Cell Costimulatory Receptor Research Articles

Molecular characterization of the T cell costimulatory receptors CD28 and CTLA4 in the European sea bass

For the activation of T cells, it is necessary the specific recognition of the peptide by the T cell receptors (TCR) in the surface of antigen-presenting cells (APCs) and additional signals delivered by costimulatory receptors. In fish, knowledge about the presence of these costimulatory signals is limited and functional evidence almost absent. Thus, in this study, we have identified the stimulatory CD28 and the inhibitory cytotoxic T-lymphocyte-associated protein 4 (CTLA4) coreceptors in the European sea bass (Dicentrarchus labrax), and evaluated their transcription. In parallel, the transcription encoding for the T cell markers CD8α and CD4 was also evaluated. Both coreceptors showed the canonical architecture including a signal peptide, an immunoglobulin domain, a transmembrane region and a cytosolic tail. Protein predictions and phylogenetic tree identify them as true mammalian orthologues of CD28 and CTLA4. We found these genes constitutively expressed in all studied organs of European sea bass with high expression in lymphoid organs (thymus, spleen and head-kidney) and liver. The molecular expression pattern of these genes was up-regulated in head-kidney leucocytes stimulated with T mitogens as concanavalin A and phytohemagglutinin (PHA), but not with the B cell mitogen lipopolysaccharide (LPS). Fish challenged with nodavirus (NNV) evidenced a differential and opposing regulation of the cd28 and ctla4 transcription levels in the brain, the target organ for viral replication, and head-kidney. While cd28 transcription tends to decrease over the infection time in both organs the expression of the ctla4 gene tends to increase. Interestingly, the coreceptor expression is highly and significantly correlated to the transcription of the T cell markers. Our results highlight the important role of CD28 and CTLA4 as costimulatory receptors of T cells in European sea bass but further studies are deserved.

To B, or not to B: Is calcium the answer?

B lymphocytes are an important component of the adaptive and innate immune system because of their ability to secrete antibodies and to present antigens to T cells, which is critical for immune responses to many pathogens. Abnormal B cell function is the cause of diseases including autoimmune, paraneoplastic, and immunodeficiency disorders. The development, survival, and function of B cells depend on signaling through the B cell receptor (BCR) and costimulatory receptors. One of the signaling pathways induced by antigen binding to the BCR is store-operated Ca2+ entry (SOCE), which depends on the Ca2+ channel ORAI1 and its activators stromal interaction molecule (STIM) 1 and 2. A recent study by Berry et al. [1] reports that B cells lacking STIM1 and STIM2 fail to survive and proliferate because abolished SOCE results in impaired expression of two key anti-apoptotic genes and blunted activation of mTORC1 and c-Myc signaling. The associated Ca2+ regulated checkpoints of B cell survival and proliferation can be bypassed, at least partially, by costimulation through CD40 or TLR9. This study provides important new insights on how SOCE controls B cell function.

CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival

SUMMARYDurable humoral immunity against epidemic infectious disease requires the survival of long-lived plasma cells (LLPCs). LLPC longevity is dependent on metabolic programs distinct from short-lived plasma cells (SLPCs); however, the mechanistic basis for this difference is unclear. We have previously shown that CD28, the prototypic T cell costimulatory receptor, is expressed on both LLPCs and SLPCs but is essential only for LLPC survival. Here we show that CD28 transduces pro-survival signaling specifically in LLPCs through differential SLP76 expression. CD28 signaling in LLPCs increased glucose uptake, mitochondrial mass/respiration, and reactive oxygen species (ROS) production. Unexpectedly, CD28-mediated regulation of mitochondrial respiration, NF-κB activation, and survival was ROS dependent. IRF4, a target of NF-κB, was upregulated by CD28 activation in LLPCs and decreased IRF4 levels correlated with decreased glucose uptake, mitochondrial mass, ROS, and CD28-mediated survival. Altogether, these data demonstrate that CD28 signaling induces a ROS-dependent metabolic program required for LLPC survival.

1O Inducible T cell co-stimulatory (ICOS) receptor agonist, GSK3359609 (GSK609) alone and combination with pembrolizumab: Preliminary results from INDUCE-1 expansion cohorts in head and neck squamous cell carcinoma (HNSCC)

INDUCE-1 is a first-in-human study investigating GSK609 alone (mono) and in combination (combo) with other regimens including pembrolizumab (pembro). The study consists of dose escalation (DE) and expansion phases. Findings from DE and PK/PD mono cohorts demonstrated that a range of GSK609 doses (≥0.1-1 mg/kg) have biological and clinical activity supporting the mechanism of action of a non-T cell depleting IgG4 ICOS agonist antibody as a clinical target. Eligible patients (pts) for HNSCC expansion cohorts (EC) had recurrent or metastatic disease, ≤5 prior lines of therapy, measurable disease, and no active autoimmune disease. Pts received 1 mg/kg GSK609 in the mono EC and 0.3 mg/kg GSK609 + 200 mg pembro in the combo EC until disease progression or unacceptable toxicity, up to 2 years (yrs). Disease assessments were performed every 9 weeks (wks) through wk 54 then every 12 wks. Overall response rate (ORR) was assessed for futility in ≥10 pts/EC, analyzed by prior PD-1/L1 treatment status (naïve vs. experienced). PD-L1 expression was determined by 22C3 pharmDx assay. As of 16 April 2019, 12/17 PD-1/L1-experienced pts in mono and 29/34 PD-1/L1-naïve pts in combo head and neck squamous cell carcinoma (HNSCC) ECs had at least one disease assessment (evaluable population). In the mono EC, median age was 56 yrs (range: 27-73); 88% were male; 82% received ≥1 prior line in the metastatic setting. In the combo EC, median age was 61 yrs (range: 33-77); 85% were male; 68% received ≥1 prior line in the metastatic setting. ORR was 8% (95% CI: 0.2%, 38.5%) and 28% (95% CI: 12.7%, 47.2%) in mono (1/8 pts) and combo (8/29 pts) ECs, respectively. Median PFS in the combo EC was 5.6 months (95% CI: 2.4, NR). Treatment-related adverse events in the overall mono (n=208) and combo populations (n=178) were consistent with that previously reported (Hansen et al. ESMO, 2018). PD-L1 IHC testing is ongoing. Preliminary data demonstrate GSK609 has single agent activity in PD-1/L1 experienced HNSCC. The combo of GSK609 with pembro shows promising antitumor activity and a manageable safety profile in pts with previously treated, PD-1/L1 naive HNSCC.

Comprehensive analysis of tumor necrosis factor receptor TNFRSF9 (4-1BB) DNA methylation with regard to molecular and clinicopathological features, immune infiltrates, and response prediction to immunotherapy in melanoma.

BackgroundImmunotherapy, including checkpoint inhibition, has remarkably improved prognosis in advanced melanoma. Despite this success, acquired resistance is still a major challenge. The T cell costimulatory receptor TNFRSF9 (also known as 4-1BB and CD137) is a promising new target for immunotherapy and two agonistic antibodies are currently tested in clinical trials. However, little is known about epigenetic regulation of the encoding gene. In this study we investigate a possible correlation of TNFRSF9 DNA methylation with gene expression, clinicopathological parameters, molecular and immune correlates, and response to anti-PD-1 immunotherapy to assess the validity of TNFRSF9 methylation to serve as a biomarker.MethodsWe performed a correlation analyses of methylation at twelve CpG sites within TNFRSF9 with regard to transcriptional activity, immune cell infiltration, mutation status, and survival in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas. Furthermore, we used quantitative methylation-specific PCR to confirm correlations in a cohort of N = 115 melanoma patients’ samples (UHB validation cohort). Finally, we tested the ability of TNFRSF9 methylation and expression to predict progression-free survival (PFS) and response to anti-PD-1 immunotherapy in a cohort comprised of N = 121 patients (mRNA transcription), (mRNA ICB cohort) and a case-control study including N = 48 patients (DNA methylation, UHB ICB cohort).FindingsWe found a significant inverse correlation between TNFRSF9 DNA methylation and mRNA expression levels at six of twelve analyzed CpG sites (P ≤ 0.005), predominately located in the promoter flank region. Consistent with its role as costimulatory receptor in immune cells, TNFRSF9 mRNA expression and hypomethylation positively correlated with immune cell infiltrates and an interferon-γ signature. Furthermore, elevated TNFRSF9 mRNA expression and TNFRSF9 hypomethylation correlated with superior overall survival. In patients receiving anti-PD-1 immunotherapy (mRNA ICB cohort), we found that TNFRSF9 hypermethylation and reduced mRNA expression correlated with poor PFS and response.InterpretationOur study suggests that TNFRSF9 mRNA expression is regulated via DNA methylation. The observed correlations between TNFRSF9 DNA methylation or mRNA expression with known features of response to immune checkpoint blockage suggest TNFRSF9 methylation could serve as a biomarker in the context of immunotherapies. Concordantly, we identified a correlation between TNFRSF9 DNA methylation and mRNA expression with disease progression in patients under immunotherapy. Our study provides rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for response to immunotherapy.FundingAF was partly funded by the Mildred Scheel Foundation. SF received funding from the University Hospital Bonn BONFOR program (O-105.0069). DN was funded in part by DFG Cluster of Excellence ImmunoSensation (EXC 1023). The funders had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript; or any aspect pertinent to the study.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

UBASH3A Regulates the Synthesis and Dynamics of TCR–CD3 Complexes

The TCR-CD3 complex is a multicomponent membrane receptor, the expression of which is tightly regulated in thymocytes, as well as in mature T cells both at steady state and upon stimulation. In this study, we report novel roles for UBASH3A in TCR-CD3 synthesis and turnover. UBASH3A is a negative regulator of T cell function and plays a broad role in autoimmunity. We show that modulation of UBASH3A levels in unstimulated Jurkat cells leads to altered amounts of total cellular CD3 chains and of cell-surface TCR-CD3 complexes; in contrast, UBASH3A does not affect the level of cell-surface CD28, an important T cell costimulatory receptor. Upon TCR engagement, UBASH3A enhances the downmodulation of cell-surface TCR-CD3. Mass spectrometry and protein-protein interaction studies uncover novel associations between UBASH3A and components of several cellular pathways involved in the regulation of TCR-CD3 turnover and dynamics, including endoplasmic reticulum-associated protein degradation, cell motility, endocytosis, and endocytic recycling of membrane receptors. Finally, we demonstrate that the SH3 domain of UBASH3A mediates its binding to CBL-B, an E3 ubiquitin ligase that negatively regulates CD28-mediated signaling and, hence, T cell activation. In summary, this study provides new mechanistic insights into how UBASH3A regulates T cell activation and contributes to autoimmunity. The interaction between UBASH3A and CBL-B may synergistically inhibit T cell function and affect risk for type 1 diabetes, as both genes have been shown to be associated with this autoimmune disease.

Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

2B4 but not PD-1 blockade improves mortality in septic animals with preexisting malignancy.

In addition to its well-known beneficial effects for the treatment of several types of cancer, PD-1 blockade has shown encouraging results in preclinical models of sepsis and in a recent clinical trial in sepsis. Because cancer is the most common comorbidity in septic patients, here we aimed to determine the efficacy of PD-1 checkpoint blockade in the setting of sepsis complicated with preexisting malignancy. In a model of established lung cancer followed by cecal ligation and puncture-induced (CLP-induced) sepsis, PD-1 blockade exhibited no therapeutic effect on sepsis survival. This diminished efficacy of PD-1 blockade in cancer septic animals (septic animals with cancer) was characterized by a reduction in both the quality and quantity of PD-1+ responder cells. Specifically, CD8+ T cells isolated from cancer septic animals exhibited decreased CD28 expression and a reduction in the CXCR5+PD-1+ subset. In addition, flow cytometric analysis of T cells isolated from cancer septic animals revealed 2B4 as another possible checkpoint under these conditions. Administration of anti-2B4 to cancer septic animals significantly improved sepsis survival and was associated with increased T cell costimulatory receptor expression and decreased coinhibitory receptor expression. These results illustrate functions of coinhibitory receptors in the setting of sepsis complicated with cancer.

1119PD - Inducible T cell costimulatory (ICOS) receptor agonist, GSK3359609 (GSK609) alone and in combination with pembrolizumab (pembro): Preliminary results from INDUCE-1 expansion cohorts (EC) in head and neck squamous cell carcinoma (HNSCC)

1119PD - Inducible T cell costimulatory (ICOS) receptor agonist, GSK3359609 (GSK609) alone and in combination with pembrolizumab (pembro): Preliminary results from INDUCE-1 expansion cohorts (EC) in head and neck squamous cell carcinoma (HNSCC)

Molecular and metabolic pathways mediating curative treatment of a non-Hodgkin B cell lymphoma by Sindbis viral vectors and anti-4-1BB monoclonal antibody

BackgroundLimitations to current therapies for treating non-Hodgkin B cell lymphoma include relapse, toxicity and high cost. Thus, there remains a need for novel therapies. Oncolytic viral (OV) therapy has become a promising cancer immunotherapy because of its potential effectiveness, specificity and long-lasting immunity. We describe and characterize a novel cancer immunotherapy combining Sindbis virus (SV) vectors and the agonistic monoclonal antibody (mAb) to the T cell costimulatory receptor, 4-1BB (CD137).MethodsA20 lymphoma was transfected with luciferase and tumor cells were inoculated to BALB/c mice. Tumor growth was monitored by IVIS imaging. Tumor bearing mice were treated with Sindbis virus, α4-1BB Ab or SV plus α4-1BB Ab. On day 7 after treatment, splenocytes were harvested and surface markers, cytokines, and transcription factors were measured by flow cytometry or Elispot. Splenic T cells were isolated and RNA transcriptome analysis was performed. Tumor cured mice were rechallenged with tumor for testing immunological memory.ResultsSV vectors in combination with α4-1BB monoclonal antibody (mAb) completely eradicated a B-cell lymphoma in a preclinical mouse model, a result that could not be achieved with either treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFNγ production, T cell proliferation, migration, and glycolysis. In addition, all mice that survived after treatment developed long lasting antitumor immunity, as shown by the rejection of A20 tumor rechallenge. We identified the molecular pathways, including upregulated cytokines, chemokines and metabolic pathways in T cells that are triggered by the combined therapy and help to achieve a highly effective anti-tumor response.ConclusionsOur study provides a novel, alternative method for B cell lymphoma treatment and describes a rationale to help translate SV vectors plus agonistic mAb into clinical applications.

Abstract 1219: Generation of anti-OX40 antibody with different immunization strategies

Abstract Variety of immunogen such as recombinant protein, DNA or cells has been used for animal immunization to develop hybridoma antibody. However, the effectiveness among different types of immunogen in generating functional antibodies especially antibody with agonistic activity has not been systematically investigated. OX40 (CD134) is a T cell costimulatory receptor that is found primarily on activated CD4+ and CD8+ T cells, regulatory T (Treg) cells and natural killer (NK) cells. Activation of OX40 by its natural ligand or an agonist antibody augments CD4 and CD8 T cell expansion, differentiation, and survival. Agonistic anti-OX40 antibody holds promise as therapeutics for cancer immunotherapy. In the present study, different immunization strategies (recombinant protein, cell and DNA) have been used to generate anti-OX40 antibody. Antigen specific immune response, number of binders, epitope bins and the functional activities obtained from different immunogens were analyzed and compared. Based on cross reactivity and cell based functional assay results, 25 clones from protein immunization, 22 from DNA immunization and 29 from cell based immunization were expanded and purified. Among the purified antibodies, antibodies obtained from DNA immunization showed stronger agonist activity in both Jurkat OX40-NFAT luciferase reporter activity and primary T cell activation assay. The antibodies obtained from DNA immunization also demonstrated in vivo efficacy and significantly inhibited tumor growth in MC38 syngeneic mouse model using human OX-40 knock-in mice. In summary, we have evaluated different immunization strategies using OX-40 as a case study and successfully generated high potency high affinity agonistic anti-OX40 antibody that support further development. Citation Format: Xiaojuan Chai, Wenfang Xin, Teddy Yang, Qing Duan, Louis Liu. Generation of anti-OX40 antibody with different immunization strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1219.

Abstract 2552: Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses

Abstract Cancer immunotherapies have shown remarkable clinical activity across several tumor types but responses have been modest in breast cancers. This is attributed to low levels of neo-antigens in breast tumors and immunosuppressive nature of mammary tumor microenvironment. Here we investigated if we can counteract these obstacles to an effective immunotherapy by inducing senescence in breast cancer cells. Senescence is one of the physiological stress response programs characterized by loss of proliferative potential and secretion of a variety of pro-inflammatory markers which promotes identification and removal of senescent cells by the immune cells. Here we hypothesized that senescence will stimulate anti-tumor immune response by establishing immune-permissive microenvironment. We utilized immunocompetent and humanized PDX-bearing mice to investigate senescence-induced modulation of tumor secretome proteome, transcriptome and immune cell recruitment and activity. An FDA-approved small molecule CDK4/6 inhibitor was used to induce senescence. We detected induction of multiple markers of senescence after CDK4/6 inhibitor treatment of cells and tumors. Senescent cells and tumors had increased secretion of chemokines CCL4, CCL5, CXCL9, CXCL10 and CXCL11 which facilitated T cell homing into the tumor. This was a result of their transcriptional induction by NF-κB. Consequently, we observed the dynamic enrichment of tumor immune microenvironment with T cells in tumors treated with CDK4/6 inhibitor. Furthermore, the recruitment of adoptively transferred ex-vivo activated anti-tumor T cells was significantly enhanced in tumors that were treated with CDK4/6 inhibitor. Finally, CDK4/6 inhibitor-treated tumors became responsive to T cell-activating immunotherapy with antibody agonists of T cell co-stimulatory receptors OX40 and 4-1BB. In summary, our findings suggest that CDK4/6 inhibition can facilitate T cell recruitment into the mammary tumors which can lead to an improved immunotherapy response. This pre-clinical study provides rationale for clinical development of CDK4/6 inhibitors and T cell agonists and uncovers the potential of senescence-inducing approach for sensitizing tumors to immune therapy. Citation Format: Ashlyn Blevins, Stacey Mont, Hunter Lawrence, Nabil Saleh, Sheau-Chiann Chen, Ann Richmond, Anna Vilgelm. Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2552.

TCR and CD28 Concomitant Stimulation Elicits a Distinctive Calcium Response in Naive T Cells.

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.

Abstract A21: Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway

Abstract Purpose: Agonist antibodies targeting the T cell co-stimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across multiple preclinical models of cancer. In the clinic, however, development of these agents has been stymied by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist driven anti-tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immune competent mice, with or without co-administration of checkpoint blockade, via measurement of serum transaminase levels, through imaging of liver immune infiltrates, and via qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27, which is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage and their removal dramatically exacerbates 4-1BB agonist hepatitis. Co-administration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation of myeloid cells to produce Interleukin-27. Co-administration of CTLA-4 and/or CCR2 blockade may minimize hepatitis but yields equal or greater antitumor immunity. Citation Format: Ashvin R. Jaiswal, Todd Bartkowiak, Casey R. Ager, Renee Chin, Chao Hsien Chen, Pratha Budhani, Midan Ai, Matthew J. Reilley, Manu M. Sebastian, David Hong, Michael A. Curran. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A21.

OX40 Stimulation Enhances Protective Immune Responses Induced After Vaccination With Attenuated Malaria Parasites.

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11ahiCD44hi) CD8+ and CD4+ T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4+ T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

Abstract 3818: A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor

Abstract Targeting PD-L1-overexpressing cells with therapeutic antibodies is a clinically validated strategy for the treatment of multiple solid tumors. In order to increase efficacy, PD-1/PD-L1 blocking agents are currently being tested in combination with additional immune checkpoint modulators (ICMs). However, such combination therapies are associated with considerable treatment-related adverse events, resulting in a narrow therapeutic window and thereby limiting treatment efficacy. To maximize potency and improve the safety of ICM combination approaches, we sought to design a multi-specific molecule bearing two ICM domains that depletes PD-L1-overexpressing cancer cells via selective recruitment and stimulation of tumor-reactive effector T cells in the tumor microenvironment. The molecule's design consists of three monovalent antibody variable-domain fragments (Fvs) specific for PD-L1, human serum albumin and a T cell costimulatory receptor fused in a single-chain (PD-L1/HSA/costim tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that agonism of the costimulatory receptor on effector cells can only arise when the molecule concomitantly binds to PD-L1 on the surface of target cells. The addition of a half-life-extending anti-SA domain, meanwhile, not only enables convenient dosing but also should promote delivery of the molecule to tumor microenvironments. Here, we demonstrate the successful production of a novel multi-specific antibody that potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of cells that overexpress PD-L1, as confirmed using a transgenic Jurkat reporter T cell-line in co-cultures with HT29 and HCC827 cancer cell-lines. We show that the molecule elicits such costimulation considerably more selectively and potently than a first-generation, clinical-stage, anti-costim IgG. Furthermore, the tri-specific is incapable of stimulating T cells in the absence of primary T cell activation. Finally, in an HCC827 xenograft model in hPBMC-supplemented NOG mice, the tri-specific slowed tumor growth and enhanced intratumoral CD8+ T cell activation to a greater extent than monospecific IgG variants of the anti-PD-L1 and anti-costimulatory receptor domains. These data support the hypothesis that broadening the therapeutic window for a promising, clinically validated ICM-based treatment strategy can be achieved by combining multiple ICM domains in an Fc-less, monovalent multi-specific that selectively forms immunological synapses between cancer cells and activated effector cells. Citation Format: Tea Gunde, Sebastian Meyer, Stefan Warmuth, Alexandre Simonin, Christian Hess, Matthias Brock, Timothy Egan, David M. Urech. A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3818.

Abstract 3790: Human tumor-infiltrating monocytes/macrophages do not predominantly inhibit tumor-specific effector T cell responses in early-stage lung cancer: The role of macrophage versus tumor PD-L1

Abstract Our current understanding of tumor-associated monocyte/macrophage lineage cells (MMLC) as being immunosuppressive is based largely on murine tumor models that generated a widespread interest in targeting these cells therapeutically. Unfortunately, this approach has not yet been successful in many clinical trials. One potential explanation is that there are fundamental differences between mice and humans in tumor evolution and immunity that might impact the function of human MMLC, limiting the success of myeloid cell-related therapies. Moreover, despite recent successes with checkpoint blockade therapy, the contribution of PD-L1 expressed on MMLC to T cell suppression in humans remains unclear. Thus, the overall purpose of this study was to determine the phenotypic composition of monocyte-macrophage cell lineage in a large cohort of early stage (resected) human lung tumors and delineate how different cell populations of this lineage regulate the effector phase of tumor-specific T cell responses. In addition we also explored the role of MMLC-expressed PD-L1 by interrogating the tripartite functional interactions between tumor-specific effector T cells, PD-L1+ or PD-L1- tumor cells, and TAM that possess a varied surface PD-L1 expression. Phenotypically, we find that (i) MMLC are not the predominate cell population of infiltrating leukocytes within the early-stage lung tumors, (ii) tumor MMLC consist primarily of tumor-associated macrophages (TAM), (iii) the TAM phenotype is complex and does not fit the conventional M1 and M2 phenotype denominations, (iv) TAM are able to co-express T cell co-inhibitory and co-stimulatory receptors. Functionally, MMLC are not primarily suppressive, but have diverse effects, including stimulation of the effector phase of tumor-specific T cell responses. Mechanistically, TAM-expressed PD-L1 (in contrast to tumor-expressed PD-L1), does not inhibit the interaction between tumor-specific effector T cells and tumor cells, but does protect TAMs expressing a tumor antigenic peptide/MHC class I complex from being killed by effector T cells recognizing the cognate peptide on the tumor. These data provide new insights into the phenotype of tumor MMLC and functional cross-talk between TAM, tumor-specific T cells, and tumor cells. Our results show that the prevailing premise that TAMs are predominantly immunosuppressive does not apply to early-stage human lung cancer and thus the current MMLC-targeted approaches would not be helpful in majority of early-stage lung cancer patients. Citation Format: Jason Stadanlick, Abhishek Rao, Sunil Singhal, Michael Annunziata, Pratick Bhojnagarwala, Shaun Obrien, Edmund Moon, Edward Cantu, Gwenn Danet-Desnoyers, Hyun-Jeong Ra, Leslie Litzky, Wayne W. Hancock, Steven M. Albelda, Evgeniy Eruslanov. Human tumor-infiltrating monocytes/macrophages do not predominantly inhibit tumor-specific effector T cell responses in early-stage lung cancer: The role of macrophage versus tumor PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3790.

Abstract 607: Inhibition of CDK4/6 and Pi3Kγ modulates mammary tumor immune microenvironment to enhance response to immunotherapy

Abstract An antitumor immune response is critical in achieving immunotherapy-driven tumor regression. Despite remarkable clinical advances in immunotherapies, patients with breast cancer have been only moderately responsive. In this study, we tested whether CDK4/6 inhibition in combination with Pi3Kγ inhibition can augment the immunotherapy response. Tumor growth was inhibited in the MMTV-PyMT breast cancer model when treated with palbociclib (CDK4/6i) in combination with anti-CD137, an antibody agonist of T cell co-stimulatory receptor 4-1BB. We also assessed the effect of palbociclib alone in a postsurgical model, where treatment began 5 days after injecting 37,000 MMTV-PyMT cells into the mammary fat pad, and show a significant inhibition of both average tumor weight and volume. In addition, we demonstrate that palbociclib can be effective in reducing total tumor burden in the PyMT-FVB spontaneous breast cancer model. Furthermore, mice treated with palbociclib in this model had an increase in CD3+ cell infiltrate in tumor lung metastasis. We used the Pi3Kγ-null mice treated with palbociclib or vehicle control to assess the effect of dual inhibition of CDK4/6 and Pi3Kγ on MMTV-PyMT tumors. We found an increase in CD45+ cells within the tumor microenvironment and an increase in MHC class II+, antigen-presenting macrophages upon palbociclib treatment. Additionally, gene expression analysis of chemokines in palbociclib-treated MCF-7 cells showed an increased expression of CCL4, CCL5, CXCL9, CXCL10, and CXCL11, each of which is implicated in T-cell tumor-homing. Overall, our results suggest that dual inhibition of CDK4/6 and Pi3Kγ may enhance tumor immunogenicity. Drugs targeting CDK4/6 and Pi3Kγ are of high clinical relevance and our findings indicate that they may be used in combination with immunotherapies for the treatment for breast cancer. Citation Format: Stacey Mont, Kevin Black, Sheau-Chiann Chen, Gregory D. Ayers, Ann Richmond, Anna E. Vilgelm. Inhibition of CDK4/6 and Pi3Kγ modulates mammary tumor immune microenvironment to enhance response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 607.

Abstract 3752: Preclinical pharmacology of MP0310: A 4-1BB/FAP bispecific DARPin drug candidate promoting tumor-restricted T-cell costimulation

Abstract Agonistic antibodies against the T cell costimulatory receptor 4-1BB (CD137) have proved to be very efficacious anti-tumor agents in preclinical animal models. However, clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. Here we describe the generation of a tumor-targeted 4-1BB agonist aimed at inducing more effective triggering of 4-1BB without associated systemic toxicity. Tumor targeting is achieved via fibroblast activation protein (FAP) which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. Drug candidate MP0310 comprises DARPin domains binding to 4-1BB and FAP and is devoid of an antibody Fc domain. Compared to first generation monoclonal antibodies targeting 4-1BB, MP0310 shows high potency in vitro and less systemic activation in vivo. In vitro reporter and T cell assays indicate that MP0310 is a potent T cell co-stimulator whose activity is restricted to the presence of FAP-expressing cells. In humanized mouse xenograft studies, FAP-targeted 4-1BB activation induced potent co-stimulation of CD8 T cells leading to tumor growth inhibition. On the other hand, the DARPin molecule did not induce effects associated with strong systemic activation such as hepatotoxicity or exacerbation of graft versus host disease observed in such models, unlike the first generation FcγR-dependent 4-1BB antibodies. In addition, no systemic activation of T cell proliferation was observed in the absence of FAP-positive tumors. In healthy cynomolgus monkeys, administration of MP0310 did not induce systemic stimulation of memory T cell proliferation in contrast to an anti-4-1BB antibody despite MP0310 being fully cross-reactive to cyno 4-1BB and binding effectively to cyno FAP. Therefore, we conclude that the tumor-restricted co-stimulation of 4-1BB may prevent toxicities caused by systemic 4-1BB activation and provide a safe and effective way to boost anti-tumor T cell responses. This could allow more effective dosing and better combination therapies with checkpoint inhibitors and other immune stimulating drugs. MP0310 is in preparation to enter clinical development. Citation Format: Alexander Link, Julia Hepp, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Taylor, Laurent Juglair, Alexander Titz, Mirela Matzner, Ralph Bessey, Christof Zitt, Guy Lemaillet, Joerg Herbst, Keith M. Dawson, Hong Ji, Victor Levitsky, Dan Snell, Michael T. Stumpp, Andreas Harstrick, Elmar vom Baur. Preclinical pharmacology of MP0310: A 4-1BB/FAP bispecific DARPin drug candidate promoting tumor-restricted T-cell costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3752.