Stimulation Of CD4 Research Articles

Association between Immunosenescence, Mitochondrial Dysfunction and Frailty Syndrome in Older Adults

Aging is associated with changes in the immune system, increased inflammation and mitochondrial dysfunction. The relationship between these phenomena and the clinical phenotype of frailty is unclear. Here, we evaluated the immune phenotypes, T cell functions and mitochondrial functions of immune cells in frail and robust older subjects. We enrolled 20 frail subjects age- and gender-matched with 20 robust controls, and T cell phenotype, response to immune stimulation, cytokine production and immune cell mitochondrial function were assessed. Our results showed that numbers of CD4+ and CD8+ T cells were decreased in frail subjects, without impairment to their ratios. Memory and naïve T cells were not significantly affected by frailty, whereas the expression of CD28 but not that of ICOS was decreased in T cells from frail subjects. T cells from robust subjects produced more IL-17 after CD28 stimulation. Levels of serum cytokines were similar in frail subjects and controls. Mitochondrial bioenergetics and ATP levels were significantly lower in immune cells from frail subjects. In conclusion, we suggest that changes in T cell profiles are associated with aging rather than with frailty syndrome; however, changes in T cell response to immune stimuli and reduced mitochondrial activity in immune cells may be considered hallmarks of frailty.

Effects of Bacterial Metabolites on CAR-T Cell Therapy

Introduction Cancer immunotherapy by chimeric antigen receptor modified T cells (CAR-T cells) has shown promising clinical efficacy in hematological malignancies. Nevertheless, the success of the therapy is limited, and patients suffer from relapse. Bacterial metabolites were described as important host intrinsic regulators of innate immune pathways like cGAS/STING and RIG-I/MAVS. However, their role on CAR-T cell therapies and mechanism of action is currently unknown. We investigated the CAR-T cell intrinsic and extrinsic role of intestinal derived interferon-linked bacterial metabolites on cytotoxicity and exhaustion. Methods Based on previously generated data we chose two bacterial derived metabolites showing beneficial effects on survival of patients after allogeneic stem cell transplantation. Anti-CD19-CD28z CAR-T cells were treated with metabolites for 72 hours and cytokine production as well as expression of extracellular activation markers were assessed by flow cytometry. To determine the killing capacity of metabolite treated CAR-T cells on CD19+ Nalm6 ALL blasts, in vitro bioluminescence-based cytotoxicity assays at a 1:8 E:T ratio over 24 hours were performed. Expression of extracellular and transcriptional exhaustion markers were analyzed by flow cytometry after anti-CD3/CD28 stimulation as well as after repetitive challenge with tumor cells. Results Our data suggest an immunosuppressive effect of one of the metabolites via direct modulation of CAR-T cells as well as innate immune cells. We could show that treatment with metabolite 1 leads to CAR-T cell activation, activation induced cell death (AICD) and increased expression of exhaustion markers (PD-1, LAG-3 and TIM3) in antiCD19-CD28z CAR-T cells, leading to impaired cancer cell cytotoxicity in vitro in a concentration dependent fashion. To better investigate the complex interaction between innate immune cells and CAR-T cells we further set up an in vitro coculture assay of CAR-T cells and macrophages. Our data suggest effects of these metabolites on the polarization of macrophages modulating the CAR-T cell function and immune response. Additionally, we performed polyclonal knockouts in CAR-T cells of type I interferon related genes (Stimulator of interferon genes (STING), Retinoic acid-inducible gene I (RIG-I), Interferon-α/β receptor(IFNAR1), C/EBP Homologous Protein (CHOP)) and the metabolite sensor Aryl-hydrocarbon-receptor (AHR) in order to narrow down the mechanism of action of these metabolites on the T cell level, which revealed putative involvement of the AHR signaling pathway, but mostly independence from type I IFN pathways. Further, we could show distinct, metabolite specific cytokine secretion of monocyte derived macrophages and CAR-T cells after stimulation with the two metabolites. Conclusions Our data suggest a direct, CAR-T cell intrinsic and innate immune cell mediated immunosuppressive effect of metabolite 1. These findings will now be validated in vivo and the mechanism of action besides AHR-agonism will be further elucidated utilizing single cell RNAseq.

Attenuation of Graft-Versus-Host-Disease Via Genetic or Pharmacologic Inhibition of the RNA Splicing Factor RBM39

Recent studies have identified pharmacologic modulation of RNA splicing via degradation of the splicing factor RBM39 to mediate anticancer effects and enhance responses to immune checkpoint blockade through generation of novel RNA-splicing derived immunogenic peptides in tumors. However rigorous evaluation of the role of RBM39 in immune cells has not been performed. Here we identify that RBM39 modulates exon recognition in T cells to regulate their function. Although essential for the survival of hematopoietic precursors and tumors, RBM39 is dispensable for peripheral T cell survival. Functional evaluation of the T cell alloantigen response in allogeneic hematopoietic stem transplantation (allo-HSCT) revealed that Rbm39 loss attenuated graft-versus-host-disease (GVHD) and improved overall survival while partially sparing graft-versus-tumor (GVT) effects. To rigorously evaluate the role of RBM39 in immune cells, we generated an Rbm39 conditional knockout mouse by floxing Rbm39 exon 7, which encodes its first RNA recognition motif. Pan-hematopoietic deletion of Rbm39 resulted in rapid complete failure of hematopoiesis and impaired hematopoietic stem cell (HSC) self-renewal. We next analyzed the phenotype of animals with T cell specific deletion of RBM39 and compared this with the effects of pharmacologic RBM39 degradation on T cells. Strikingly, in contrast to the profound effects of genetic RBM39 loss in HSCs and the inability of cancer cells to tolerate RBM39 deletion, RBM39 deletion under CD4-cre (deletion early in thymopoiesis) or distal Lck-cre (late in thymopoiesis) yielded modestly fewer viable peripheral T cells, which skewed towards a CD44+ effector/memory phenotype. This observation was also recapitulated with extended treatment of mice with the RBM39 degrader E7820 (in phase II trials currently; clinicaltrials.gov NCT05024994). Deep RNA-seq analysis of splenic CD3+ T cells from distal Lck-cre Rbm39fl/fl mice revealed widespread decreased efficiency of RNA splicing in Rbm39 null T cells compared to controls, indicative of altered RNA splicing in T cells with Rbm39 deletion. Rbm39 KO T cells experienced global increases in exon skipping at genomic regions where upstream introns had reduced GC content. These data identify that RBM39 is required in a cell and tissue-specific manner for cell survival and further highlights the selectivity of therapeutic approaches targeting RBM39. We next assessed the functional consequences of pharmacologic RBM39 degradation or genetic RBM39 deletion in T cells. Consistent with observations in genetic mouse models, T cells could tolerate extensive pharmacologic RBM39 degradation without loss of proliferative capacity in response to PMA/Ionomycin or anti-CD3+CD28 stimulation. Similarly, genetic Rbm39 null T cells had an intact proliferative response to PMA/Ionomycin. Furthermore, when we evaluated the effects of RBM39 loss in the MHC-mismatched C57BL/6 => Balb/c allo-HSCT system, Rbm39 deletion in donor T cells under either CD4- or distal Lck-cre profoundly reduced GVHD severity and improved overall survival in recipients (Fig. A). These genetic findings were recapitulated via pharmacologic degradation of RBM39 in the same HSCT system (Fig. B). Similar experiments performed in the setting of Balb/c hosts engrafted with A20 lymphoma cells revealed partial preservation of GVT activity using Rbm39 KO T cells. Single cell RNA sequencing analyses of CD4-cre Rbm39fl/fl primary T cells revealed upregulation of the integrin beta1 subunit in both CD4 and CD8 T cells. This suggests alteration of T cell trafficking away from GVHD target organs as one mechanism by which RBM39 loss may alter T cell alloreactivity in allo-HSCT. Our results identify cell-type specific roles for RBM39 in hematopoiesis and immune cells and suggest a therapeutic approach for GVHD via direct modulation of RNA splicing. Of note, as the RBM39 degrader E7820 is currently in phase II clinical trials for relapsed myeloid neoplasms, our previous studies and this data identify a treatment for myeloid neoplasms in the post-transplant setting, where RBM39 degraders mediate both direct and immune-mediated anti-tumor effects while also attenuating GVHD via modulation of RNA splicing in T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-52. HARNESSING A B CELL THERAPY TO PROMOTE ANTI-GLIOBLASTOMA HUMORAL RESPONSE

Abstract Glioblastoma (GBM) continues to retain its dismal prognosis despite numerous new therapeutic modalities to target various aspects of the tumor. There is a need to identify new, and even personalized, targetable GBM specific antigens. Our B-cell-based vaccine (BVax) is generated by isolating immune experienced B cells, identified by 4-1BBL, from murine secondary lymphoid organs or patient blood. We have shown this subset of B cells to have anti-tumoral potential in GBM. These cells are strengthened with BAFF, CD40, and IFNg stimulation to form BVax and then activated in vitro to form plasmablasts. Immunoprecipitation-mass spectrometry is performed using BVax-derived antibodies and tumor lysate from the paired patient specimen. We have identified unique antigens bound by BVax-derived antibodies. Given the significant therapeutic benefit we have seen using BVax-derived antibodies in preclinical murine models, we hypothesize that targeting these unique antigens would have significant therapeutic benefit in GBM patients. We focused on antigens that showed a survival benefit with lower expression using CGGA database analysis. We then confirm the presence and prevalence of the antigens within tumor cells in the patient’s tissue using immunohistochemistry, identifying some antigens in up to 15% of tumor cells. Using our brain tumor bank repository, we screened other GBM patient’s tissue for the presence of these markers to determine the broader applicability of targeting each antigen. We have identified proteins key in extracellular matrix formation which promote tumor growth and progression in cancer, including the ability to induce epithelial-mesenchymal transition. Simultaneously we have developed a computational algorithm to predict which antigens BVax-derived antibodies will bind using single cell RNA sequencing data from GBM patient-derived tumor and BVax, which may have broader applicability to a larger number of patients. Both techniques serve as exciting platforms to identify new therapeutic targets in GBM.

Changes in Immune Function during Initial Military Training.

Initial military training (IMT) is a transitionary period wherein immune function may be suppressed and infection risk heightened due to physical and psychological stress, communal living, and sleep deprivation. This study characterized changes in biomarkers of innate and adaptive immune function, and potential modulators of those changes, in military recruits during IMT. Peripheral leukocyte distribution and mitogen-stimulated cytokine profiles were measured in fasted blood samples, Epstein-Barr (EBV), varicella zoster (VZV), and herpes simplex 1 (HSV1) DNA was measured in saliva by quantitative polymerase chain reaction as an indicator of latent herpesvirus reactivation, and diet quality was determined using the healthy eating index measured by food frequency questionnaire in 61 US Army recruits (97% male) at the beginning (PRE) and end (POST) of 22-wk IMT. Lymphocytes and terminally differentiated cluster of differentiation (CD)4+ and CD8+ T cells increased PRE to POST, whereas granulocytes, monocytes, effector memory CD4+ and CD8+ T cells, and central memory CD8+ T cells decreased ( P ≤ 0.02). Cytokine responses to anti-CD3/CD28 stimulation were higher POST compared with PRE, whereas cytokine responses to lipopolysaccharide stimulation were generally blunted ( P < 0.05). Prevalence of EBV reactivation was higher at POST ( P = 0.04), but neither VZV nor HSV1 reactivation was observed. Diet quality improvements were correlated with CD8+ cell maturation and blunted proinflammatory cytokine responses to anti-CD3/CD28 stimulation. Lymphocytosis, maturation of T-cell subsets, and increased T-cell reactivity were evident POST compared with PRE IMT. Although EBV reactivation was more prevalent at POST, no evidence of VZV or HSV1 reactivation, which are more common during severe stress, was observed. Findings suggest increases in the incidence of EBV reactivation were likely appropriately controlled by recruits and immune-competence was not compromised at the end of IMT.

FcγRs and Their Relevance for the Activity of Anti-CD40 Antibodies.

Inhibitory targeting of the CD40L-CD40 system is a promising therapeutic option in the field of organ transplantation and is also attractive in the treatment of autoimmune diseases. After early complex results with neutralizing CD40L antibodies, it turned out that lack of Fcγ receptor (FcγR)-binding is the crucial factor for the development of safe inhibitory antibodies targeting CD40L or CD40. Indeed, in recent years, blocking CD40 antibodies not interacting with FcγRs, has proven to be well tolerated in clinical studies and has shown initial clinical efficacy. Stimulation of CD40 is also of considerable therapeutic interest, especially in cancer immunotherapy. CD40 can be robustly activated by genetically engineered variants of soluble CD40L but also by anti-CD40 antibodies. However, the development of CD40L-based agonists is biotechnologically and pharmacokinetically challenging, and anti-CD40 antibodies typically display only strong agonism in complex with FcγRs or upon secondary crosslinking. The latter, however, typically results in poorly developable mixtures of molecule species of varying stoichiometry and FcγR-binding by anti-CD40 antibodies can elicit unwanted side effects such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of CD40 expressing immune cells. Here, we summarize and compare strategies to overcome the unwanted target cell-destroying activity of anti-CD40-FcγR complexes, especially the use of FcγR type-specific mutants and the FcγR-independent cell surface anchoring of bispecific anti-CD40 fusion proteins. Especially, we discuss the therapeutic potential of these strategies in view of the emerging evidence for the dose-limiting activities of systemic CD40 engagement.

Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy

BackgroundImmune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI...

Changes In Leukocyte Distribution And T-cell Reactivity During Initial Military Training

PURPOSE: Physiological stressors present during initial military training (IMT) may increase risk of illness and infection by suppressing immune function. This study aimed to improve insight into those relationships by characterizing changes in markers of innate and adaptive immune function during IMT. METHODS: Sixty U.S. Army infantry recruits (97% male) provided fasted blood and saliva samples at the beginning (PRE; week 0) and end (POST; week 22) of IMT. Peripheral leukocyte distribution was measured by flow cytometry, and T-cell and innate cell reactivity were measured by mitogen (anti-CD3/CD28 or lipopolysaccharide (LPS))-stimulated cytokine profiles in 48-hr whole blood culture. Latent reactivation of Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and herpes simplex virus 1 (HSV1) was measured in saliva by qPCR. RESULTS: Lymphocyte percentage increased (+28%) while granulocytes (-15%) and monocytes (-13%) decreased PRE to POST (all P < 0.01). This lymphocytosis was driven by an increase in natural killer cells (+19%), and accompanied by decreases in B-cells (-9%) and T-cells (-5%; all P < 0.01). Within T-cell subsets, terminally differentiated CD4+ (+22%) and CD8+ (+30%) cells increased while those of effector memory CD4+ cells (-11%), effector memory CD8+ cells (-54%) and central memory CD8+ cells (-15%) decreased (all P ≤ 0.02). T-cell production of most measured cytokines following anti-CD3/CD28 stimulation increased from PRE to POST (P < 0.05). Conversely, stimulation with LPS resulted in reduced production of most measured cytokines (P < 0.05). Prevalence of EBV reactivation was higher POST (n=30) relative to PRE (n=19; P = 0.01), but neither VZV nor HSV1 reactivation was observed at either time point. CONCLUSION: Lymphocytosis, maturation of CD4+/CD8+ T-cell subsets, and heightened T-cell reactivity at POST collectively suggests an appropriate immune response to pathogen challenges during IMT. Though EBV reactivation was increased at POST, no evidence of VZV or HSV1 reactivation, which are more common during severe stress, was observed. Findings suggest that EBV reactivation during IMT was likely appropriately controlled in recruits and that immune-competence was relatively uncompromised at the end of IMT. Supported by USAMRDC. Authors’ views not official DoD or Army policy

Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Faslpr/lpr Mice.

Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFNγ), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2+ lymphocytes (miR-182−/−B6/lpr and miR-183C−/-B6/lpr). The miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/−B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183Cfl/flB6/lpr controls. In vitro activated splenocytes from miR-182−/−B6/lpr mice and miR-183C−/−B6/lpr mice showed reduced ability to produce lupus-associated IFNγ. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4+ cells of miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182−/−B6/lpr and miR-183C−/-B6/lpr mice significantly increased IFNγ expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFNγ in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4+, CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells in the miR-183C−/−B6/lpr and miR-182−/−B6/lpr mice and their respective littermate controls, miR-183Cfl/flB6/lpr and miR-182fl/flB6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFNγ in in vitro activated splenocytes from B6/lpr mice.

Normal mesenchymal stem cells can improve the abnormal function of T cells in psoriasis via upregulating transforming growth factor-β receptor.

Psoriasis, a chronic inflammatory skin disease, is a refractory disorder. Previous studies have shown that the imbalance of the T-helper (Th)17/regulatory T cells (Treg) results in the immune imbalance of T cells in psoriatic patients, and that mesenchymal stem cells display an immunosuppressive role by promoting the differentiation of T cells into Treg, leading to a reduction in the proportion of Th17/Treg. Utility of mesenchymal stem cells is becoming a new approach for the treatment of immune disorders. Following co-culture of dermal mesenchymal stromal cells (DMSC) and CD3+ T cells with or without transforming growth factor (TGF)-β receptor inhibitor, the biological function and relative signal pathway of CD3+ T cells were assessed by flow cytometry, transwell, real-time polymerase chain reaction and western blotting, respectively. Normal DMSC were more potent than psoriatic DMSC in inhibition of CD3+ T-cell proliferation, and stimulation of CD3+ T-cell apoptosis than psoriasis DMSC. Moreover, normal DMSC decreased the ratio of Th17/Treg, while enhancing the immunosuppressive effect of Tregs on effector T cells. However, TGF-β receptor (TGF-βR)inhibitor attenuated the effect of normal DMSC on CD3+ T cells and Th17/Treg ratio. Additionally, the normal DMSC were more potent than the psoriatic DMSC in increasing TGF-β receptors and activation of TGF-β/SMAD pathway in psoriatic CD3+ T cells. In conclusion, normal DMSC can partially improve the biological function and immunosuppressive ability of psoriatic CD3+ T cells, possibly via upregulating the TGF-β receptors.

Abstract 2122: CRD1600 is a potent HPK-I inhibitor with robust immune modulatory properties

Abstract Background: Haematopoietic Progenitor Kinase (HPK-1, MAP4K1) is a member of the STE20 family of serine/threonine kinases, expressed predominantly in hematopoietic cells. The kinase activity of HPK-1 provides an inhibitory signal that dampens immune responses1. Upon activation of T-cell and/or B-cell receptors, activated HPK-1 phosphorylates the adaptor protein SLP76 at Ser376 in T cells or BLNK at Thr152 in B-cells to trigger proteasomal degradation of the adaptors and disrupt downstream T-cell or B-cell activating signalosome complexes. Blocking HPK-1 extends immune action and since HPK-1 expression is primarily restricted to immune cells, it is a compelling drug target for immune modulation. Anti-tumor efficacy has been demonstrated in HPK-1 kinase dead knock-in mice, HPK-1 knockout mice or using small molecule pharmacological inhibitors of HPK-1 either as monotherapy or in combination regimens2. Methods: Curadev used a pharmacophore based approach to discover novel small molecule HPK-1 inhibitors. SAR studies were carried out using a screening funnel that included efficacy assessment by monitoring in vitro enzymatic and cellular activity, anti-target cross-kinase specificity by assessing cytotoxicity in fresh murine splenocytes and effector mobilization by monitoring IL2 production and human T-cell proliferation under anti-CD3/CD28 stimulation. Results: Curadev’s medicinal chemistry campaign yielded several highly potent HPK-1 inhibitors with sub-nanomolar to nanomolar potency in in vitro human HPK-1 enzyme assays which were active in the cellular SLP76 assay. A single dose of the lead compound CRD1600 to mice treated with an anti-CD3 antibody caused systemic enhancement of pro-inflammatory cytokines such as IL2 and reduced pSLP76 levels in the spleen. These pharmacodynamic effects correlated with plasma levels of CRD1600. Conclusions: Inhibition of HPK-1 is a promising therapeutic modality that could augment the effects of existing anti-cancer treatments including immunotherapy. CRD1600 is a potent, selective HPK-1 inhibitor that has been selected from a rich portfolio of active compounds and shows in vivo activity against the target. Further development of the compound is ongoing.

Abstract 3579: T cell intrinsic DNA damage and repair response as a novel marker associated with clinical response to PD-1 blockade

Abstract Despite the high tumor mutational burden (TMB), immune check point blockade (ICB) still fails in about half of microsatellite instability-high (MSI-H) cancer patients, suggesting underlying immune dysregulation. Immune profiling of peripheral blood from chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab identified a rapid proliferative CD8 T cell response 2-4 weeks post PD-1 blockade (N = 32). This immunological response, however, did not correlate with clinical response, suggesting additional parameters may be relevant. We focused on DNA damage and repair (DDR) in T cells as potential novel parameters. DDR has been extensively studied in the context of inducing cell death in highly-proliferating tumor cells. However, despite induction of rapid proliferation of T cells upon ICB, whether T cell-intrinsic DDR impacts T cell function, and how the coordination of DDR affects immunological and clinical response to ICBs have been largely unexplored. We hypothesized that the T celI-intrinsic DDR responses to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response. To test the hypothesis, we developed a novel high-dimensional cytometry platform. This DDR-Immune platform enables simultaneous analysis of T cell differentiation state with changes in major DDR pathways at single-cell resolution. The DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage, such as γ-irradiation (IR), UV irradiation (UV) or proliferative stress (i.e. anti-CD3/CD28 stimulation). For example, terminally differentiated effector T cells accumulated higher DNA damage and cell death. In contrast, stem cell memory (TSCM) and regulatory T cells (Treg) displayed high DDR with less cell death, suggesting that effective cell-intrinsic DDR against genotoxic stress in T cells confers a survival advantage. We applied the platform to MSI-H uterine cancer cohort to test if T cell-intrinsic DDR distinguish the clinical response. Indeed, we found that in clinical responders but not clinical non-responders, Ki67+ CD8 T cells responding to PD-1 blockade exhibited rapid induction of DDR represented as a spike increase of phosphorylated-ATM (pATM). This likely indicates the T cell ‘fitness’ response to proliferative stress induced by PD-1 blockade. Furthermore, patients with higher induction of pATM in responding CD8 T cells at the peak of the immunological response to PD-1 blockade had longer progression-free survival (PFS). Collectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome to ICB and has potential application to other cancer therapies including chemotherapy and radiotherapy. Citation Format: Yuki Muroyama, Sasikanth Manne, Derek A. Oldridge, Nils Wellhausen, Allison R. Greenplate, Lakshmi Chilukuri, Divij Mathew, Caiyue Xu, Ramin S. Herati, Alexander C. Huang, Dmitriy Zamarin, Claire F. Friedman, E. John Wherry. T cell intrinsic DNA damage and repair response as a novel marker associated with clinical response to PD-1 blockade [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 3579.

Abstract 3869: A human in-vitro T cell exhaustion model for assessing immuno-oncology therapies

Abstract Chronic stimulation of CD8 T cells either as a result of viral infection or within the tumor microenvironment can lead to a state of exhaustion, whereby T cells no longer make a productive effector response. Remobilisation of exhausted tumor specific CD8 T cells (TEX) presents a possible therapeutic strategy for driving anti-tumor T cell responses. The success of biologics targeting receptors and ligands associated with exhausted T cells such as PD-1/PD-L1 and CTLA-4 has spurred an acceleration of research to unravel the molecular processes that drive or maintain the exhausted state with the goal of developing treatments that target them. Effective therapeutic development necessitates reliable human in-vitro models that closely mimic disease conditions. To achieve this we adapted an in-vitro TEX model via application of flowing cytometry and time-lapse imaging techniques to kinetically assess TEX cell phenotype and function. Persistent and repeated rounds of polyclonal human CD8 T cell stimulation recapitulated distinctive characteristics of exhausted T cells including high expression of checkpoint receptors and upregulation of transcription factors known to suppress immune activation. Over 8 days and 4 rounds of stimulation TEX cells lost effector capabilities, producing lower levels of pro-inflammatory cytokines and became refractory to re-stimulation through the TCR. In functional cytotoxicity assays TEX cells exhibited substantial deficits in target killing compared to donor matched Teff cell counterparts. Checkpoint receptor blockade partially restored Teff function by enhancing immune mediated target cell death. Overall, this in-vitro TEX assay models key aspects of CD8 T cell exhaustion and offers a versatile and accessible platform to test novel therapeutics targeting exhaustion pathways either individually or in combination with checkpoint inhibitors to test if they fully restore effector function. Citation Format: Paulina Goral, Louise Brackenbury, Robert Nunan, Rhiannon Jenkinson. A human in-vitro T cell exhaustion model for assessing immuno-oncology therapies [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 3869.

Abstract 5559: Development of a novel PD-L1xCD40 bispecific antibody with excellent efficacy and safety profile for cancer therapy

Abstract CD40 agonistic antibody is emerging as a front-line immunotherapy for patients with cancer. However, despite its remarkable curative effects, serious immune-related adverse reactions limit the clinical application of CD40 agonists. Crosslinking-dependent activation is one of the promising approaches to diminish the adverse effects of CD40 agonists. Agonistic CD40 stimulation on antigen presenting cells (APCs) not only promotes activation of T cells, but also results in PD-L1 upregulation on APCs, thus creating a negative feedback loop to dampen the anti-tumor immune response. Therefore, we hypothesized that the combination of CD40 agonist and blockade of PD-1/PD-L1 interaction may further induce a synergistic effect on T cell activation and anti-tumor activity. Here, we report a novel PD-L1xCD40 Bispecific Antibody (BsAb) designed to have synergistic curative effects as well as improved safety profiles. The PD-L1/CD40 BsAb was generated using the Harbour HBICE࣪® platform. It exhibited superior binding activities to both PD-L1 and CD40, respectively. CD40 activation was fully relied on the PD-L1 crosslinking. The PD-L1xCD40 BsAb showed potent tumor growth inhibition in preclinical efficacy models. Meanwhile, it showed much improved safety profile compared to a reference anti-CD40 agonistic antibody in the preclinical tox models. Overall, the PD-L1xCD40 BsAb demonstrated remarkable in vivo anti-tumor efficacy and improved safety profiles in preclinical models, which presents it as a promising candidate for the development of next-generation cancer immune therapeutics. Citation Format: Haishan luo, Zihong Meng, Jimmy Rong, Yuandong Wang, Yunxing Yang, Fei Chen, Youhong Wang, Victor Chen, Gang Deng, Yebo He, Xin Gan, Yiping Rong. Development of a novel PD-L1xCD40 bispecific antibody with excellent efficacy and safety profile for cancer therapy [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 5559.

Abstract 4261: CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses

Abstract CD27 is a member of the TNF-receptor superfamily, highly expressed on CD4+ and CD8+ T cells as well as on NK and NKT cells. It plays a key role on T cell proliferation and differentiation after stimulation with its ligand CD70. The co-stimulatory signal of CD27 on T cell is mediated via the NFκB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B. CD27/CD70 co-stimulation has the potential to boost immunity by T-cell activation, clonal expansion and enhanced differentiation into antigen specific cytotoxic and memory T cells. CD27/CD70 also influences the innate immune response via a direct activation of the NK cells and a subsequent secretion of interferon-gamma (IFN-γ). Therefore, CD27 signaling promotes cytotoxic T cell based anti-tumor immunity. With its central role in an immunological response, CD27 is a promising target for antitumor therapy. Previous works have demonstrated the efficacy of an agonistic CD27 antibody in controlling tumor growth and metastasis in different mice models including melanoma, renal cell carcinoma, breast cancer and lymphomas. This anti-tumor effect is mediated in part by an effective recruitment of IFN-γ producing CD8+ T cells within the tumor. Moreover, CD27 stimulation of Tumor Infiltrating Lymphocytes (TILs) can lower their threshold of activation and provide a broader repertoire of Ag-reactive T cells within the tumor. We have selected a lead therapeutic antibody from our library of 147 fully human anti-CD27 monoclonal antibodies generated in the Trianni mice. After confirming its binding potency and selectivity as well as its cross-reactivity with Non-Human Primate (NHP)-CD27 but not with the mouse-CD27, this lead candidate demonstrated strong agonistic proprieties. This was shown by its ability to induce a strong NFκB signal as well as to induce T cell proliferation and activation with secretion of pro-inflammatory cytokines. This antibody demonstrated agonistic proprieties without cross-linking confirming its potency. T cell activation observed after treatment with our anti-CD27 antibody only occurs in the presence of TCR engagement, preventing the risk of spontaneous activation of naïve T cells in vivo. The ability of our CD27 monoclonal antibody to increase an immune response was confirmed in a Mixed Lymphocyte Reaction assay with multiple donors. The role played by the NK cells and their activation via CD27 antibody was also demonstrated. To evaluate the anti-tumor functions of our lead antibody as a single agent or in combination with other immuno-therapies in vivo we have used human CD27 transgenic mice. We have demonstrated in the MB49 bladder tumor model as well as the EG7 thymoma model that our lead antibody induces a strong single agent anti-tumor activity and these tumors were totally controlled in combination with an anti-PD1 antibody. We are now analyzing the pharmacokinetic and pharmacodynamic of our antibody as well as its safety in a NHP model Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Shaarwari Sridhar, David Peckham, Jessica Cross, Nathan Eyde, Emily Frazier, Neda Kabi, Remington Lance, Kurt Lustig, Mei Xu, Tarcha Eric, Shawn Iadonato. CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses [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 4261.

An Adaptable Antibody‐Based Platform for Flexible Synthetic Peptide Delivery Built on Agonistic CD40 Antibodies

AbstractThe agonistic potentials of therapeutic anti‐CD40 antibodies have been profiled in relation to antibody isotype and epitope specificity. Still, clinical impact relies on a well‐balanced clinical efficacy versus target‐mediated toxicity. As CD40‐mediated immune activation must rely on a combination of stimulation of antigen‐presenting cells (APCs) alongside antigen presentation, for efficient T cell priming, alternative approaches to improve the therapeutic outcome of CD40‐targeting strategies should focus on providing optimal antigen presentation together with CD40 stimulation. Herein, a bispecific antibody targeting CD40 as a means to deliver cargo (i.e., synthetic peptides) into APCs through a non‐covalent, high‐affinity interaction between the antibody and the cargo peptide, further referred to as the Adaptable Drug Affinity Conjugate (ADAC) technology, has been developed. The ADAC platform demonstrated a target‐specific CD4+ and CD8+ T cell expansion in vitro and significantly improved peptide‐specific CD8+ T cell proliferation in vivo. In addition, the strategy dramatically improved the in vitro and in vivo half‐life of the synthetic peptides. Future applications of ADAC involve pandemic preparedness to viral genetic drift as well as neoepitope vaccination strategies where the bispecific antibody is an off‐the‐shelf product, and the peptide antigen is synthesized based on next‐generation sequencing data mining.

Optimization of CD19 chimeric antigen receptor T cell establishment and observation of the killing effect in vitro and in vivo

Objective: To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Method: Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. Results: The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % vs (47.00±13.46) % , P<0.001; bone marrow: (1.82±0.29) % vs (37.30±1.44) % , P<0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. Conclusion: This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.

IMMU-18. Targeting Antigen Presenting Cells to improve virotherapy efficacy in Diffuse Midline Gliomas

With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the non-responsive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in immunocompetent mice bearing orthotopic DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages, and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 (using an agonistic antibody) on the tumor APCs results in a remodeling of the tumor immune compartment towards a proinflammatory scenario and a more efficient T-cell infiltration. Of importance, the combination therapy extends survival of treated mice as compared to single treatments or non-treated mice. In addition, we observe a complete regression of tumors in more than 40% of treated mice and the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

DuoBody-CD40x4-1BB induces dendritic-cell maturation and enhances T-cell activation through conditional CD40 and 4-1BB agonist activity

BackgroundDespite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational...