Systemic T-cell Activation Research Articles

Adding Metastasis-Directed Therapy to Standard-of-Care Systemic Therapy for Oligometastatic Breast Cancer (EXTEND): a Multicenter, Randomized Phase II Trial

PurposePrior evidence suggests a progression-free survival (PFS) benefit from adding metastasis-directed therapy (MDT) to standard-of-care (SOC) systemic therapy for patients with some oligometastatic solid tumors. Randomized trials testing this hypothesis in breast cancer have yet to be published. We sought to determine whether adding MDT to SOC systemic therapy improves PFS in oligometastatic breast cancer. MethodsEXTEND is a multicenter phase II randomized basket trial testing the addition of MDT to SOC systemic therapy in patients with ≤5 metastases (NCT03599765). Patients were randomized 1:1 to MDT (definitive local treatment to all sites of disease, plus SOC systemic therapy) or to SOC systemic therapy only. Primary endpoint was PFS, and secondary endpoints included overall survival (OS), time to subsequent line of systemic therapy, and time to the appearance of new metastases. Exploratory analyses included quality of life (QOL) and systemic immune response measures. ResultsFrom September 2018 through July 2022, 22 and 21 patients were randomized to the MDT and no-MDT arms, respectively. At a median follow-up of 24.8 months, PFS was not improved with the addition of MDT to SOC systemic therapy (median PFS 15.6 months MDT vs 24.9 months no-MDT [hazard ratio {HR} 0.91; 95% CI 0.34–2.48, p=0.86]). Similarly, MDT did not improve OS, time to subsequent line of systemic therapy, or time to the appearance of new metastases (all p>0.05). No significant differences were found in QOL measures, systemic T-cell activation, or T-cell stimulatory cytokine concentration. ConclusionAmong patients with oligometastatic breast cancer, the addition of MDT to SOC systemic therapy did not improve PFS. These findings suggest that MDT may have no systemic benefit in otherwise unselected oligometastatic breast cancer patients, although this trial was limited by a heterogenous and small sample size and overperformance of both treatment arms.

Systemic T-cell activation and IFN-γ activity in indeterminate severe hepatitis are reminiscent of hemophagocytic lymphohistiocytosis: Implications for T-cell– and IFN-γ–directed therapies

BackgroundSevere hepatitis cases in children are increasingly recognized, however, the exact etiology remains unknown in a significant proportion of patients. Cases of indeterminate severe hepatitis (iSH) may progress to indeterminate pediatric acute liver failure (iPALF), hence understanding the immunobiology is critical to preventing disease progression. Hemophagocytic lymphohistiocytosis (HLH) is a systemic hyperinflammatory disorder associated with T-cell and macrophage activation with liver injury. ObjectivesWe hypothesized a high proportion of patients with iSH demonstrate systemic T-cell activation similar to HLH prior to developing iPALF and that the degree of T-cell activation in iSH might correlate with outcomes. MethodsFrom 2019-2022, 14 patients with iSH and 7 patients with PALF of known, non-immune etiology were prospectively enrolled. We compared immune signatures of iSH, HLH, known PALF, and healthy controls. ResultsWe found that patients with iSH have increased CD8+ T-cell activation and high interferon-γ activity similar to HLH. The amplitude of CD8+ T-cell activation was predictive of iSH progression to iPALF. We also found that in patients with iSH, ferritin had only modest elevation. However, age-normalized plasma soluble interleukin-2 receptor (sIL-2R) to ferritin level ratio can distinguish iSH from known PALF and HLH. As a proof of concept, we report that in three patients with steroid refractory iSH, emapalumab, an IFN-γ blocking antibody used in combination with steroids, improved liver function and may have prevented progression to PALF. ConclusionsOur data suggests flow-based T-cell activation markers could help in early identification and risk stratification for targeted intervention in patients with iSH.

Irreversible electroporation of localised prostate cancerdownregulates immune suppression andinduces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase(PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory Tcells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) Tcells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Impact of Recombinant VSV-HIV Prime, DNA-Boost Vaccine Candidates on Immunogenicity and Viremia on SHIV-Infected Rhesus Macaques.

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques. To improve the immunogenicity of these VSV-HIV Env vaccine candidates, we generated chimeric Envs containing the transmembrane and cytoplasmic tail of the simian immunodeficiency virus (SIV), which increases surface Env on the particle. Additionally, the Ebola virus glycoprotein was added to the VSV-HIV vaccine particles to divert tropism from CD4 T cells and enhance their replications both in vitro and in vivo. Animals were boosted with DNA constructs that encoded matching antigens. Vaccinated animals developed non-neutralizing antibody responses against both the HIV Env and the Ebola virus glycoprotein (EBOV GP) as well as systemic memory T-cell activation. However, these responses were not associated with observable protection against simian-HIV (SHIV) infection following repeated high-dose intra-rectal SHIV SF162p3 challenges.

Abstract 5294: Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy

Abstract Introduction: Tumor-specific recruitment of co-stimulatory bispecific antibodies (bsAbs) has emerged as a promising therapeutic strategy. Here, we investigated pH-selective CD28xVISTA bsAbs to act selectively within the acidic tumor microenvironment (TME). Our CD28xVISTA bsAbs are designed for tripartite “trans-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. Experimental Procedures: We evaluated various CD28xVISTA bsAbs, focusing on a prototype 1+2 format with monovalent CD28 binding, bivalent VISTA binding and Fc receptor interaction null mutations (BS2). BS2 was tested for T-cell trans-activation using Jurkat-IL-2-luciferase reporter cells in the presence of HEK293 cells expressing membrane bound OKT3-scFv and CHO cells expressing human VISTA. BS2’s potency was further evaluated in an xCelligence-based human T-cell killing assay that dynamically monitors growth of LNCaP prostate cancer cells co-cultured with VISTA+ Kasumi-3 cells, alone or in combination with a CD3xPSMA bispecific T-cell engager. T-cell activation and proliferation were also measured using flow cytometry with CD3, CD4, CD8 and CD25 markers. Additionally, we modified BS2 to introduce pH-selective VISTA binding, limiting its activity to the TME, and tested this version in a syngeneic mouse model in combination with anti-murine PD-1 (anti-mPD-1). Finally, cytokine release from human PBMCs, co-cultured with human umbilical vein endothelial cells (HUVECs) and treated with the pH-selective BS2 (or TGN1412 as a positive control), was measured using a bead-based multiplex immune assay. Results: Our data show that the combination of anti-mPD-1 and our prototype CD28xVISTA bsAb (BS2) with pH-selective VISTA binding significantly inhibited tumor growth in vivo. In vitro, CD28xVISTA co-stimulation in trans by BS2 potentiated the activity of a CD3xPSMA bispecific T-cell engager in our human T-cell killing assay. Cytokine release assessments demonstrated negligible induction of inflammatory cytokines, indicating a favorable safety profile for this antibody. Conclusion: Our study demonstrates the feasibility of cis and trans CD28 co-stimulation using a CD28xVISTA bsAb (BS2). This approach, which bypasses the need for tumor-associated antigens (TAAs) required by other CD28xTAA bispecifics in development, suggests a potentially safer alternative for T-cell engagement and stimulation. Moreover, our novel myeloid-directed TME-selective co-stimulation strategy with a CD28xVISTA bsAb may broaden the potential for T-cell engagement approaches in solid tumors by enabling rational combinations with existing CD3xTAA T-cell engagers without competing for the same target. Citation Format: Thomas Thisted, Zhi-Gang Jiang, Zuzana Biesova, Adejumoke Onumajuru, Yuliya Kleschenko, Kanam Malhotra, Vikas Saxena, Arnab Mukherjee, F. Donelson Smith, Edward H. van der Horst. Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5294.

Peripheral T-Cell Priming and Micrometastatic Disease Control with Metastasis-Directed Therapy: Multidimensional Immunogenomic Profiling of Oligometastatic Prostate Cancer in the EXTEND Trial

Comprehensive metastasis-directed therapy (MDT) for oligometastatic prostate cancer extended progression-free survival (PFS) and time to new lesion formation in the intermittent hormone therapy (HT) basket of EXTEND. To better understand the mechanism of MDT benefit, we pooled the intermittent and continuous HT baskets of EXTEND and tested the hypothesis that adding MDT to HT would program systemic T-cells to control micrometastatic disease. A total of 174 men were randomized to HT with or without MDT to up to 5 sites of metastases. HT was given for 6 months (intermittent basket, n = 87) or indefinitely (continuous basket, n = 87). Peripheral blood samples were drawn at enrollment, at the end of MDT, at 3 months follow-up (3 mo F/U), and at progression and then analyzed by flow cytometry, T-cell receptor (TCR)-β CDR3 variable region sequencing, multiplex cytokine profiling, and next-generation circulating tumor DNA (ctDNA) sequencing. TCR clonal expansion was determined using a published betabinomial model. Repertoire changes were assessed by Morisita's index, and dominant TCR repertoire motifs were characterized with ImmunoMap. Associations between blood markers and PFS were evaluated with Cox regression adjusted hazard ratios (aHR) accounting for randomization arm and stratifying for intermittent vs continuous HT. Randomization to MDT+HT was associated with T-cell activation, proliferation, and clonal expansion. This response was first observed at end-MDT as upregulated expression of T-cell activation and inhibition markers (i.e., ICOS, Tim-3, and LAG-3) and increases in highly proliferative CD4+ and CD8+ Ki67hi T-cells (all P<0.05). TCR sequencing of 7,678,911 T-cells revealed that MDT+HT was associated with TCR clonal expansion, remodeling of the TCR repertoire, and changes in dominant TCR motifs at end-MDT and 3 mo F/U (all P<0.05). Observed T-cell priming could be driven by signaling networks of canonical T-cell stimulatory cytokines (IL-2, IL-12, and IL-15), which were upregulated at end-MDT and persisted at 3 mo F/U (all P<0.05). This modulation of T-cell phenotype, clonotype, and cytokine concentrations was not observed in the HT-monotherapy arm. At end-MDT, systemic T-cell responses were associated with improved PFS, most notably CD8+ T-cell expression of LAG-3 (aHR 0.22, 95% CI 0.03-0.91) and high TCR clonal expansion (aHR 0.13, 95% CI 0.02-0.52). High ctDNA burden at end-MDT correlated with worse PFS (aHR 1.41, 95% CI 1.04-2.54), as did CD8+ T-cell expression of inhibitory receptor TIGIT at 3 mo F/U (aHR 1.03, 95% CI 1.01-1.06). The addition of MDT to HT induced systemic T-cell activation and expansion, which was not observed in the HT-only arm. This systemic immune response was independently associated with improved PFS. In addition to cytoreduction of macroscopic disease, MDT-induced immune education may be an important complementary mechanism of micrometastatic control in oligometastatic prostate cancer.

Frequency of HLA-DR+CD38hi T cells identifies and quantifies T-cell activation in hemophagocytic lymphohistiocytosis, hyperinflammation, and immune regulatory disorders

Quantifying T-cell activation is essential for the diagnosis and evaluation of treatment response in various hyperinflammatory and immune regulatory disorders, including hemophagocytic lymphohistiocytosis. Plasma soluble IL-2 receptor (sIL-2R) is a well-established biomarker for evaluating systemic T-cell activation. However, the limited availability of sIL-2R testing could result in delayed diagnosis. Furthermore, high sIL-2R levels may not always reflect T-cell activation. To address these limitations, this study investigated whether cell surface markers of T-cell activation, HLA-DR, and CD38, as assessed by flow cytometry, could be used to quantify systemic T-cell activation in a variety of inflammatory disease states and examine its correlation with sIL-2R levels. Results for sIL-2R, CXCL9, and ferritin assays were obtained from patient's medical records. Frequency of HLA-DR+CD38high(hi) T-cells was assessed in different T-cell subsets using flow cytometry. In this study's cohort, activation in total CD8+ T (r=0.65; P< .0001) and CD4+ (r= 0.42; P< .0001) T-cell subsets significantly correlated with plasma sIL-2R levels. At the disease onset, the frequency of HLA-DR+CD38hi T cells in CD8+ T (r= 0.65, P< .0001) and CD4+ T (r= 0.77; P< .0001) effector memory (TEM) compartments correlated strongly with sIL-2R levels. Evaluation of T-cell activation markers in follow-up samples also revealed a positive correlation for both CD4+ TEM and CD8+ TEM activation with sIL-2R levels; thus, attesting its utility in initial diagnosis and in evaluating treatment response. The frequency of HLA-DR+CD38hi T-cells in the CD8+ TEM compartment also correlated with plasma CXCL9 (r= 0.42; P= .0120) and ferritin levels (r= 0.32; P=.0037). This study demonstrates that flow cytometry-based direct T-cell activation assessed by HLA-DR+CD38hi Tcells accurately quantifies T-cell activation and stronglycorrelates with sIL-2R levels across a spectrum ofhyperinflammatory and immune dysregulation disorders.

HLA-DR+ CD38high Expression in T Cells Is Excellent in Quantifying the Amplitude of T-Cell Activation in a Spectrum of Hyperinflammatory Disorders Including HLH

Introduction: T cell activation is the hallmark of several hyperinflammatory states such as HLH and immune dysregulation disorders like LRBA deficiency. Identifying T-cell activation and quantifying the amplitude of T-cell activation is critical for diagnosis, management, and monitoring treatment response in patients with hyperinflammatory and immune regulatory disorders. Soluble Interleukin 2 Receptor (sIL2R) is one of the established systemic T-cell activation markers for diagnosing and monitoring HLH and other hyperinflammatory disorders. However, the clinical availability of sIL2R testing is limited, and it may not be a reliable marker of T-cell activation in patients with eosinophilia and T-cell lymphoma. A recent study by Chaturvedi et.al has demonstrated the utility of flow-based T-cell activation assessment in differentiating HLH from sepsis. However, its utility in defining and quantifying T-cell activation across the hyperinflammatory spectrum and its correlation with sIL2R has not yet been established. Methods: A total of 125 samples were collected either at disease onset (n = 81) or at follow-up (n = 44) from 85 human subjects. Paired plasma sIL2R and T-cell activation markers were prospectively evaluated in patients with suspected HLH (n = 79) or immune dysregulation disorders (n = 46). Out of 85 patients, 21 patients were diagnosed with either primary HLH (p-HLH), Epstein-Barr virus infection-associated HLH (EBV-HLH), malignancy-associated HLH (m-HLH), or macrophage activation syndrome (MAS). The rest of the patients were diagnosed with either sepsis, multisystem inflammatory syndrome in children (MIS-C), hyperinflammatory conditions, hepatitis, or acute liver failure. Flow cytometry-based T-cell activation was evaluated in different T-cell sub-compartments by the co-expression of HLA-DR and CD38 surface markers. Plasma sIL2R levels and CXCL9 (a chemokine driven by IFN-g) were determined independently in CLIA certified clinical diagnostic laboratory. Research personnel who analyzed the flow cytometry data were blinded to the corresponding sIL2R test results and clinical diagnosis. Longitudinal follow-up was performed for a subset of patients with HLH or immune dysregulation with evidence of T-cell activation. We also evaluated T-cell activation in 30 healthy controls (HC) to establish a reference range. In addition, the correlation between T-cell activation markers with plasma levels of sIL2R and CXCL9 was also investigated. Results: Evaluation of T-cell activation measured by HLADR+CD38high in HCs and different hyperinflammatory/ immune regulatory disorders revealed that HCs have less than 8% of T-cell activation in CD8+ T effector memory (TEM) and less than 4% for activation in the CD4+ TEM compartments. In patients with suspected hyperinflammatory/ immune regulatory disorders, we identified significant correlation of direct T-cell activation with plasma sIL2R level in different T-cell subsets, CD3 (r = 0.44, p < 0.0001), CD4 (r = 0.41, p < 0.0001), CD8 (r = 0.45, p <0.001), CD4 TEM (T effector memory) (r =0.53, p < 0.001), and CD8 TEM (r =0.53, p < 0.0001). The strongest correlation between HLADR+CD38high with sIL2R level was noted in CD4+ TEM and CD8+ TEM compartments (Figure 1). The correlation is even more robust at disease onset (r = 0.69, p <0.0001 for CD4+ TEM and r = 0.64, p < 0.0001 for CD8+ TEM). However, at follow up, only activation in CD8+ TEM correlated with sIL2R levels (r = 0.4, p = 0.0069). Moreover, T-cell activation in CD8+ TEM correlated well with CXCL9 and ferritin levels. Among the cohort of patients with the confirmed final diagnosis, we found that both p-HLH and EBV-HLH have CD8+ TEM activation > 70%, whereas MAS and m-HLH have a median T cell activation of 37.8% (10.5-67) and 48.1% (4.9-80.6) respectively. Immune dysregulation patients had T-cell activation range from 8.0 to 46.0 % and sepsis group has 0% to 10.8% (Figure 2). A similar trend was also validated using corresponding plasma sIL2R levels in different hyperinflammatory states. Conclusions: Flow cytometry-based direct assessment of HLADR+CD38high in T cells subsets can reliably quantify T-cell activation and strongly correlate with sIL2R level across a spectrum of different hyperinflammatory and immune dysregulations disorders. If validated, this could be used as a real-time assessment of T-cell activation at disease onset and monitoring response to therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IFN-γ Is Protective in Cytokine Release Syndrome-associated Extrapulmonary Acute Lung Injury.

The mechanisms by which excessive systemic activation of adaptive T lymphocytes, as in cytokine release syndrome (CRS), leads to innate immune cell-mediated acute lung injury (ALI) or acute respiratory distress syndrome, often in the absence of any infection, remains unknown. Here, we investigated the roles of IFN-γ and IL-17A, key T-cell cytokines significantly elevated in patients with CRS, in the immunopathogenesis of CRS-induced extrapulmonary ALI. CRS was induced in wild-type (WT), IL-17A- and IFN-γ knockout (KO) human leukocyte antigen-DR3 transgenic mice with 10 μg of the superantigen, staphylococcal enterotoxin B, given intraperitoneally. Several ALI parameters, including gene expression profiling in the lungs, were studied 4, 24, or 48 hours later. Systemic T-cell activation with staphylococcal enterotoxin B resulted in robust upregulation of several chemokines, S100A8/A9, matrix metalloproteases, and other molecules implicated in tissue damage, granulocyte as well as agranulocyte adhesion, and diapedesis in the lungs as early as 4 hours, which was accompanied by subsequent neutrophil/eosinophil lung infiltration and severe ALI in IFN-γ KO mice. These pathways were significantly underexpressed in IL-17A KOmice, which manifested mildest ALI and intermediate in WTmice. Neutralization of IFN-γ worsened ALI in WT and IL-17A KO mice, whereas neutralizing IL-17A did not mitigate lung injury in IFN-γ KO mice, suggesting a dominant protectiverole for IFN-γ in ALI and that IL-17A is dispensable. Ruxolitinib, a Janus kinase inhibitor, increased ALI severity in WT mice. Thus, our study identified novel mechanisms of ALIin CRS and its differential modulation by IFN-γ and IL-17A.

Activation-induced pyroptosis contributes to the loss of MAIT cells in chronic HIV-1 infected patients

BackgroundMucosal-associated invariant T (MAIT) cells are systemically depleted in human immunodeficiency virus type 1 (HIV-1) infected patients and are not replenished even after successful combined antiretroviral therapy (cART). This study aimed to identify the mechanism underlying MAIT cell depletion.MethodsIn the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.ResultsSingle-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4+ T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D (GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12 (IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.ConclusionsActivation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients, which could potentiate disease progression and poor immune reconstitution.

MFG-E8 Reduces Aortic Intimal Proliferation in a Murine Model of Transplant Vasculopathy.

Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.

Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

80. Heme Oxygenase-1 Gene promoter and associations with inflammation and subclinical vascular disease in Ugandan adolescents with and without HIV

Abstract Background Heme oxygenase-1 (HO-1) is a cytoprotective enzyme with potent anti-inflammatory, and anti-oxidant effects. The HO-1 response is modulated by functional polymorphisms (a dinucleotide (GT)n repeat length variation) in the HO-1 gene promoter region which have been associated with cardiovascular disease (CVD) susceptibility in adults. HO-1 polymorphisms and their associations with markers of inflammation and CVD in Ugandan adolescents with (HIV+) and without HIV (HIV-) have not been investigated. Methods We included 177 children (92 HIV+, 85 HIV-) enrolled in an ongoing observational cohort study at the Joint Clinical Research Center, Kampala, Uganda. All HIV+ participants were on ART. HO-1 (GT)n allele genotypes were determined by PCR of the (GT)n repeat region followed by fragment size determination on a capillary sequencer in DNA extracted from blood samples. Allele designations were assigned by number of (GT)n repeats: S &amp;lt; 27, M 27-34, or L &amp;gt; 34 repeats. We measured mean common carotid artery intima-media thickness (IMT) as a marker of CVD, markers of systemic inflammation (hsCRP, IL6, sTNFRI), monocyte activation (sCD14 and sCD163), and T-cell activation (expression of CD38 and HLA-DR on CD4+ and CD8+) , oxidized lipids, markers of gut integrity and fungal translocation (BDG). Results Median age (IQR) was 13 (11, 14), 44% were females, 86% had viral load &amp;lt; 20 copies/mL. 19% had a short allele genotype, 37% had a medium allele genotype and 44% had a long allele genotype (Figure). The shortest and longest allele length correlated with lower IMT in HIV- only (r=-0.36 and -0.30, respectively, p≤ 0.01 for both). Among biomarkers, only the medium allele correlated with oxidized lipids in HIV+ and with hsCRP and BDG in HIV- (p≤ 0.05). After adjusting for age, sex, and BMI, the presence of a long allele was associated with lower IMT. This was no longer significant after adjusting for markers of inflammation or oxidized lipids (Table). Heme Oxygenase-1 genotype allele length frequency in Ugandan cohort of HIV+ and HIV- youth 1: Models are adjusted for age (years), sex (male vs female), BMI (kg/m2) and HIV status (positive vs negative) 2: Models are adjusted for age (years), sex (male vs female), BMI (kg/m2), sCD14 (pg/mL) and HIV status (positive vs negative) 3: Models are adjusted for age (years), sex (male vs female), BMI (kg/m2), high sensitivity C reactive protein (ng/mL) and HIV status (positive vs negative) 4: Models are adjusted for age (years), sex (male vs female), BMI (kg/m2), oxidized lipids and HIV status (positive vs negative) Conclusion These findings underscore the potential of the HO pathways in modulating future risk for CVD in adolescents through inflammation. HIV status in this setting, likely influences the associations with the genotype with the risk of CVD. Further studies to validate our findings in this population are required. Disclosures Grace A. McComsey, MD, Gilead (Consultant, Advisor or Review Panel member)Janssen (Consultant, Advisor or Review Panel member)Merck (Consultant, Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member)Redhill (Research Grant or Support)Roche (Grant/Research Support)Tetraphase, Astellas (Research Grant or Support)Theratechnologies (Consultant)Vanda (Grant/Research Support)ViiV/GSK (Scientific Research Study Investigator, Advisor or Review Panel member)

Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

Lipidome association with vascular disease and inflammation in HIV+ Ugandan children.

HIV infection and antiretroviral therapy (ART) have both been linked to dyslipidemia and increased cardiovascular disease (CVD). The relationships among the lipidome, immune activation, and subclinical vascular disease in children with perinatally acquired HIV (PHIV) have not been investigated. Serum lipid composition, including 13 lipid classes constituting 850 different lipid species were measured by direct infusion-tandem mass spectrometry in samples from 20 ART-treated PHIV and 20 age-matched and sex-matched HIV- Ugandan children. All participants were between 10 and 18 years of age with no other known active infections. PHIVs had HIV-1 RNA level 50 copies/ml or less. In addition, common carotid artery intima--media thickness (IMT), as well as plasma marker of systemic inflammation (hsCRP, IL6, sTNFRa I), monocyte activation (soluble CD14 and CD163), and T-cell activation (expression of CD38 and HLA-DR on CD4+ and CD8+) were evaluated. Median age (Q1, Q3) of study participants was 13 years (11, 15), 37% were boys, 75% were on an NNRTI-based ART regimen. The concentrations of cholesterol ester, LCER, phosphatidylcholines, and sphingomyelin lipid classes were significantly increased in serum of PHIV compared with HIV (P≤0.04). Biomarkers associated with CVD risk including hsCRP, sCD163, and T-cell activation were directly correlated with lipid species in PHIV (P ≤ 0.04). Contents of free fatty acids including palmitic (16 : 0), stearic (18 : 0), and arachidic acid (20 : 0) were positively correlated with IMT in PHIV. Serum lipidome is altered in young virally suppressed PHIV on ART. A direct association between inflammation and lipid species known to be associated with CVD was observed.

STING Activated Tumor-Intrinsic Type I Interferon Signaling Promotes CXCR3 Dependent Antitumor Immunity in Pancreatic Cancer.

Background & AimsPancreatic ductal adenocarcinoma (PDA) is a lethal chemoresistant cancer that exhibits early metastatic spread. The highly immunosuppressive PDA tumor microenvironment renders patients resistant to emerging immune-targeted therapies. Building from our prior work, we evaluated stimulator of interferon genes (STING) agonist activation of PDA cell interferon-α/β-receptor (IFNAR) signaling in systemic antitumor immune responses.MethodsPDA cells were implanted subcutaneously to wild-type, IFNAR-, or CXCR3-knockout mice. Tumor growth was monitored, and immune responses were comprehensively profiled.ResultsHuman and mouse STING agonist ADU-S100 reduced local and distal tumor burden and activated systemic antitumor immune responses in PDA-bearing mice. Effector T-cell infiltration and inflammatory cytokine and chemokine production, including IFN-dependent CXCR3-agonist chemokines, were elevated, whereas suppressive immune populations were decreased in treated tumors. Intratumoral STING agonist treatment also generated inflammation in distal noninjected tumors and peripheral immune tissues. STING agonist treatment of type I IFN–responsive PDA tumors engrafted to IFNAR-/- recipient mice was sufficient to contract tumors and stimulate local and systemic T-cell activation. Tumor regression and CD8+ T-cell infiltration were abolished in PDA engrafted to CXCR3-/- mice treated with STING agonist.ConclusionsThese data indicate that STING agonists promote T-cell infiltration and counteract immune suppression in locally treated and distant tumors. Tumor-intrinsic type I IFN signaling initiated systemic STING-mediated antitumor inflammation and required CXCR3 expression. STING-mediated induction of systemic immune responses provides an approach to harness the immune system to treat primary and disseminated pancreatic cancers.

Engineering of a trispecific tumor-targeted immunotherapy incorporating 4-1BB co-stimulation and PD-L1 blockade

ABSTRACT Co-stimulatory 4–1BB receptors on tumor-infiltrating T cells are a compelling target for overcoming resistance to immune checkpoint inhibitors, but initial clinical studies of 4–1BB agonist mAbs were accompanied by liver toxicity. We sought to engineer a tri-specific antibody-based molecule that stimulates intratumoral 4–1BB and blocks PD-L1/PD-1 signaling without systemic toxicity and with clinically favorable pharmacokinetics. Recombinant fusion proteins were constructed using scMATCH3 technology and humanized antibody single-chain variable fragments against PD-L1, 4–1BB, and human serum albumin. Paratope affinities were optimized using single amino acid substitutions, leading to design of the drug candidate NM21-1480. Multiple in vitro experiments evaluated pharmacodynamic properties of NM21-1480, and syngeneic mouse tumor models assessed antitumor efficacy and safety of murine analogues. A GLP multiple-dose toxicology study evaluated its safety in non-human primates. NM21-1480 inhibited PD-L1/PD-1 signaling with a potency similar to avelumab, and it potently stimulated 4–1BB signaling only in the presence of PD-L1, while exhibiting an EC50 that was largely independent of PD-L1 density. NM21-1480 exhibited high efficacy for co-activation of pre-stimulated T cells and dendritic cells. In xenograft models in syngeneic mice, NM21-1480 induced tumor regression and tumor infiltration of T cells without causing systemic T-cell activation. A GLP toxicology study revealed no evidence of liver toxicity at doses up to 140 mg/kg, and pharmacokinetic studies in non-human primates suggested a plasma half-life in humans of up to 2 weeks. NM21-1480 has the potential to overcome checkpoint resistance by co-activating tumor-infiltrating lymphocytes without liver toxicity.

Mitigating the risk of cytokine release syndrome in a Phase I trial of CD20/CD3 bispecific antibody mosunetuzumab in NHL: impact of translational system modeling

Mosunetuzumab, a T-cell dependent bispecific antibody that binds CD3 and CD20 to drive T-cell mediated B-cell killing, is currently being tested in non-Hodgkin lymphoma. However, potent immune stimulation with T-cell directed therapies poses the risk of cytokine release syndrome, potentially limiting dose and utility. To understand mechanisms behind safety and efficacy and explore safety mitigation strategies, we developed a novel mechanistic model of immune and antitumor responses to the T-cell bispecifics (mosunetuzumab and blinatumomab), including the dynamics of B- and T-lymphocytes in circulation, lymphoid tissues, and tumor. The model was developed and validated using mosunetuzumab nonclinical and blinatumomab clinical data. Simulations delineated mechanisms contributing to observed cell and cytokine (IL6) dynamics and predicted that initial step-fractionated dosing limits systemic T-cell activation and cytokine release without compromising tumor response. These results supported a change to a step-fractionated treatment schedule of mosunetuzumab in the ongoing Phase I clinical trial, enabling safer administration of higher doses.

Abstract 2268: The PD-L1 and OX40 bispecific antibody IBI327 induces immune activation and improves cancer immunotherapy

Abstract Background PD-L1 is a B7 family member which is constantly expressed on anti-tumors, and could be upregulated by INF-γ. OX40, as a member of TNFR superfamily, is expressed primarily on activated T cell. Recent studies highlighted the therapeutic efficacy of PD-L1 and OX40, which utilize the patients' own immune system to destroy cancerous cell. PD-L1 is known as a negative regulatory molecule, which inhibits T cell function and suppresss anti-tumor immunity. Meanwhile, OX40 as a T cell engager can initiate the T cell activation, and reinvigorate T cell effector function and block Treg suppressive function. PD-L1 blockade antibody and OX40 activating antibody have been shown synergistic effect in tumor regression. To address this mechanism and enhance tumor growth inhibition, we developed PD-L1 and OX40 bispecific antibody (IBI327), which fuses PD-L1 VHH antibody at the C terminal of OX40 antibody IgG2 Fc. By targeting two molecules that are overexpressed in the tumor and activated T cells respectively, there is a potential to increase localization to the tumor area compared to monospecific antibodies. This in turn may reduce the risk of systemic T-cell activation and improve the efficacy. By targeting PD-L1 and OX40 simultaneously, IBI327 is directed to the tumor area where it induces enhanced immune activation and improves cancer immunotherapy. Methods In this study the design a novel bispecific antibody (IBI327), which fuses PD-L1 VHH antibody at the C terminal of OX40 antibody IgG2 Fc. IBI327 bridges hOX40 positive cells with hPD-L1 positive cells and anti-OX40 anti-PD-L1 and IgG were tested in MLR assay and T cells activation assay. The effect of IBI327 on the tumor growth potential Lovo (colon), H292 (lung) and MJ (colon) cell lines was tested. Results Our data shows that IBI327 has high affinity to human PD-L1 and OX40, and anti-PD-L1 shows higher affinity than OX40. Meanwhile, IBI327 retains both PD-L1 and OX40 functions independently. Further, this molecule bridge PD-L1 expressed Tumors or DCs to T cells and activate T effect cell immune function. The vivo studies shows that IBI327 confers a significant tumor regression in humanized model, and induces more CD8 T cell infiltration. Conclusion These results suggest that a PD-L1 OX40 bispecific antibody induces enhanced immune activation and improve cancer immunotherapy. Our study supports that PD-L1 and OX40 bispecific antibody could be a new option for PD-L1 positive or weakly positive patients in clinical. Citation Format: Bingliang Chen, Zhihui Kuang, Yiming Li, Shuaixiang Zhou, Zhihai Wu, Min Wu, Michael Yu, Junjian Liu. The PD-L1 and OX40 bispecific antibody IBI327 induces immune activation and improves cancer immunotherapy [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 2268.