Costimulatory CD86 Research Articles

Agonistic anti-CD27 antibody ameliorates EAE by suppressing IL-17 production.

CD27/CD70 costimulation enhances T-cell survival, memory formation and Th1-cell differentiation and effector function. In addition to promoting Th1 responses, CD27 signaling has been shown to exert a negative regulatory role on IL-17 production, resulting in increased sensitivity of CD27 KO mice to EAE. By inducing EAE in full CD27 KO mice, and in a novel, T-cell specific CD27 KO mouse strain (CD4-Cre x CD27flox/flox ), we demonstrate herein that CD27 engagement by its natural ligand (CD70) suppresses IL-17 production in a cell autonomous fashion. We further show that CD27 engagement by an agonistic antibody given after EAE induction or at symptom onset similarly suppresses IL-17 production by activated CD4+ T cells infiltrating the inflamed CNS while IFN-γ production was unaffected, leading to an amelioration of inflammatory-related symptoms. These findings propose CD27 costimulation as a potential candidate for therapeutic manipulation to treat autoimmune and autoinflammatory diseases characterized by excessive IL-17 production.

Improving the anti-acute myeloid leukemia activity of CD123-specific engager T cells by MyD88 and CD40 costimulation.

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.

Phenotypic characterization of human peripheral γδT-cell subsets in glioblastoma.

The antitumoral contribution of γδT cells depends on their activation and differentiation into effectors. This depends on different molecules and membrane receptors, which conditions their physiology. This study aimed to determine the phenotypic characteristics of γδT cells in glioblastoma (GBM) according to five layers of membrane receptors. Among ten GBM cases initially enrolled, five of them who had been confirmed by pathological examination and ten healthy controls underwent phenotyping of peripheral γδT cells by flow cytometry, using the following staining: αβTCR, γδTCR, CD3, CD4, CD8, CD16, CD25, CD27, CD28, CD45, CD45RA, CD56, NKG2D, CD272(BTLA), and CD279(PD-1). Compared with the controls, the results showed no significant change in the number of γδT cells. However, there was a decrease of double-negative (CD4- CD8- ) Tγδ cells and an increase of naive γδT cells, a lack of CD25 expression, a decrease of the expression of CD279, and a remarkable, but not significant, increase in the expression of the CD27 and CD28 costimulation markers. Among the γδT cell subsets, the number of Vδ2 decreased in glioblastoma and showed no significant difference in the expression of CD16, CD56, and NKG2D. In contrast, the number of Vδ1 increased in glioblastoma with overexpression of CD16, CD56, and NKG2D. Our results showed that γδT cells are prone to adopt a pro-inflammatory profile in the glioblastoma context, which suggests that they might be a potential tool to consider in T cell-based immunotherapy in glioblastoma. However, this requires additional investigation on a larger sample size.

Quantitative proteomics reveals protein dysregulation during T cell activation in multiple sclerosis patients compared to healthy controls

BackgroundMultiple sclerosis (MS) is an autoimmune, neurodegenerative disorder with a strong genetic component that acts in a complex interaction with environmental factors for disease development. CD4+ T cells are pivotal players in MS pathogenesis, where peripherally activated T cells migrate to the central nervous system leading to demyelination and axonal degeneration. Through a proteomic approach, we aim at identifying dysregulated pathways in activated T cells from MS patients as compared to healthy controls.MethodsCD4+ T cells were purified from peripheral blood from MS patients and healthy controls by magnetic separation. Cells were left unstimulated or stimulated in vitro through the TCR and costimulatory CD28 receptor for 24 h prior to sampling. Electrospray liquid chromatography-tandem mass spectrometry was used to measure protein abundances.ResultsUpon T cell activation the abundance of 1801 proteins was changed. Among these proteins, we observed an enrichment of proteins expressed by MS-susceptibility genes. When comparing protein abundances in T cell samples from healthy controls and MS patients, 18 and 33 proteins were differentially expressed in unstimulated and stimulated CD4+ T cells, respectively. Moreover, 353 and 304 proteins were identified as proteins exclusively induced upon T cell activation in healthy controls and MS patients, respectively and dysregulation of the Nur77 pathway was observed only in samples from MS patients.ConclusionsOur study highlights the importance of CD4+ T cell activation for MS, as proteins that change in abundance upon T cell activation are enriched for proteins encoded by MS susceptibility genes. The results provide evidence for proteomic disturbances in T cell activation in MS, and pinpoint to dysregulation of the Nur77 pathway, a biological pathway known to limit aberrant effector T cell responses.

Properdin produced by dendritic cells contributes to the activation of T cells

The complement system does not only play an important role in the defence against microorganism and pathogens, but also contributes to the regulation of innate and adaptive immunity. Especially activation fragments C3a and C5a and complement activation at the interface of antigen presenting cell (APC) and T cell, were shown to have a role in T cell activation and proliferation. Whereas most complement factors are produced by the liver, properdin, a positive regulator of the C3 convertase, is mainly produced by myeloid cells. Here we show that properdin can be detected in myeloid cell infiltrate during human renal allograft rejection. In vitro, properdin is produced and secreted by human immature dendritic cells (iDCs), which is further increased by CD40-L-matured DCs (mDCs). Transfection with a specific properdin siRNA reduced properdin secretion by iDCs and mDCs, without affecting the expression of co-stimulatory markers CD80 and CD86. Co-culture of properdin siRNA-transfected iDCs and mDCs with human allogeneic T cells resulted in reduced T cell proliferation, especially under lower DC-T cell ratio’s (1:30 and 1:90 ratio). In addition, T cell cytokines were altered, including a reduced TNF-α and IL-17 secretion by T cells co-cultured with properdin siRNA-transfected iDCs. Taken together, these results indicate a local role for properdin during the interaction of DCs and allogeneic T cells, contributing to the shaping of T cell proliferation and activation.

CD137 costimulation is associated with reduced herpetic stromal keratitis and with developing normal CD8+ T cells in trigeminal ganglia.

Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

Mycobacterium tuberculosis Methyltransferase Rv1515c Can Suppress Host Defense Mechanisms by Modulating Immune Functions Utilizing a Multipronged Mechanism.

Mycobacterium tuberculosis (M. tb) gene Rv1515c encodes a conserved hypothetical protein exclusively present within organisms of MTB complex and absent in non-pathogenic mycobacteria. In silico analysis revealed that Rv1515c contain S-adenosylmethionine binding site and methyltransferase domain. The DNA binding and DNA methyltransferase activity of Rv1515c was confirmed in vitro. Knock-in of Rv1515c in a model mycobacteria M. smegmatis (M. s_Rv1515c) resulted in remarkable physiological and morphological changes and conferred the recombinant strain with an ability to adapt to various stress conditions, including resistance to TB drugs. M. s_Rv1515c was phagocytosed at a greater rate and displayed extended intra-macrophage survival in vitro. Recombinant M. s_Rv1515c contributed to enhanced virulence by suppressing the host defense mechanisms including RNS and ROS production, and apoptotic clearance. M. s_Rv1515c, while suppressing the phagolysosomal maturation, modulated pro-inflammatory cytokine production and also inhibited antigen presentation by downregulating the expression of MHC-I/MHC-II and co-stimulatory signals CD80 and CD86. Mice infected with M. s_Rv1515c produced more Treg cells than vector control (M. s_Vc) and exhibited reduced effector T cell responses, along-with reduced expression of macrophage activation markers in the chronic phase of infection. M. s_Rv1515c was able to survive in the major organs of mice up to 7 weeks post-infection. These results indicate a crucial role of Rv1515c in M. tb pathogenesis.

Lenalidomide bypasses CD28 co-stimulation to reinstate PD-1 immunotherapy by activating Notch signaling

Programmed cell death protein 1 (PD-1) checkpoint blockade therapy requires the CD28 co-stimulatory receptor for CD8+ Tcell expansion and cytotoxicity. However, CD28 expression is frequently lost in exhausted Tcells and during immune senescence, limiting the clinical benefits of PD-1 immunotherapy in individuals with cancer. Here, using a cereblon knockin mouse model that regains invivo Tcell response to lenalidomide, an immunomodulatory imide drug, we show that lenalidomide reinstates the anti-tumor activity of CD28-deficient CD8+ Tcells after PD-1 blockade. Lenalidomide redirects the CRL4Crbn ubiquitin ligase to degrade Ikzf1 and Ikzf3 in Tcells and unleashes paracrine interleukin-2 (IL-2) and intracellular Notch signaling, which collectively bypass the CD28 requirement for activation of intratumoral CD8+ Tcells and inhibition of tumor growth by PD-1 blockade. Our results suggest that PD-1 immunotherapy can benefit from a lenalidomide combination when treating solid tumors infiltrated with abundant CD28- Tcells.

Optimal CD8+ T cell effector function requires costimulation-induced RNA-binding proteins that reprogram the transcript isoform landscape

Boosting T cell activation through costimulation directs defense against cancer and viral infections. Despite multiple studies targeting costimulation in clinical trials, the increased potency and reprogramming of T cells endowed by costimulation is poorly understood. Canonical dogma states that transcription mediates T cell activation. Here, we show that the spliceosome, controlling post-transcriptional alternative splicing and alternative polyadenylation, is the most enriched pathway in T cells after CD134/CD137 costimulation. Costimulation of CD8+ T cells significantly increases expression of 29 RNA-binding proteins while RNA-seq uncovers over 1000 differential alternative splicing and polyadenylation events. Using in vivo mouse and in vitro human models, we demonstrate that RNA-binding protein Tardbp is required for effector cytokine production, CD8+ T cell clonal expansion, and isoform regulation after costimulation. The prospect of immune response optimization through reprogramming of mRNA isoform production offered herein opens new avenues for experimentally and therapeutically tuning the activities of T cells.

CD137 Costimulation Enhances the Antitumor Activity of Vγ9Vδ2-T Cells in IL-10-Mediated Immunosuppressive Tumor Microenvironment.

Although γδ-T cell-based tumor immunotherapy using phosphoantigens to boost γδ-T cell immunity has shown success in some cancer patients, the clinical application is limited due to the rapid exhaustion of Vγ9Vδ2-T cells caused by repetitive stimulation from phosphoantigens and the profoundly immunosuppressive tumor microenvironment (TME). In this study, using a cell culture medium containing human and viral interleukin-10 (hIL-10 and vIL-10) secreted from EBV-transformed lymphoblastoid B cell lines (EBV-LCL) to mimic the immunosuppressive TEM, we found that the antitumor activity of Vγ9Vδ2-T cells was highly suppressed by endogenous hIL-10 and vIL-10 within the TME. CD137 costimulation could provide an anti-exhaustion signal to mitigate the suppressive effects of IL-10 in TME by suppressing IL-10R1 expression on Vγ9Vδ2-T cells. CD137 costimulation also improved the compromised antitumor activity of Vγ9Vδ2-T cells in TME with high levels of IL-10 in Rag2-/- γc-/- mice. In humanized mice, CD137 costimulation boosted the therapeutic effects of aminobisphosphonate pamidronate against EBV-induced lymphoma. Our study offers a novel approach to overcoming the obstacle of the hIL-10 and vIL-10-mediated immunosuppressive microenvironment by costimulating CD137 and enhancing the efficacy of γδ-T cell-based tumor therapy.

Molecular and Functional Analyses of the Primordial Costimulatory Molecule CD80/86 and Its Receptors CD28 and CD152 (CTLA-4) in a Teleost Fish.

The moderate activation of T cells in mammals requires the costimulatory molecules, CD80 and CD86, on antigen-presenting cells to interact with their respective T cell receptors, CD28 and CD152 (CTLA-4), to promote costimulatory signals. In contrast, teleost fish (except salmonids) only possess CD80/86 as their sole primordial costimulatory molecule. However, the mechanism, which underlies the interaction between CD80/86 and its receptors CD28 and CD152 still requires elucidation. In this study, we cloned and identified the CD80/86, CD28, and CD152 genes of the grass carp (Ctenopharyngodon idella). The mRNA expression analysis showed that CD80/86, CD28, and CD152 were constitutively expressed in various tissues. Further analysis revealed that CD80/86 was highly expressed in IgM+ B cells. Conversely, CD28 and CD152 were highly expressed in CD4+ and CD8+ T cells. Subcellular localization illustrated that CD80/86, CD28, and CD152 are all located on the cell membrane. A yeast two-hybrid assay exhibited that CD80/86 can bind with both CD28 and CD152. In vivo assay showed that the expression of CD80/86 was rapidly upregulated in Aeromonas hydrophila infected fish compared to the control fish. However, the expression of CD28 and CD152 presented the inverse trend, suggesting that teleost fish may regulate T cell activation through the differential expression of CD28 and CD152. Importantly, we discovered that T cells were more likely to be activated by A. hydrophila after CD152 was blocked by anti-CD152 antibodies. This suggests that the teleost CD152 is an inhibitory receptor of T cell activation, which is similar to the mammalian CD152. Overall, this study begins to define the interaction feature between primordial CD80/86 and its receptors CD28 and CD152 in teleost fish, alongside providing a cross-species understanding of the evolution of the costimulatory signals throughout vertebrates.

Abstract 628: Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Abstract Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [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 628.

Abstract CT041: Monotherapy dose escalation of davoceticept (ALPN-202), a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies (NEON-1)

Abstract Background: Strong preclinical rationale has emerged for combining checkpoint inhibition (CPI) with T cell costimulatory agonists, particularly CD28, a critical T cell costimulatory molecule recognized as a key target of checkpoint inhibition. Davoceticept (ALPN-202) is a variant CD80 vIgD-Fc fusion that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints. It has demonstrated superiority to CPI-only therapies in vitro and in in vivo tumor models, while demonstrating favorable preclinical safety. Methods: This is a first-in-human, cohort-based, open-label dose escalation and expansion study of davoceticept in adults with advanced solid tumors or lymphoma (NCT04186637). Patients with cancers refractory to standard therapies (including approved CPIs), or cancers without available standard or curative therapy are eligible. After two single-subject cohorts, a standard 3+3 dose escalation has been implemented with two dose schedules in parallel, Q1W and Q3W. Objectives include evaluation of safety and tolerability, PK, PD and preliminary anticancer activity. Disease assessments are evaluated by RECIST v1.1 for solid tumors. A prior presentation discussed the initial first 5 cohorts of the Q1W schedule; this presentation includes the remainder of dose escalation, including the Q3W schedule. Results: As of December 2021, 57 adults with various advanced solid tumors, most commonly colorectal and pancreatic, received davoceticept monotherapy, which was well tolerated through 10 mg/kg Q3W. Davoceticept demonstrated dose-dependent PK and target saturation. Immune-related adverse events (irAEs), occurred in 20/54 (35%), mostly involving the skin, endocrine and gastrointestinal systems. All but 2 of the irAE were grade 1-2. One DLT, chronic active gastritis grade 3, was observed at 3 mg/kg Q3W. Among 47 evaluable, unconfirmed partial responses were observed in 2 (colorectal carcinoma and renal cell carcinoma). Stable disease, defined as first scan at 6 weeks, was observed in 22; 9 (41%) demonstrated tumor volume reduction, 2 (9%) demonstrated SD for > 6 months, and 1 has an ongoing SD at 8 months. At doses above 0.1 mg/kg, pharmacodynamic ex vivo analyses demonstrated agonistic engagement of CD28 on T cells, and flow cytometry demonstrated increased circulating activated (ICOS+) and proliferating (Ki-67+) T cells, increased central memory T cells, and reduced regulatory T cells, consistent with active CD28 engagement. Conclusion: First-in-human dose escalation with davoceticept was well tolerated at doses capable of engaging CD28 costimulation in vivo, with early signs of activity in a largely treatment-refractory, non-immunogenic tumor population. These findings suggest a potential additive benefit of combining CD28 agonism with checkpoint inhibition and identify biologically active dose regimens of davoceticept for subsequent single agent development, and demonstrate further rationale for combination development. Expansion cohorts, including cutaneous melanoma and PD-L1-positive cancers, are planned. Citation Format: Michael Millward, Justin C. Moser, Diwakar Davar, Mark Voskoboynik, Nehal J. Lakhani, Rachel E. Sanborn, Jaspreet Grewal, Ajita Narayan, Mario Sznol, Keren Schieber, Hany Zayed, Christine Dela Cruz, Graciela Perez, Stanford L. Peng. Monotherapy dose escalation of davoceticept (ALPN-202), a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies (NEON-1) [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 CT041.

Abstract 5569: CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors

Abstract The therapeutic efficacy of CAR-T cells in solid tumors, unlike in hematologic tumor, is greatly limited by the insufficient infiltration and persistence of CAR-T cells as well as the immunosuppressive tumor microenvironment. The aim of this study was to overcome these obstacles by introducing an αPDL1/CD28 switch-receptor construct and by seeking combinatorial strategies for CAR-T cells in solid tumors. We found that in non-transduction T cells, the cytokine release and T cell proliferation were repressed in response to PD-L1 protein, while T cells that express αPDL1/CD28 switch-receptor showed enhanced functions, indicating that αPDL1/CD28 could revert PD-L1 inhibition into CD28 costimulation. CAR-T cells with αPDL1/CD28 switch-receptor showed better effector function than that of unitary CAR-T in vitro studies and significant responses in tumor-bearing mice, with more effector memory T cells infiltrated in the tumor. On this basis, PD-1 inhibitor can further enhance the efficacy and persistence of αPDL1/CD28 CAR-T cells, especially in PDL1+ tumor models. We found in vitro studies that radiotherapy increased the expression of T-cell recruiting chemokines and promoted CAR-T cell transmigration. In tumor-bearing mice, synergistic anti-tumor efficacy of mice treated with radiotherapy and αPDL1/CD28 CAR-T cells was further observed. Finally, triple therapy with radiotherapy and anti-PD1 plus αPDL1/CD28 CAR-T cells maximized antitumor responses and induced complete cures in the tumor-bearing mice. Our study suggests that αPDL1/CD28 augments the function of CAR-T cells, and the combinatorial regime of αPDL1/CD28 CAR-T cells, radiotherapy and anti-PD1 in solid tumors could be further investigated. Citation Format: Limei Yin, Wenbo Wang, Zhuoran Yao, Jianxin Xue, Ruizhan Tong, Hui Wang, Shuangsi Liao, Laduona Wang, Yue Zheng, Feifei Na, Min Yu, Xuanwei Zhang, Youling Gong, Meijuan Huang, Xianming Mo, Chong Chen, You Lu. CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors [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 5569.

Abstract 3613: CD28 signaling controls maintenance and differentiation of PD1+CD8 T cells during chronic antigen stimulation

Abstract During chronic infections and cancer, CD8 T cells that recognize pathogens/tumors can persist in a dysfunctional state characterized by high expression of the inhibitory receptor Programmed Cell Death (PD)-1. PD-1+ CD8 T cells are heterogeneous: T cell factor (TCF)-1+PD-1+ progenitor exhausted cells (Tpex) are capable of self-renewal and give rise to the more differentiated subpopulation of TCF-1negPD-1+ CD8 T cells that can retain some effector-like function. Tpex are essential for the maintenance of the antigen-specific T cell pool during chronic stimulation, and in animal models, only these cells respond to PD-1 blockade. In cancer patients, the presence of Tpex cells in tumors has been associated with response to PD-1 targeted therapy. However, it has been reported that the presence of these cells in the tumor microenvironment requires a specific niche, and the molecular mechanisms supporting maintenance of Tpex remains to be investigated. Tpex have high CD28 expression, and we have shown that during PD-1 targeted therapies, CD28 costimulation is required for the reinvigoration of CD8 T cell responses. In this study, we sought to understand the role of sustained CD28 signaling for self-renewal and differentiation of Tpex cells. In mice chronically infected with lymphocytic choriomeningitis virus (LCMV), we abrogated CD28 signaling by blockade of anti-B7-1/B7-2, and observed a sharp reduction of virus-specific CD8 T cells (both TCF-1+ and TCF-1neg subsets). By contrast, B7-2 single blockade resulted in reduction of differentiated TCF-1neg virus-specific cells, yet the Tpex subset was not affected. We hypothesized that stronger CD28 signaling might be required for differentiation of Tpex cells into TCF-1neg, whereas weaker CD28 signaling would be sufficient for Tpex self-renewal. To address this question, we performed conditional deletion of Cd28 on PD-1+ CD8 T cells in mice with established chronic infection. Deletion of both Cd28 alleles resulted in reduction of TCF-1+ and TCF-1neg subsets. In contrast, when CD28 signaling was reduced (deletion of one allele), TCF-1neg PD-1+ CD8 T cells were decreased but the number of Tpex cells was not affected. We performed RNA sequencing and metabolic assays to better understand the role of CD28 signaling on PD-1+ CD8 T cells. These data revealed that sustained CD28 signaling during persistent antigen stimulation is required to maintain mitochondrial fitness. Our work support the novel hypothesis that CD28 signaling strength modulates cell fate decision of Tpex cells through metabolic regulation. Our data also suggest that antigen presenting cells capable of providing ligands to CD28 are required for the long-term maintenance of antigen-specific T cells during chronic stimulation. These findings provide key insights about PD-1+ CD8 T cells, with important implications for checkpoint therapy in cancer, including longevity of responses. Citation Format: Etienne Humblin, Verena Van der Heide, Dan Filipescu, Ashley Lu, Alessandra Soares-Schanoski, Myvizhi Selvan, Laila Horta, Zeynep Gumus, Emily Bernstein, Jerry Chipuk, Dirk Homann, Alice O. Kamphorst. CD28 signaling controls maintenance and differentiation of PD1+CD8 T cells during chronic antigen stimulation [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 3613.

Abstract 3432: A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors

Abstract Claudin-6 is a tetraspan membrane protein associated with tight junction formation. Its expression in normal tissues is restricted to the fetal organs but is not detected in the adult tissues. Increased expression of CLDN6 has been shown in several human malignancies such as testicular, ovarian, uterine, liver and lung adenocarcinoma, and is associated with poor prognosis in these cancer patients. Therefore, CLDN6 is a promising tumor-associated antigen (TAA) for tumor-targeting therapeutics such as CART and T cell engaging bispecific antibodies. CD137 co-stimulation has been reported to lead to extended T-cell proliferation, reactivating anergic T cells, promoting memory T cell formation and maintenance. Activating CD137 with agonistic antibodies presents a great opportunity to improve the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or overcome resistance to ICIs. Additionally, a bispecific antibody that activates CD137 signaling only in the presence of TAA would help reduce the dose-dependent hepatotoxicity that was observed in clinical trials with monoclonal anti-CD137 agonistic antibody due to the activation of CD137 signaling in liver resident Kupffer cells. NBL-028 is a novel tetravalent bispecific antibody uniquely designed to activate the CD137 co-stimulatory pathway in the tumor microenvironment through the CLDN6-mediated clustering of CD137. NBL-028 binds to Claudin 6 with high affinity and specificity, activates CD137 in a CLDN6-dependent manner with fast-on and fast-off CD137 binding properties. NBL-028 demonstrated strong CLDN6-dependent T-cell activation and T-cell mediated cytotoxicity in vitro. NBL-028 elicits potent anti-tumor effects and immunological memory in mouse models without detectable liver damages or systemic toxicity. The tumor infiltrated Lymphocyte (TIL) analysis also demonstrated that NBL-028 treatment induced CD45+ immune cells infiltration, decreased Treg/CD8 ratio and increased M1/M2 like macrophage ratio. Furthermore, NBL-028 is designed upon a proprietary molecular scaffold with superior developability features, demonstrated by stability, yield and ease to purify. NBL-028 is currently at the stage of IND-enabling activities with the aim to enter clinical studies for treating Claudin 6 positive solid tumors in 1Q 2023. Citation Format: Raymond Yu, Yong Tong, Huarui Lu, Makenzie Danton, Samantha Dillman, Joshua Dewe, Joshua Karchin, Samruddhi Patil, Danielle Lavery, Haichun Huang, James Pei, Zhong Liu, Han Li, Ming Lei. A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors [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 3432.

Abstract 5506: CD30 co-stimulatory domain enhances the efficacy of chimeric antigen receptor-engineered γδT cells

Abstract Adoptive transfer of immune killer cells genetically engineered to express chimeric antigen receptors (CARs) has demonstrated a feasible and effective approach against numerous types of cancers. Among the killer cells, γδT cells possessing a wide range of antigen-independent tumor recognition and low risk of developing GVHD offer a chance to develop CAR-engineered allogenic ‘off-the-shelf’ cellular therapeutics. Successful tumor eradication primarily depends on providing optimized signals affecting the proliferation, persistence, antitumor activity and cytokine secretion of CAR-γδT cells. We describe here a novel CD30 costimulatory domain for CAR signaling, which is known to be involved in cell anti-apoptosis, activation and proliferation. Conventional αβT cells and innate γδT cells activated by a CD30-ζ signaling-CAR displayed similar levels of cytolytic efficacies and cytokine secretion against antigen-expressing cancer cell lines as those of other CAR containing commonly used costimulatory domains CD28, CD27, 4-1BB, OX40, and ICOS. Furthermore, the third-generation CARs including the CD30 costimulatory domain retained potent antitumor efficacies independent of their position or type of partner costimulatory domain. More significantly, CAR-γδT cells containing CD30-ζ signaling revealed effective cytolytic activities against several solid cancer cell lines based on its antigen-recognizing antibody fragments. Our findings suggest that the CD30 costimulatory domain could be an attractive candidate for developing CAR-engineered killer cell therapeutics and may be applicable for the design of numerous antigen-recognizing CAR constructs, with an emphasis on targeting solid tumors. Citation Format: Hyun-Il Cho, Chung-Hyo Kang, Sang-Eun Lee, In-Sil Song, Jung-Min Ha, Seon-Duk Lee, Hyun-Jung Sohn, Tai-Gyu Kim. CD30 co-stimulatory domain enhances the efficacy of chimeric antigen receptor-engineered γδT cells [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 5506.

Abstract 571: Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. While CAR T cells can successfully treat hematopoietic malignancies, CAR T cells have not shown success in treating solid tumors. There are many factors that may enhance CAR T cell efficacy and safety for solid tumor treatment, including addition of costimulatory domains to increase T cell functions and switching the T cell type used from alpha beta to gamma delta T cells. We developed a novel CAR (chimeric PD-1, chPD1) that targets the programmed death 1 receptor (PD-1) ligands expressed on many types of solid tumors. The aim of this study was to compare anti-tumor efficacy and safety of chPD1-expressing gamma delta and alpha beta T cells in murine breast and ovarian cancer models. Inclusion of different costimulatory domains, CD28, 4-1BB, and Dap-10, in the chPD1 receptor was also compared. There were no differences in T cell viability and proliferation when comparing costimulatory domains or T cell type. Additionally, there were no significant differences in killing of murine breast and ovarian cancer cell lines. ChPD1-CD28 T cells had increased secretion of proinflammatory and cytokine release syndrome (CRS)-inducing cytokines, including IFN-gamma, IL-2, IL-6, GM-CSF, and nitric oxide, compared to chPD1-41BB and chPD1-Dap10 receptors. Additionally, alpha beta chPD1 T cells had increased cytokine secretion of inflammatory cytokines compared to chPD1 gamma delta T cells. Furthermore, while gamma delta and alpha beta T cells expressing chPD1 receptors with a Dap10 domain had similar anti-tumor efficacy in vivo, chPD1-Dap10 gamma delta T cells had the lowest serum levels of inflammatory cytokines IFN-gamma, IL-6, and GM-CSF. Therefore, inclusion of Dap10 domains in gamma delta T cells may be a safer and more efficacious option for CAR T cell therapy of solid tumors. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells [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 571.

Abstract 2001: Delta-24-RGDOX oncolytic adenovirus improves the survival by modulating the immune system in DMG models

Abstract Purpose of Study: Diffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children, with a peak of incidence between 5-10 years old. The median survival of DMG patients is only 9 months, being the leading cause of pediatric death caused by a brain tumor. On this project we set to characterize the efficacy of Delta-24-RGDOX, an oncolytic adenovirus based on Delta-24-RGD platform, which has demonstrated safety and a therapeutic benefit in different pediatric tumors, armed with the ligand of OX40. The binding of OX40 to OX40L leads to the co-stimulation of CD4 and CD8 cells, generating effector and memory T cells. Therefore, the aim of this project is to improve the antitumor effect of the virus, providing a greater co-stimulation in the tumor. Experimental Procedures: Murine and human DMG cell lines were used. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. OX40L expression was measured by flow cytometry and qPCR. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system. The adenovirus was administered once into the tumor using the same procedure. Tumor immune populations were analyzed by flow cytometry. Results: We first confirmed the oncolytic effect of Delta-24-RGDOX in DMG murine and human cell lines in vitro. The virus was able to infect the cells, produce viral proteins, and to cause cell death in a dose-dependent manner. In addition, we observed effective viral replication in human cell lines but not in mouse cells, as previously described. We then measured OX40L expression in infected cells both at protein and mRNA level, observing that at 50 PFU/cell almost 100% of cells expressed the ligand on their membrane. More importantly, the OX40L was functional as it activated CD8 lymphocytes in vitro. Once we confirmed that the viral administration in vivo was safe, we assess the efficacy of the virus in murine DMG models. We observed a significant survival benefit in mice bearing NP53 tumor treated with Delta-24-RGDOX, which lead to 30% of long-term survivors (P=0.003, median OS PBS 25.5 days vs 35.5 days for treated mice). More importantly, we administered the virus in an already stablished tumor model using XFM cells and also obtained a significant improve of survival (P=0.018, median OS PBS 9 days vs 12.5 treated mice). Finally, we analyzed the immune mechanisms underlying the survival benefit, both in the tumor and in spleen. We observed a significant increase of activated immune cells in the tumor microenvironment 7 days after the viral administration. In addition, splenocytes from virus-treated mice were also significantly more activated. Conclusions: These data show that Delta-24-RGDOX adenovirus expresses a functional OX40L that can modulate the immune response, leading to a significantly improved survival outcome in DMG models. Citation Format: Virginia Laspidea, Sara Labiano, Sumit Gupta, Hong Jiang, Iker Ausejo-Mauleon, Daniel de la Nava, Marc García-Moure, Javier Marco-Sanz, Reyes Hernández-Osuna, Oren J Becher, Juan Fueyo, Ana Patiño-García, Candelaria Gomez-Manzano, Marta M Alonso. Delta-24-RGDOX oncolytic adenovirus improves the survival by modulating the immune system in DMG models [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 2001.

Abstract 573: Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models

Abstract Adoptive cell transfer (ACT) involving autologous tumor-specific lymphocytes (TIL) has shown promising results in the treatment of advanced cancer. Poor in vitro cell expansion, inefficient T-cell migration and T cell exhaustion in the tumor microenvironment greatly limits the broader application of this approach. The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is highly expressed in T cells, where it limits cell activation following TCR engagement, therefore functioning as an important intracellular checkpoint that limits T cell mediated anti-tumor responses. We have developed NX-0255 a highly potent small molecule inhibitor of CBL-B that increases TCR-mediated activation of T cells in the presence or absence of CD28 co-stimulation. Here we used the OT-1 ACT/E.G7-OVA lymphoma and the Pmel-1 ACT/B16 melanoma tumor models to investigate whether adding NX-0255, during the in vitro expansion of tumor-specific T cells would have favorable effects on their ability to reject tumors following adoptive transfer in vivo. OT-1 CD8+ T cells were expanded in vitro using anti-CD3 stimulation and either NX-0255 alone, IL-2 alone, or NX-0255 combined with IL-2 and subsequently transferred in vivo to mice bearing established E.G7-OVA. ACT of OT-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 demonstrated superior anti-tumor activity against E.G7-OVA lymphoma tumors, resulting in significant improvement in survival when compared to mice receiving OT-1 CD8+ T cells cultured with NX-0255 alone or IL-2 alone. Importantly, OT-1 CD8+ T cells cultured in the presence of NX-0255 or the combination of NX-0255 plus IL-2 were found at an increased frequency in the blood and in the tumor and persisted longer in circulation when compared to cells cultured with IL-2 alone. A lower percentage of the tumor-infiltrating OT-1 cells were triple-positive for exhaustion markers PD1+TIM3+LAG3+ expression when cultured with NX-0255 alone or the combination of NX-0255 and IL-2 compared to culture with IL-2 alone. Similarly, ACT of Pmel-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 provides a robust and durable anti-tumor response compared to mice receiving Pmel-1 CD8+ T cells cultured with IL-2 alone in the aggressive B16-OVA model. Flow cytometry analysis showed that Pmel-1 CD8+ T cells cultured in the presence of NX-0255 plus IL-2 were found at an increased frequency in the blood and showed an increased central-memory phenotype (CD44+CD62L+) after adoptive transfer when compared to cells cultured with IL-2 alone. Collectively, these data suggest that adding the CBL-B inhibitor, NX-0255, during the in vitro expansion of tumor-specific T cells increases the frequency and absolute numbers of less exhausted CD8+ memory T-cells, increasing their in vivo persistence and ability to infiltrate the tumor. Citation Format: Marilena Gallotta, Jose Gomez Romo, Alexandra Borodovsky, Austin Tenn-McClellan, Jennifer Stokes, Jennifa Gosling, Gwenn M. Hansen, Arthur Sands, Ryan Rountree, Cristiana Guiducci. Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models [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 573.