Teff Proliferation Research Articles

Sonodynamic Cytokine Nanocomplexes with Specific Stimulation towards Effector T Cell for Combination Cancer Immunotherapy

AbstractCytokine therapy mediates the interaction between immune cells and non‐immune cells in the tumor microenvironment (TME), forming a promising approach in cancer therapy. However, the dose‐dependent adverse effects and non‐selective stimulation of cytokines limit their clinical use. We herein report a sonodynamic cytokine nano‐immunocomplex (SPNAI) that specifically activates effector T cells (Teffs) for antitumor immunotherapy. By conjugating anti‐interleukin‐2 (anti‐IL‐2) antibodies S4B6 on the semiconducting polymer nanoparticles to afford SPNA, this nanoantibody SPNA can bind with IL‐2 to form SPNAI which can block the interaction between IL‐2 and regulatory T cells (Tregs), selectively activating Teffs in TME. Moreover, SPNAI generates 1O2 to trigger immunogenic cell death of cancer cells upon sono‐irradiation, which promotes the maturation of dendritic cells and the proliferation of Teffs. This SPNAI‐mediated combination sonodynamic immunotherapy thus elevates the ratio of Teffs/Tregs in TME, resulting in inhibition of tumor growth, suppression of lung metastasis and prevention of tumor relapse.

Sonodynamic Cytokine Nanocomplexes with Specific Stimulation towards Effector T cell for Combination Cancer Immunotherapy.

Cytokine therapy mediates the interaction between immune cells and non-immune cells in tumor microenvironment (TME), forming a promising approach in cancer therapy. However, the dose-dependent adverse effects and non-selective stimulation of cytokines limit their clinical use. We herein report a sonodynamic cytokine nano-immunocomplex (SPNAI) that specifically activates effector T cells (Teffs) for antitumor immunotherapy. By conjugating anti-interleukin-2 (anti-IL-2) antibodies S4B6 on the semiconducting polymer nanoparticles to afford SPNA, this nanoantibody SPNA can bind with IL-2 to form SPNAI which can block the interaction between IL-2 and regulatory T cells (Tregs), selectively activating Teffs in TME. Moreover, SPNAI generates 1O2 to trigger immunogenic cell death of cancer cells upon sono-irradiation, which promotes the maturation of dendritic cells and the proliferation of Teffs. This SPNAI-mediated combination sonodynamic immunotherapy thus elevates the ratio of Teffs/Tregs in TME, resulting in inhibition of tumor growth, suppression of lung metastasis and prevention of tumor relapse.

Regulatory T-cell dysfunctions are associated with increase in tumor necrosis factor α in autoimmune hemolytic anemia and participate in Th17 polarization.

Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. The involvement of CD4 T-helper cells has been scarcely explored, with most findings extrapolated from animal models. Here, we performed quantification of both effector T lymphocytes (Teff) and regulatory T cells (Treg), associated with functional and transcriptomic analyses of Treg in human wAIHA. We observed a shift of Teff toward a Th17 polarization concordant with an increase in serum interleukin-17 concentration that correlates with red blood cell destruction parameters, namely lactate dehydrogenase and bilirubin levels. A decrease in circulating Treg, notably effector Treg, associated with a functional deficiency, as represented by their decrease capability to inhibit Teff proliferation, were also observed. Treg deficiency was associated with a reduced expression of Foxp3, the master transcription factor known to maintain the Treg phenotype stability and suppressive functions. Transcriptomic profiling of Treg revealed activation of the tumor necrosis facto (TNF)-α pathway, which was linked to increased serum TNF-α concentrations that were twice as high as in controls. Treg transcriptomic profiling also suggested that post-translational mechanisms possibly accounted for Foxp3 downregulation and Treg dysfunctions. Since TNF-α participates in the rupture of immune tolerance during wAIHA, its inhibition could be of interest. To this end, the effects of fostamatinib, a SYK inhibitor, were investigated in vitro, and we showed that besides the inhibition of erythrocyte phagocytosis by monocytes, fostamatinib is also able to dampen TNF-α production, thus appearing as a promising multitargeting therapy in wAIHA (clinicaltrials gov. Identifier: NCT02158195).

P53 promotes the expansion of regulatory T cells via DNMT3a- and TET2- mediated Foxp3 expression in sepsis.

Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes. Regulatory T cells (Tregs) contribute to immune suppression by inhibiting effector T cell (Teff) proliferation and differentiation. We aimed to investigate the role of p53 in Treg expansion after sepsis. We constructed a sepsis model in wild-type (WT) and p53f/f/CD4-Cre+ mice by cecal ligation and puncture (CLP) and evaluated the proportions of CD4+CD25+ Foxp3+ Tregs by flow cytometry. The expression levels of forkhead/winged helix transcription factor p3 (Foxp3), DNA methyltransferase enzyme (DMNT)3a and ten-eleven translocation (TET)2 were examined using quantitative real-time PCR and Western blot analysis. Treg-specific demethylation region (TSDR) methylation sites in cells were analyzed by bisulfite-sequencing PCR. Furthermore, the direct binding of p53 to the Dnmt3a and TET2 promoters was illustrated using a luciferase assay. The suppressive ability of Tregs was indicated by enzyme-linked immunosorbent assay analysis of cytokine levels and the proliferation of cocultured Teffs. Finally, mortality rates after CLP were compared among WT and p53f/f/CD4-Cre+ mice. The proportion of CD4+CD25+ Foxp3+ Tregs was significantly reduced in p53f/f/CD4-Cre+ mice compared to WT mice after CLP. The enhanced expression of Foxp3 in WT mice was downregulated in the p53f/f/CD4-Cre+ group. We found decreased DMNT3a and increased TET2 levels after CLP. However, the dysregulation of DNMT3a and TET2 was significantly reversed in p53f/f/CD4-Cre+ mice. TSDR underwent increased demethylation in p53f/f/CD4-Cre+ mice. Luciferase activity indicated direct binding of p53 to the promoter regions of DNMT3a and TET2 to regulate their transcription. Consequently, Tregs from p53f/f/CD4-Cre+ CLP mice exhibited limited suppressive ability, as indicated by the reduced production of transforming growth factor-β and interleukin 10 (IL-10). In the coculture system, Teffs showed preserved production of IL-2, differentiation into Th1 cells and proliferation in the presence of Tregs isolated from p53f/f/CD4-Cre+ CLP mice. Finally, the mortality rate of the p53f/f/CD4-Cre+ group after CLP was significantly reduced in comparison to that of the WT group. p53 appears to be critical for Foxp3 expression and consequent Treg expansion by regulating the induction of DNMT3a and TET2, thereby resulting in Foxp3-TSDR demethylation in the context of sepsis.

Research Highlights.

Research Highlights.

Pancreatic islet-specific engineered Tregs exhibit robust antigen-specific and bystander immune suppression in type 1 diabetes models.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in type 1 diabetes (T1D) mouse models. Tregs that are specific for pancreatic islets are more potent than polyclonal Tregs in preventing disease. However, the frequency of antigen-specific natural Tregs is extremely low, and ex vivo expansion may destabilize Tregs, leading to an effector phenotype. Here, we generated durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4+ T cells isolated from patients with T1D, we generated islet-specific EngTregs that suppressed effector T cell (Teff) proliferation and cytokine production. EngTregs suppressed Teffs recognizing the same islet antigen in addition to bystander Teffs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teffs or diabetogenic polyclonal Teffs in recipient mice. These data demonstrate the potential of antigen-specific EngTregs as a targeted therapy for preventing T1D.

Abstract 2550: Tumor microenvironment (TME) is more suppressive in hepatocellular carcinoma (HCC) than in colorectal cancer liver metastasis (CRLM): CXCR4 antagonism as strategy to revert T regulatory cells (Tregs) suppressive activity

Abstract Background: Hepatocellular carcinoma (HCC) and colorectal cancer liver metastasis (CRLM) represent the two most occurring liver cancer. Aim of the project was to analyze the impact of TME on HCC versus CRLM. In particular, Tregs were evaluated in tumor (TT), peritumoral (PT) tissue and peripheral blood (PB) of liver tumor to identify immune mechanisms and therapeutic targets. Methods: Tregs were characterized on PB/PT/TT cell suspension from 40 HCC and 34 CRLM by flow cytometry (FOXP3, CD25, CD4, CD45RA, CTLA-4, CXCR4, PD-1, ICOS and ENTPD1). PB-, PT- and TT-Tregs were isolated by Dynabeads Regulatory T-cell kit and tested for the capability to inhibit CFSE-labeled autologous T effector (Teff) proliferation. Tregs were also treated with Pep R29, a new CXCR4 antagonists, before coculturing with Teffs. Quantitative real-time PCR was performed to assess the expression of cyto-chemokines in PT and TT samples (IL-15, Arginase, CCL5, CXCL12). Results: In HCC/CRLM-PB higher number of CXCR4 (p<0,001/p<0,05), CTLA-4 (p<0,001/p<0,05), PD-1 (p<0,001/p<0,05), ICOS (p<0,05/0,06% vs 0,03%) and ENTPD-1 (p<0,001/p<0,05) expressing Tregs was detected as compared to healthy donors (HD). In HCC/CRLM-TT higher number of FOXP3hiCD45RA-Activated Tregs subpopulation (p<0,01/p<0,05) and higher CXCR4-positive Tregs (p<0,05) were observed as compared to PT. Moreover, Tregs from HCC showed higher expression of ICOS (p<0,05) and ENTPD1 (p<0,05) as compared to CRLM. In terms of function, TT-Tregs exert a stronger immunosuppressive effect on Teffs proliferation than PT-Tregs (p<0,05) as shown by IL-35 cytokine secretion (150 vs 15 pg/mL/p<0,05). Interestingly, HCC-Tregs more powerfully inhibit Teffs proliferation at the tumor site and also in periphery. Expression of TME cyto-chemokines responsible of immunity promotion (IL-15) or inhibition (CXCL12, CCL5, Arginase) and tumor features, such as epithelial to mesenchymal transition (EMT) are under evaluation. Preliminary data showed higher CCL5, Tregs recruiting, in HCC as compared to CRLM suggesting a more suppressive TME in HCC. Based on high % of CXCR4-positive Tregs in HCC and CRLM (PB-HCC/CRLM: p<0,001 and TT-HCC/CRLM: p<0,01), ex vivo PB-Tregs were treated with Pep R29. CXCR4 targeting re-induced Teffs proliferation (PB-HCC -/+ Pep R29:12% vs 32%, p<0,001; PB-CRLM-/+ Pep R29:18,3% vs 49%, p<0,01) and decreased Tregs-derived IL-35 secretion (PB-HCC -/+ Pep R29: 106vs 16,5pg/mL, p<0,05; -/+ Pep R29:70 vs 0,6pg/mL, p<0,01). Conclusions: These data suggest a more suppressive TME in HCC compared to CRLM and highlighted CXCR4 antagonism as a potential strategy to overcome Tregs-mediated TME immunosuppressive function in HCC and CRLM patients. Citation Format: Daniela Castaldo, Sara Santagata, Giuseppina Rea, Maria Napolitano, Crescenzo D'Alterio, Rita Guarino, Vittorio Albino, Andrea Belli, Carmen Cutolo, Francesco Izzo, Stefania Scala. Tumor microenvironment (TME) is more suppressive in hepatocellular carcinoma (HCC) than in colorectal cancer liver metastasis (CRLM): CXCR4 antagonism as strategy to revert T regulatory cells (Tregs) suppressive activity [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 2550.

Abstract 5523: LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model

Abstract Objective: Although immunotherapies, e.g., anti-PD-(L)1 has achieved great success in treating cancers, a large portion of patients still cannot benefit from it, due largely to primary refractory or acquired resistance leading to unsatisfied ORR or relapse. TNFR2 is a receptor for TNF-α, expressed highly selective in immune cells, and some tumor cells, transducing survival and immunostimulatory signal to cells expressing this receptor. Targeting TNFR2 may modulate this signaling, therefore a humanized anti-TNFR2 antibody, LBL-019, was developed for monotherapy or in combination with other anti-tumor therapies. Methods: LBL-019 is a monoclonal antibody of IgG1 subtype. The binding affinity to TNFR2 was determined by Fortebio. The epitope of TNFR2 was identified by differential binding to a serial human/mouse chimeric TNFR2 proteins. The binding of TNF-α to either TNFR2 protein or TNFR2 expressed in cells was examined by using ELISA and flow cytometry. The in vitro activity of LBL-019 was measured using several in vitro assays, e.g., cell proliferation and cytokine release in T cells, ADCC and CDC assays. The anti-tumor activity of LBL-019 was investigated in a model of human TNFR2 transgenic mice (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-019 was measured as 5.32 nM to human and 3.62 nM to cynomolgus TNFR2. It was found that TNFR2 was expressed in immune cells including T cells and monocytes examined in human PBMC, which was upregulated when T cells were activated. TNFR2 was also found highly expressed in some tumor cell lines and tumor TILs (from in vivo studies described below). The epitope of TNFR2 that LBL-019 binds was identified and mapped out CRD1 domain. The binding of TNF-α with either TNFR2 protein or TNFR2 expressed in immune cells was blocked by LBL-019. The growth of CD8+ T cells and CD4+ T cells was significantly enhanced, when incubated with LBL-019, along with elevated IFN-γ release. In a model of human TNFR2 transgenic mice implanted with MC38-OVA cells, LBL-019 was shown significant tumor growth inhibition (TGI) in a dose dependent manner as compared with vehicle treated mice (p<0.01). It appeared that the antibody with wild type Fc showed a better anti-tumor activity than that with silenced Fc, suggesting that Fc mediated NK killing of tumor cells or Treg may have contributed in part to the observed anti-tumor efficacy in this model. In addition, LBL-019 was also exhibited a synergy of anti-tumor activity, when dosed with anti-PD-1 (TGI reaching 79.51%). Conclusions: LBL-019, a humanized anti-TNFR2 antibody, shows great anti-tumor efficacy in a mouse tumor model, likely due to the mechanisms involving enhanced proliferation of Teff, elevated IFN-γ release, and possibly killing of tumor cells and Treg. The data support further development of LBL-019 as a novel therapeutic antibody for cancer, and clinical trial is expected to launch early 2022. Citation Format: Huan Lin, Xiao Huang, Yurong Qin, Yujia Dang, Peng Zhang, Jianming Sun, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model [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 5523.

PD-L1 is expressed on human activated naive effector CD4+ T cells. Regulation by dendritic cells and regulatory CD4+ T cells.

The T cell expression of various co-signalling receptors from the CD28 immunoglobulin superfamily (Inducible T cell co-stimulator (ICOS), Programmed cell death 1(PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), B and T lymphocyte attenuator (BTLA) or from the tumour necrosis factor receptor superfamily (glucocorticoid-induced TNFR family related (GITR), 4-1BB, and CD27), is essential for T cell responses regulation. Other receptors (such as T cell immunoglobulin and mucin domain-containing protein 3, T cell immunoglobulin and T cell immunoglobulin and ITIM domain (TIGIT), and lymphocyte activation gene 3) are also involved in this regulation. Disturbance of the balance between activating and inhibitory signals can induce autoimmunity. We have developed an in vitro assay to simultaneously assess the function of naive CD4+ effector T cells (TEFFs), dendritic cells (DCs) and regulatory T cells (TREGs) and the expression of co-signalling receptors. By running the assay on cells from healthy adult, we investigated the regulation of activated T cell proliferation and phenotypes. We observed that TEFFs activated by DCs mainly expressed BTLA, ICOS and PD-1, whereas activated TREGs mainly expressed TIGIT, ICOS, and CD27. Strikingly, we observed that programmed death-ligand 1 (PD-L1) was significantly expressed on both activated TEFFs and TREGs. Moreover, high PD-L1 expression on activated TEFFs was correlated with a higher index of proliferation. Lastly, and in parallel to the TREG-mediated suppression of TEFF proliferation, we observed the specific modulation of the surface expression of PD-L1 (but not other markers) on activated TEFFs. Our results suggest that the regulation of T cell proliferation is correlated with the specific expression of PD-L1 on activated TEFFs.

Abstract 692: Optimized antagonist anti-PD-1/IL-7 bispecific antibody to sustain exhausted T cell function and to disarm Treg suppressive activity

Abstract Although anti PD(L)-1 therapy lead to impressive responses, the majority of patient are unresponsive or fail to develop a durable response after treatment. Immunocytokines have the potential to strengthen PD-(L)1 therapy by promoting T cell survival, however, their clinical developments are limited by a shortened half-life and systemic toxicity. To redirect immunotherapy to tumor specific T cells, we propose to selectively deliver IL-7 to PD1+ T cells using a bispecific anti-PD1 fused to IL-7 (BiCKI®IL-7). Intratumoral progenitor exhausted CD8+ T cells (Tpex) coexpressing PD1/IL-7R+ were described as the key subset responsive to anti PD-(L)1; but their efficacies are transient as Tpex rapidly undergo apoptosis limiting the full efficacy of anti-PD-(L)1. BICKI®IL7 was specifically designed to preferentially reinvigorate effector functions of PD1+IL-7R+ tumor-specific T cells, to sustain long-term anti-tumor response and counteract immune resistance in refractory patients. BICKI®IL7 was created by the fusion of a flexible linker and human IL-7 to the Fc portion of a high affinity antagonist anti-PD1 antibody. BICKI®IL7 constructed with wild-type IL-7 exhibited a high binding and potency (pSTAT5 IL-7 signaling) irrespective of PD1 expression. As our goal is to selectively activate the IL-7 signaling into PD1+ T cells but not PD1 negative cells, we fused different IL-7 muteins having 2 to 30,000-fold lower affinity for CD127/CD132 complex. One BICKI®IL7 mutein construction was selected for its loss of potency on PD1- cells and its optimal cis-binding/redirection and cis-activity on PD1+ cells. Furthermore, BICKI®IL7 mutein fully antagonized the PD-L1/2 interactions and inhibitory signaling to a similar extent to the naked anti PD1. Interestingly, BICKI®IL7 mutein synergistically activates TCR signaling while IL-7+anti-PD1 combo has no additive effect. Using a chronic antigen stimulation model, we demonstrated that BICKI®IL-7 efficiently activates and sustains the proliferation of early and fully exhausted T cells, and also confirmed the synergy. In parallel, we also explored the effect of the BICKI®IL7 mutein on Tregs in coculture assay. BICKI®IL7 mutein preferentially stimulated CD8 Teff over Treg as opposed to IL-2 & IL-15 and, also abrogated Treg suppression by restoring Teff IFN-γ secretion and proliferation. In vivo, BICKI®IL-7 mutein enhanced anti-tumor responses in mesothelioma model and promoted the proliferation and generation of memory CD8 T cells to higher extent to anti-PD1 treatment. Taken together, our data validate the therapeutic potential of providing IL-7 signal to strengthen PD1 therapy and prevent immunoresistance by sustaining T cell response and overcoming Treg suppression. The bispecific BiCKI®IL-7 mutein can preferentially deliver and activate IL-7 into PD1+ tumor-specific T cells limiting the risk of I-O/I-O immunotoxicity. Citation Format: Aurore Morello, Justine Durand, Margaux Seite, Virginie Thepenier, Géraldine Teppaz, Emmanuelle Wilhelm, Arianne Desselle, Caroline Mary, Nicolas Poirier. Optimized antagonist anti-PD-1/IL-7 bispecific antibody to sustain exhausted T cell function and to disarm Treg suppressive activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 692.

Targeting JAK2 Kinase Promotes Foxp3 Stability and Function of CD8iTregs in Prevention of Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T cells (Teff) have a beneficial effect on graft-versus-leukemia (GVL), they cause mortality and morbidity due to GHVD. Regulatory T cells (Tregs; CD4+Foxp3+) have been demonstrated to efficiently prevent GHVD by suppressing Teff proliferation. In addition to CD4+Foxp3+ Tregs, CD8+Foxp3+induced Tregs (CD8 iTregs) have also been identified not only inhibit Teff function, but also possess the GVL activity. However, instability of CD8 iTregs is an important barrier for GVHD clinical applications. Previous studies have revealed that Janus kinase2 (JAK2) signal transduction is an essential mediator of immune response. JAK2 activation is a positive regulator of Th1 and Th17 differentiation whereas JAK2 acts as a negative regulator in Tregs as its activation leads to Foxp3 instability.In this current study, we attempted to promote Foxp3 stability in CD8 iTregs by targeting JAK2 kinase using JAK2 conditional knockout mice (JAK2fl/flCD4Cre). The enhanced stability of CD8 iTregs could be a potential Treg-based therapy to prevent GVHD onset while maintaining GVL activity. In the generation of alloreactive iTregs in vitro, we found that a significant higher frequency of Foxp3+ cells could be induced from JAK2-/-CD8 T cells than WT CD8 T cells (Fig. 1A). We also observed that JAK2-/- CD8 iTregs exhibited higher levels of canonical Treg markers, Neuropilin-1 (NRP1), which is known to express on natural Tregs (nTregs) and has been proposed to associate with Treg stability (Fig. 1B). We initially examined in vitro stability of JAK2-/- CD8 iTregs in the presence of proinflammatory cytokines. Strikingly, JAK2-/-CD8 iTregs retained much higher Foxp3 than WT CD8 iTregs. Thus, we further investigated the function of JAK2-/- CD8 iTregs in preventing GVHD after allo-HSCT. Upon iTreg transfer into MHC-mismatched C57BL/6 (B6) to BALB/c model, JAK2-/- CD8 iTregs could significantly alleviate GVHD with a marked reduction of recipient's mortality (Fig. 1C).To understand the immune mechanisms of JAK2-/- CD8 iTregs in vivo, spleen, liver and mesenteric lymph nodes from recipient mice received JAK2-/- CD8 iTregs were analyzed on day 7 and 14 after allo-HSCT. JAK2-/- CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintain higher Foxp3 expression compared to WT counterparts (Fig. 1D). Consistently, lower levels of inflammatory cytokines (IFNγ, TNFα, IL-17 and IL-6) were detected from serum of mice received JAK2-/- CD8 iTregs than those of WT iTregs. For translational application, we evaluated the systemic effect of specific JAK2 kinase inhibitor (Pacritinib; PAC) on CD8 iTregs using a GVL model (B6 to BDF1). Mice transplanted with WT CD8iTregs plus P815 mastocytoma and then treated with Pacritinib could significantly prolong the survival and exhibited the GVL activity (Fig. 1E). In conclusion, our studies provide a strong rationale and means to stabilize CD8 iTregs by targeting JAK2 kinase, and stabilizedCD8 iTregs have a therapeutic potential for controlling GVHD in clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions

How early events in effector Tcell (TEFF) subsets tune memory Tcell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using invivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of Tcell fate and shape the quantity and quality of TMEM responses.

Improvement of Treg immune response after treatment with tocilizumab in giant cell arteritis.

ObjectivesTo study the percentage, suppressive function and plasticity of Treg in giant cell arteritis (GCA), and the effects of glucocorticoids and tocilizumab.MethodsBlood samples were obtained from 40 controls and 43 GCA patients at baseline and after treatment with glucocorticoids + IV tocilizumab (n = 20) or glucocorticoids (n = 23). Treg percentage and phenotype were assessed by flow cytometry. Suppressive function of Treg was assessed by measuring their ability to inhibit effector T‐cell (Teff) proliferation and polarisation into Th1 and Th17 cells.ResultsTreg (CD4+CD25highFoxP3+) frequency in total CD4+ T cells was decreased in active GCA patients when compared to controls (2.5% vs. 4.7%, P < 0.001) and increased after treatment with tocilizumab but worsened after treatment with glucocorticoids alone. Treg lacking exon 2 of FoxP3 were increased in GCA patients when compared to controls (23% vs. 10% of total Treg, P = 0.0096) and normalised after treatment with tocilizumab + glucocorticoids but not glucocorticoids alone. In GCA patients, Treg were unable to control Teff proliferation and induced ˜50% increase in the amount of IL‐17+ Teff, which was improved after in vitro blockade of the IL‐6 pathway by tocilizumab.ConclusionThis study reports quantitative and functional disruptions in the regulatory immune response of GCA patients and demonstrates that, unlike glucocorticoids, tocilizumab improves Treg immune response.

Abstract 2230: MG1131, a novel TIGIT-targeting monoclonal antibody, enhances anti-tumor immune responses by modulating NK and T cell activity

Abstract As the recent rise of immune checkpoint inhibitors has resulted in durable clinical outcomes in cancer patients, the need for a newer and more widely effective target for immunotherapy has been vigorously sought in the field; T cell immunoreceptor with Ig and ITIM domain (TIGIT) is an immune checkpoint molecule expressed on CD8+ T cells, CD4+ T cells, NK cells, and regulatory T cells (Treg). TIGIT induces exhaustion of effector T cells (Teff) and NK cells in the tumor microenvironment via engagement with its ligand PVR (CD155) which is dominantly expressed on malignant tumor cells. After initial screening processes from a synthetic library, MG1131 clone was chosen based on the strongest binding affinity to human TIGIT and the superior PVR-blocking activity. MG1131 was cross-reactive with the cynomolgus monkey TIGIT, but not with the mouse TIGIT. Our in vitro efficacy data demonstrated that MG1131 activated NF-κB signaling in T cells, and enhanced NK-mediated tumor killing activities in a PVR-dependent manner. In vitro Treg suppression assays showed that MG1131 inhibited immunosuppressive functionality of Treg, leading to restoration of proliferation and IFN-γ secretion capacities of Teff even in the presence of Treg. In addition, expression levels of TIGIT on CD8+ T cells from PBMCs of cancer patient samples were higher compared with other immune inhibitory molecules such as PD-1, Tim-3, CTLA-4, or LAG-3, and the blockade of TIGIT by MG1131 resulted in increased IFN-γ secretion. Thus, our data indicate that MG1131 is a promising candidate for cancer immunotherapy by modulating NK and T cell functions. Citation Format: Jeewon Lee, Munkyung Kim, Hye-mi Nam, Joong Hyuk Sheen, Eun Jung Song, Hye In Yum, So Jung Lim, Hye-Young Park. MG1131, a novel TIGIT-targeting monoclonal antibody, enhances anti-tumor immune responses by modulating NK and T cell activity [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 2230.

Therapeutic potential of CKD-506, a novel selective histone deacetylase 6 inhibitor, in a murine model of rheumatoid arthritis

ObjectivesHistone deacetylase (HDAC) 6 promotes inflammation. We investigated the anti-arthritic effects of CKD-506, a novel HDAC6 inhibitor, in vitro and in a murine model of arthritis as a novel treatment option for rheumatoid arthritis (RA).MethodsHDAC6 was overexpressed in mouse peritoneal macrophages and RAW 264.7 cells, and the effects of a HDAC6 inhibitor CKD-506 on cytokine production and activity of NF-κB and AP-1 signaling were examined. Peripheral blood mononuclear cells (PBMCs) from RA patients and fibroblast-like synoviocytes (FLS) were activated in the presence of CKD-506. Next, regulatory T cells (Tregs) were induced from RA patients and co-cultured with healthy effector T cells (Teffs) and cell proliferation was analyzed by flow cytometry. Finally, the effects of the inhibitor on the severity of arthritis were assessed in a murine model of adjuvant-induced arthritis (AIA).ResultsOverexpression of HDAC6 induced macrophages to produce TNF-α and IL-6. The inhibitory effect of CKD-506 was mediated via blockade of NF-κB and AP-1 activation. HDAC6 inhibition reduced TNF-α and IL-6 production by activated RA PBMCs. CKD-506 inhibited production of MMP-1, MMP-3, IL-6, and IL-8 by activated FLS. In addition, CKD-506 inhibited proliferation of Teffs directly and indirectly by improving iTreg function. In AIA rats, oral CKD-506 improved clinical arthritis in a dose-dependent manner. A combination of sub-therapeutic CKD-506 and methotrexate exerted a synergistic effect.ConclusionThe novel HDAC6 inhibitor CKD-506 suppresses inflammatory responses by monocytes/macrophages, improves Treg function, and ameliorates arthritis severity in a murine model of RA. Thus, CKD-506 might be a novel and effective treatment option for RA.

Treg suppressor function within allografts is required for tolerance

Abstract The role of regulatory CD4+Foxp3+ T cells (Treg) suppressor function within allografts in tolerance remain unclear. To directly address this, we first used a model where immune reactions are restrained to the graft only. Effector T cells (Teff) alone, or with Treg (2–3×106 each) were transferred to B6 mice lacking secondary lymphoid organs (SLO−; splenectomized LTβR−/−) 5 days after islet allograft (Balb/c). Adoptive transfer of Teff alone induced acute rejection (10d MST). In contrast, Teff + Treg co-transfers significantly prolonged graft survival, with 65% of recipients surviving long-term (&amp;gt;100d MST). Interestingly, Treg did not affect Teff proliferation or accumulation within allografts, but significantly reduced MHC-II expression on DCs and IFN-γ in Teff. Secondly, given that S1PR1 signaling is necessary for lymphocyte exit from SLO, we prevented Treg migration to allografts during tolerance by removing S1PR1 expression in endogenous Treg specifically. Tamoxifen-fed B6.Foxp3EGFP-Cre/Ert2.S1PR1fl/fl (S1PR1KO-Treg) or B6.S1PR1fl/fl (WT-Treg) mice received Balb/c islets. Untreated mice rejected allografts acutely (18d MST). Interestingly, while anti-CD45RB treatment induced tolerance in WT-Treg control mice (95d MST), all S1PR1KO-Treg mice rejected their graft (28d MST; p&amp;lt;0.05 vs WT-Treg). Also, these results correlated with a 25% decrease in Treg infiltrating islet allografts and with a 22% increase in Treg in lymph nodes on day 10 in S1PR1KO-Treg + anti-CD45RB compared to WT-Treg + anti-CD45RB recipients. Overall, our data demonstrate that Treg can suppress rejection by Teff within allografts without prior activation in SLO, and suggest that Treg suppressor function within allografts is required for tolerance.

Overexpressed interleukin-6 functionally impaired CD4+CD25+ regulatory T cells in patients with oral lichen planus: an in vitro study

Objectives Our previous studies have shown that interleukin-6 (IL-6) is overexpressed in the saliva of patients with oral lichen planus (OLP) and is positively correlated with severity of disease. IL-6 may be associated with immune imbalance in patients with OLP. The purpose of the present study was to investigate the inhibitory effect of IL-6 on regulatory T cells (Treg) derived from patients with OLP. Study Design The diagnosis of OLP was acquired on the basis of both clinical and pathologic manifestations. Peripheral blood and its cultured supernatants were detected by using cytometric beads assay. The expression of IL-6 in local lesions was detected by using immunohistochemistry. The inhibition effect of OLP-derived or healthy-derived Treg on the proliferation of T effector cells (Teff) was detected by using flow cytometry; The OLP-derived Treg inhibition effect was detected after upregulating or neutralizing IL-6. Results Plasma IL-6 concentration in 31 patients with OLP was 10660.00 ± 7205.00 fg/mL, which was significantly higher than that in the healthy control group (325.70 ± 32.28 fg/mL; n = 11; P Conclusions IL-6 was elevated in peripheral blood and local lesions in patients with OLP. In vitro, the inhibition effect of Treg on proliferation of autologous Teff in patients with OLP was significantly lower than that in healthy controls. This phenotype could be reversed in vitro when neutralizing IL-6. IL-6 may be an important factor affecting the immune homeostasis in OLP.

1223P - GI101, a novel triple-targeting bispecific CD80-IgG4-IL2variant fusion protein, elicits synergistic anti-tumour effects in preclinical models

BackgroundDespite mounting interest in combination immunotherapy including immune checkpoint inhibitors (ICIs) to improve anti-tumor responses, clinical translation has been disappointing. To address the significant unmet needs remaining, we designed GI101 comprising the extracellular domain of CD80 acting as a dual checkpoint (CTLA4/PD-L1) inhibitor, together with a long acting IL2v that preferentially binds to the IL2Rβ. MethodsAffinity of GI101 for IL2Rs, CTLA4, and PD-L1 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay and in vivo immune profiling. In vivo efficacy of GI101 were performed in a tumor-bearing mouse model. ResultsGI101 exhibits high affinity to both CTLA4 (Kd, 2.9nM), PD-L1 (Kd, 8.5nM), and preferential binding to IL2Rβ (Kd, 28.4nM). GI101 induces the robust stimulation of in vitro and in vivo CD8+ T and NK cell proliferation without a significant increase in Treg cells. GI101 has high binding affinity to CTLA4 acting as a decoy ligand, thereby enhancing the interaction between endogenous CD80 and CD28, leading to the activation of T cells. GI101 elicits improved restoration of immune functions compared to a CD80-Fc in in vitro settings using human PBMCs co-cultured with PD-L1hi tumor cells. In the CT26 tumor-bearing mice, GI101 was superior at inhibiting tumor growth when compared to a combination of aPD-1/aCLTA4 in association with a profound increase in CD8+ T and NK cells without causing an increase in Tregs in the TME. In addition, a dose-dependent anti-tumor effect was observed in CT26 syngeneic models. Furthermore, isolated mortality was observed in the aPD-1/aCTLA4 combo-treated group whereas GI101-treated group has no evidence for toxicity associated with IL2 activity including vascular leakage syndrome and cytokine storm. ConclusionsThe complementary modes of action of GI101 via dual checkpoint blockade with IL2 activity to enhance the proliferation and activation of Teff and NK cells is projected to translate into superior clinical efficacy and safety as indicated even in ‘cold tumor’ models. GI101 has promising potential to replace first-generation ICIs as a monotherapy or in combination with chemo/radiotherapies and other immunotherapies. Legal entity responsible for the studyGI innovation. FundingGI innovation. DisclosureJ.C. Park: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. K.H. Pyo: Research grant / Funding (institution): Yonsei Medical Center. Y.J. Song: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. W.J. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. J. Kim: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. H.N. Ji: Full / Part-time employment: GI innovation. S.S. Park: Full / Part-time employment: GI innovation. Y.J. Koh: Full / Part-time employment: GI innovation. K. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. B. Cho: Research grant / Funding (institution): Yonsei Medical Center. B. Chun: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. Y.M. Oh: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. B. Yang: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. S.Y. Nam: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. M.H. Jang: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation.

Abstract 2402: GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human

Abstract Introduction: At 52.5% identity in their extracellular domains (ECDs), mouse and human GITR (CD357) sequences are only modestly conserved. Thus transitioning promising murine anti-tumor activity data with the anti-GITR DTA-1 and 2F8 Mabs to clinical trials with the non-crossreactive anti-human GITR Mab TRX518 poses a considerable limitation. Here we investigated three elements of the GITR mouse and human antibodies to address this limitation: 1) the functional epitopes targeted by anti-human GITR (TRX-518) and anti-murine GITR (DTA-1 and 2F8) antibodies; 2) their biological activities on primary T cells; and 3) the role of anti-GITR Fc receptor engagement. Results and Conclusions: Alanine scanning, amino acid substitutions and flow cytometry experiments were performed to define critical residues important for the binding of all three Mabs to their respective GITR targets. Swaps of 5-8 of these critical residues showed both the loss of murine and the gain of human directed Mab binding when residues critical for TRX18 binding were substituted into murine GITR. Likewise, the substitution of residues critical for DTA-1/2F8 binding into human GITR resulted in loss of TRX518 binding and the gain of 2F8/DTA-1 binding. We then mapped these critical residues to 3D structural models of the murine and human GITR ECDs built by contact-map guided fragment assembly simulations. Regions defined by these critical residues showed a high degree of overlap, indicating structural conservation even without amino acid identity. Thus the epitopes defined by both DTA-1 and 2F8 are homologous to those defined on human GITR by TRX518. In in vitro standard proliferation/suppression assays with effector T cells (Teff) and regulatory T cells (Tregs) isolated from human healthy donors and naïve mice respectively, TRX518, similarly to DTA-1, significantly increased proliferation, activation and pro-inflammatory cytokine production of human and murine Teff cultured alone or in the presence of Tregs. These observations indicate that Mabs directed to similar mouse and human GITR ECD epitopes result in similar biological activities. Lastly, we questioned the importance of a functional Fc domain in anti-GITR Mabs in order to translate these in vitro effects into anti-tumor activity in vivo. To this end, we compared the anti-tumor activity of DTA-1 and its aglycosyl version in the aggressive and poorly immunogenic B16 melanoma model. Both Fc and non-Fc binding DTA-1 were able to control tumor growth. This suggests that the aglycosyl-anti-GITR Mab TRX518 has the potential to be effective in the clinic despite the lack of Fc receptor engagement. These results align with our preliminary results in patients treated with TRX518 monotherapy showing reductions in Treg frequencies both in tumor biopsies and in the periphery. TRX518 would therefore appear to be well-poised for use in the treatment of solid tumor malignancies. Note: This abstract was not presented at the meeting. Citation Format: Roberta Zappasodi, Heidi Heath, Yang Zhang, Michael Haas, Min Yang, Christopher Mirabelli, Jedd D. Wolchok, Cyndi Sirard, Walter Newman, Taha Merghoub. GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human [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 2402.