αPD-L1 treats young not aged.5 αPD-L2 treated B16 and TPN61R melanoma in aged but, remarkably, not young, the first single agent anti-cancer immunotherapy exhibiting this property (figure 1). B16 tumors from aged had differential IC content (PD-1, PD-L1, CD80, PD-L2) versus tumors from young (e.g., more PD-L2+ tumor and stroma cells in aged mice; figure 2). Efficacy in young (αPD-1, αPD-L1) and aged (αPD-L2) correlated with increased tumor TCSC content (figure 3). αPD-L2 efficacy against B16 in aged mice required host IFN-γ and IL-17 (figure 4). αPD-1 efficacy against B16 in aged appeared to be host and tumor PD-L1 independent (figure 5). PD-L1KO B16 response to αPD-1 in aged also correlated with increased tumor TCSC content. Myeloid cell PD-L2 signaling inhibited aged but not young CD8+ T cell IL-2 production in vitro (figure 6).Abstract 234 Figure 1Abstract 234 Figure 2Abstract 234 Figure 3Abstract 234 Figure 4Abstract 234 Figure 5Abstract 234 Figure 6ConclusionsTreatment differences in aged versus young could depend on IC, TCSC and/or host cytokine differences (IL-17/IFN-γ). αPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that PD-L2 block is sufficient for αPD-1 efficacy in aged. PD-L2 expression differences in the tumor microenvironment could also contribute to treatment efficacy differences. PD-L2 inhibitory signaling on aged but not young CD8+ T cells is a likely mechanism for αPD-L2 efficacy in aged but not young. We are now testing the role of IL-17 in αPD-L2 efficacy as it could be upstream of IFN-γ effects, and TCSC effects in aged versus young. Our work can improve cancer immunotherapy in aged hosts and provides insights into treatment failure, including in young hosts.AcknowledgementsSouth Texas MSTP training grant (NIH T32GM113896), TL1TR002647, NIH T32AI138944, R01 CA231325, Waxman Grant, UL1 TR001120ReferencesMiller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, Yates KB, Lako A, Felt K, Naik GS, et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20(3):326–336.Moroda M, Takamoto M, Iwakura Y, Nakayama J, Aosai F. Interleukin-17A-deficient mice are highly susceptible to toxoplasma gondii infection due to excessively induced T. gondii HSP70 and interferon gamma production. Infection and immunity 2017;85(12):e00399–00317.Yi T, Zhao D, Lin C-L, Zhang C, Chen Y, Todorov I, LeBon T, Kandeel F, Forman S, Zeng D. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease. Blood, The Journal of the American Society of Hematology 2008;112(5):2101–2110.Burd CE, Liu W, Huynh MV, Waqas MA, Gillahan JE, Clark KS, Fu K, Martin BL, Jeck WR, Souroullas GP. Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma. Cancer discovery 2014;4(12):1418–1429.Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, Svatek RS, Turk MJ, Drerup JM, Li R, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146–154.Ethics ApprovalAll animal studies are approved by UTHSA IACUC.

scholarly journals Checkpoint blockade accelerates a novel switch from an NKT-driven TNFα response toward a T cell driven IFN-γ response within the tumor microenvironment

2021 ◽  
Vol 9 (6) ◽  
pp. e002269
Author(s):  
Shota Aoyama ◽  
Ryosuke Nakagawa ◽  
Satoshi Nemoto ◽  
Patricio Perez-Villarroel ◽  
James J Mulé ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
T Cell Response ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

BackgroundThe temporal response to checkpoint blockade (CB) is incompletely understood. Here, we profiled the tumor infiltrating lymphocyte (TIL) landscape in response to combination checkpoint blockade at two distinct timepoints of solid tumor growth.MethodsC57BL/6 mice bearing subcutaneous MC38 tumors were treated with anti-PD-1 and/or anti-CTLA-4 antibodies. At 11 or 21 days, TIL phenotype and effector function were analyzed in excised tumor digests using high parameter flow cytometry. The contributions of major TIL populations toward overall response were then assessed using ex vivo cytotoxicity and in vivo tumor growth assays.ResultsThe distribution and effector function among 37 distinct TIL populations shifted dramatically between early and late MC38 growth. At 11 days, the immune response was dominated by Tumor necrosis factor alpha (TNFα)-producing NKT, representing over half of all TIL. These were accompanied by modest frequencies of natural killer (NK), CD4+, or CD8+ T cells, producing low levels of IFN-γ. At 21 days, NKT populations were reduced to a combined 20% of TIL, giving way to increased NK, CD4+, and CD8+ T cells, with increased IFN-γ production. Treatment with CB accelerated this switch. At day 11, CB reduced NKT to less than 20% of all TIL, downregulated TNFα across NKT and CD4+ T cell populations, increased CD4+ and CD8+ TIL frequencies, and significantly upregulated IFN-γ production. Degranulation was largely associated with NK and NKT TIL. Blockade of H-2kb and/or CD1d during ex vivo cytotoxicity assays revealed NKT has limited direct cytotoxicity against parent MC38. However, forced CD1d overexpression in MC38 cells significantly diminished tumor growth, suggesting NKT TIL exerts indirect control over MC38 growth.ConclusionsDespite an indirect benefit of early NKT activity, CB accelerates a switch from TNFα, NKT-driven immune response toward an IFN-γ driven CD4+/CD8+ T cell response in MC38 tumors. These results uncover a novel NKT/T cell switch that may be a key feature of CB response in CD1d+ tumors.

512 Terminally exhausted CD8+ T cells potentiate the tolerogenic tumor microenvironment as functional suppressors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A548-A548
Author(s):  
Paolo Vignali ◽  
Kristin DePeaux ◽  
McLane Watson ◽  
Ashley Menk ◽  
Nicole Scharping ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Oxidative Metabolism ◽  
Advanced Melanoma ◽  
Effector T Cells ◽  
Checkpoint Blockade ◽  
Tumor Control ◽  
List Type ◽  
Murine Melanoma

BackgroundBlockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.4–8 While enrichment of this population in tumor is a negative prognostic factor,9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses.MethodsT cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery.ResultsWhen sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.11–13ConclusionsOur data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions.ReferencesWolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356.Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41.Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021.Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165.Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4.Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2.Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452.. Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724.Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.

scholarly journals Targeting Pim2 for Improving T-Cell Effector Function and Promoting Cancer Immunotherapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1720-1720
Author(s):  
Yongxia Wu ◽  
Linlu Tian ◽  
Corey Mealer ◽  
Hee-Jin Choi ◽  
Xue-Zhong Yu
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Effector Function ◽  
Cell Responses ◽  
Deficient Mice

Abstract The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinases are a highly conserved family of serine/threonine kinases. The Pim kinase family is composed of three different isoforms, Pim1, Pim2, and Pim3, which have been studied extensively in tumorigenesis and as a potential therapeutic target in various cancers. We previously reported an unexpected role of Pim2 in negatively regulates T-cell responses to alloantigen and tumor (JCI, 2015, PMID: 29781812). However, the mechanisms by which Pim2 modulates T-cell responses remain largely undefined. In the current study, using genetic Pim2-deficient mouse, we demonstrated a key role of Pim2 in regulating T-cell hemostatic and anti-tumor responses in aging, hematopoietic cell transplantation (HCT), and antigen-specific adoptive T-cell therapy (ACT). We observed that Pim2 was critical for T cells to retain quiescent in aged mice, as thymic Treg development was impaired while effector T-cell differentiation in lymphoid organs, including Tc1/Th1, Tc17/Th17 and follicular helper T cells, was increased in Pim2-deficient mice, but not in Pim1/Pim3-deficient mice. Furthermore, Pim2-deficient mice were capable to completely eradicate syngeneic breast cancer (NT2.5) growth (Figure A). During antigen specific anti-tumor response, adoptively transferred Pim2 -/- CD8 T cells showed enhanced ability for controlling established NT2.5 breast cancer and B16 melanoma (Figure B, C). Mechanistically, loss of Pim2 promoted G1 to S phase cell-cycle progression while reduced apoptosis in CD8 T cells. Pim2 -/- CD8 T cells exhibited elevated effector cytokine production while maintained higher levels of CD62L expression, leading to superior effector function, persistence and anti-tumor activity. Reduced differentiation of exhausted and suppressive subsets were observed in Pim2 -/- CD8 T cells after being adoptively transferred in tumor-bearing mice. In addition, Pim2 deficiency was associated with a higher metabolic potential, reflected by increased glycolysis and oxidative phosphorylation, which was at least partially attributed to a decreased level of autophagy in Pim2 -/- CD8 T cells. To further evaluate the clinical translation potential, we applied a Pim2-specific inhibitor (JP11646) and found that blocking Pim2 improved graft-versus-leukemia activity after autologous HCT and also enhanced CD8 T-cell mediated anti-melanoma effects after ACT in mice (Figure B, C). Furthermore, blocking Pim2 using JP11646 promoted human CD8 T-cell response during polyclonal stimulation and enhanced expansion, effector function and tumor killing ability of human melanoma antigen-specific CD8 T cells (data not shown) and CD19 CAR-T cells (Figure D). Our work demonstrated that Pim2 is a potent and distinct regulator of differentiation and maintenance of T effector cells through modulating metabolism and autophagy. Specifically target Pim2 can serve as a novel strategy for improving cancer immunotherapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets

2016 ◽  
Vol 213 (8) ◽  
pp. 1589-1608 ◽  
Author(s):  
Cindy S. Ma ◽  
Natalie Wong ◽  
Geetha Rao ◽  
Akira Nguyen ◽  
Danielle T. Avery ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
Cd4 T Cells ◽  
Clinical Manifestations ◽  
Effector Function ◽  
Cd4 T Cell ◽  
The Impact ◽  
Cell Effector Function ◽  
Cell Effector

Naive CD4+ T cells differentiate into specific effector subsets—Th1, Th2, Th17, and T follicular helper (Tfh)—that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4+ T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10–secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4+ T cell effector function in the settings of infection, vaccination, or immune dysregulation.

scholarly journals 879 A promising cancer immunotherapy target: novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A921-A921
Author(s):  
Thierry Guillaudeux ◽  
Yulia Ovechkina ◽  
Shaarwari Sridhar ◽  
David Jurchen ◽  
David Peckham ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Receptor Activation ◽  
Cytotoxic T Cell ◽  
T Cell Proliferation ◽  
List Type

BackgroundCD27 is a member of the TNF receptor superfamily and plays a critical role in T-cell activation by providing a costimulatory signal. CD27 signaling enhances T-cell proliferation, activation and differentiation of effector and memory T cells and therefore promotes cytotoxic T cell (CTL)-based anti-tumor immunity.1 Agonistic stimulation of CD27 is a promising cancer immunotherapy approach to boost specific T cell driven anti-tumor responses.MethodsIn this study, we generated a series of 147 fully human monoclonal anti-CD27 antibodies and tested their agonist properties to stimulate T cell activation.ResultsUsing a NF-κB reporter Jurkat cell line, we evaluated in vitro the ability of anti-CD27 antibodies to induce CD27 receptor activation. With this assay, five antibodies have been selected for their agonist properties. When combined with suboptimal T cell receptor (TCR) stimulation, agonist antibodies induced CD27 receptor activation with an EC50 of 1–5 ug/mL. We also used human peripheral blood T cells to characterize the CD27-mediated costimulatory effects of agonist antibodies in combination with TCR stimulation. Our anti-CD27 monoclonal antibodies boosted T cell proliferation and induced IL-2 and TNFalpha secretion only in a presence of TCR engagement. Moreover, CD27 agonists induce strong T cell proliferation in a Mixed Lymphocyte Reaction. CD27 antibodies were shown to bind human and cynomolgus monkey CD27 with a KD value of 5–20 nM as determined by BioLayer Interferometry, but do not bind to mouse CD27. In vivo experiments are currently ongoing to demonstrate the efficient anti-tumor activity of the selected CD27 agonist antibodies in different mice tumor models.ConclusionsIn conclusion, we have developed and successfully selected efficient fully human immuno-stimulatory agonist CD27 mAbs as a promising cancer immunotherapy.ReferenceHendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J Exp Med 2003;Volume 198, Number 9:1369–1380.

scholarly journals 183 Therapeutic T cells exhibit distinct vulnerability to glucose deprivation in tumors which can be overcome with an engineered glucose transporter

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A194-A194
Author(s):  
Jessica Jana ◽  
Yiyang Wang ◽  
Ashley Menk ◽  
Andrew Frisch ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Tumor Cells ◽  
Glucose Transporter ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
Cell Therapies ◽  
Effector Functions

BackgroundWhile checkpoint blockade cancer immunotherapies reawaken dormant antitumor immunity, adoptive cell therapies (ACT) bolster an immune response through infusion of expanded tumor infiltrating lymphocytes (TIL), or healthy T cell redirected to tumors via chimeric antigen receptor (CAR), or T cell receptor (TCR) expression. However, the harsh, nutrient depleted tumor microenvironment (TME) creates metabolic barriers for T cell persistence and effector function. We and others have shown that glucose availability is key for T cell effector functions through multiple non-redundant mechanisms. We thus asked whether therapeutic T cells harbor increased sensitivity for glucose and whether this could be mitigated to increase efficacy.MethodsB16 and Pten deficient, Braf mutant melanomas were used as models in C57BL/6 mice. Tumor cell glucose uptake was inhibited using stable expression of shRNA to Slc2a1, encoding Glut1. Glut1 was retrovirally overexpressed in therapeutic, Pmel-1 (gp100-specific) T cells, and phosphomimetic mutations were engineered (S226D) into Glut1, stabilizing cell surface trafficking. Glucose uptake and glycolysis were measured using fluorescent glucose tracers and Seahorse analysis, respectively.ResultsHere we sought to equip glucose sensitive therapeutic T cells with heightened ability to compete for glucose within the TME. Murine therapeutic T cells, expanded in hyperglycemic conditions in vitro, that infiltrate solid tumors compete poorly for glucose tracers compared to endogenous T cells. Knockdown or deletion of Glut1 in tumor cells sensitizes tumors to T cell therapies, but not checkpoint blockade, highlighting a role for glucose competition specifically in ACT. Overexpression of WT Glut1 in therapeutic T cells yields only modest increased glucose competition due to various modes of Glut1 regulation. We thus engineered a cell surface stabilized Glut1 construct. This construct’s competitive advantage manifests robust increases in glucose uptake and glycolytic capacity, leading to superior effector functions even in extremely hypoglycemic conditions. This enhanced effector function manifests in therapeutic efficacy in highly glycolytic melanomas and with heightened competition for glucose tracers, tumor infiltration, and effector function in vivo.ConclusionsOur study suggests that, due to the hyperglycemic conditions of their ex vivo expansion, therapeutic T cells display a distinct metabolic disadvantage when they enter the nutrient poor TME in comparison to their endogenous counterparts. Overexpression of our surface engineered Glut1 in these therapeutic T cells rescues their ability to compete with highly glycolytic tumor cells for glucose, resulting in robust glycolytic metabolism and curative response to immunotherapy for cancer.

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