Innate TCRß-chain engagement drives human T cells toward distinct memory-like effector phenotypes with immunotherapeutic potentials.

Clonotypic αß T cell responses to cargoes presented by major histocompatibility complex (MHC), MR1, or CD1 proteins underpin adaptive immunity. Those responses are mostly mediated by complementarity-determining region 3 motifs created by quasi-random T cell receptor (TCR) gene rearrangements, with diversity being highest for TCRγδ. Nonetheless, TCRγδ also displays nonclonotypic innate responsiveness following engagement of germline-encoded Vγ-specific residues by butyrophilin (BTN) or BTN-like (BTNL) proteins that uniquely mediate γδ T cell subset selection. We now report that nonclonotypic TCR engagement likewise induces distinct phenotypes in TCRαß+ cells. Specifically, antibodies to germline-encoded human TCRVß motifs consistently activated naïve or memory T cells toward core states distinct from those induced by anti-CD3 or superantigens and from others commonly reported. Those states combined selective proliferation and effector function with activation-induced inhibitory receptors and memory differentiation. Thus, nonclonotypic TCRVß targeting broadens our perspectives on human T cell response modes and might offer ways to induce clinically beneficial phenotypes in defined T cell subsets.

A T cell receptor ß chain-directed antibody fusion molecule activates and expands subsets of T cells to promote antitumor activity.

Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vß6 and Vß10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αß T cell subsets expressing distinct variable ß (Vß) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vß6/Vß10 TCRs on the same T cell, promoting expansion of human Vß6 and Vß10 CD4+ and CD8+ T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vß T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vß T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

Discovery of LYC-55716: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ) Agonist for Use in Treating Cancer.

Retinoic acid receptor-related orphan receptor γ (RORc, RORγ, or NR1F3) is the nuclear receptor master transcription factor that drives the function and development of IL-17-producing T helper cells (Th17), cytotoxic T cells (Tc17), and subsets of innate lymphoid cells. Activation of RORγ+ T cells in the tumor microenvironment is hypothesized to render immune infiltrates more effective at countering tumor growth. To test this hypothesis, a family of benzoxazines was optimized to provide LYC-55716 (37c), a potent, selective, and orally bioavailable small-molecule RORγ agonist. LYC-55716 decreases tumor growth and enhances survival in preclinical tumor models and was nominated as a clinical development candidate for evaluation in patients with solid tumors.

Preclinical investigations and a first-in-human phase I trial of M4112, the first dual inhibitor of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2, in patients with advanced solid tumors.

BACKGROUND: M4112 is an oral, potent, and selective indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) dual inhibitor. Here, we report preclinical data and first-in-human phase I data, including safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy, of M4112 monotherapy in patients with advanced solid tumors. METHODS: In preclinical studies, M4112 was administered to mice with IDO1-expressing tumors to determine tumor IDO1 and liver TDO2 inhibition. In the phase I trial, patients received doses of M4112 two times per day in 28-day cycles until progression, toxicity, or withdrawal of consent. The primary objective was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D). The primary endpoint was the incidence of dose-limiting toxicities (DLTs), treatment-emergent adverse events (TEAEs), and treatment-emergent changes in safety parameters. Other endpoints included pharmacokinetics, pharmacodynamics, and antitumor effects. RESULTS: In mice, M4112 significantly decreased the kynurenine:tryptophan ratio in the liver and tumor. Fifteen patients received M4112 at five distinct dose levels (three patients per cohort: 100, 200, 400, 600, and 800 mg two times per day orally). Initially, all doses inhibited IDO1 ex vivo, but plasma kynurenine levels returned to or exceeded baseline levels after day 15. Despite initial changes in kynurenine, there was no significant reduction of plasma kynurenine at steady state. There was one DLT (grade 3 allergic dermatitis; 800 mg two times per day) and one grade 2 QT prolongation (800 mg two times per day), resulting in dose reduction (not a DLT). M4112 was well tolerated, and neither the MTD nor the RP2D was established. TEAEs included fatigue, nausea, and vomiting. The best overall response was stable disease (n=9, 60%). CONCLUSIONS: There were no serious safety concerns at any dose. Although M4112 inhibited IDO1 activity ex vivo, plasma kynurenine levels were not reduced despite achieving target exposure.Trial registration number NCT03306420.

In Vitro Priming of Adoptively Transferred T Cells with a RORγ Agonist Confers Durable Memory and Stemness In Vivo.

Adoptive T-cell transfer therapy is an FDA- approved treatment for leukemia that relies on the ex vivo expansion and reinfusion of a patient's immune cells, which can be engineered with a chimeric antigen receptor (CAR) for more efficient tumor recognition. Type 17 T cells, controlled transcriptionally by RORγ, have been reported to mediate potent antitumor effects superior to those observed with conventionally expanded T cells. Here, we demonstrate that addition of a synthetic, small-molecule RORγ agonist during ex vivo expansion potentiates the antitumor activity of human Th17 and Tc17 cells redirected with a CAR. Likewise, ex vivo use of this agonist bolstered the antitumor properties of murine tumor-specific CD4+ and CD8+ T cells. Expansion in the presence of the RORγ agonist enhanced IL17A production without compromising IFNγ secretion in vitroIn vivo, cytokine neutralization studies revealed that IFNγ and IL17A were required to regress murine melanoma tumors. The enhanced antitumor effect of RORγ agonist treatment was associated with recovery of more donor T cells in the tumor and spleen; these cells produced elevated levels of cytokines months after infusion and expressed markers of long-lived stem and central memory cells such as Tcf7 and CD62L. Conversely, untreated cells mainly exhibited effector phenotypes in the tumor. Cured mice previously treated with agonist-primed T cells were protected from tumor rechallenge. Collectively, our work reveals that in vitro treatment with a RORγ agonist generates potent antitumor Type 17 effector cells that persist as long-lived memory cells in vivoSignificance: RORγ agonists can be used in vitro during T-cell expansion to enhance the efficacy of adoptive cell therapy (e.g., CAR-T) and to provide long-term protection against tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3888/F1.large.jpg Cancer Res; 78(14); 3888-98. ©2018 AACR.

A CD80-Biased CTLA4-Ig Fusion Protein with Superior In Vivo Efficacy by Simultaneous Engineering of Affinity, Selectivity, Stability, and FcRn Binding.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab-Resistant Tumors.

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance.

Synthetic RORγ agonists regulate multiple pathways to enhance antitumor immunity.

RORγt is the key transcription factor controlling the development and function of CD4+ Th17 and CD8+ Tc17 cells. Across a range of human tumors, about 15% of the CD4+ T cell fraction in tumor-infiltrating lymphocytes are RORγ+ cells. To evaluate the role of RORγ in antitumor immunity, we have identified synthetic, small molecule agonists that selectively activate RORγ to a greater extent than the endogenous agonist desmosterol. These RORγ agonists enhance effector function of Type 17 cells by increasing the production of cytokines/chemokines such as IL-17A and GM-CSF, augmenting expression of co-stimulatory receptors like CD137, CD226, and improving survival and cytotoxic activity. RORγ agonists also attenuate immunosuppressive mechanisms by curtailing Treg formation, diminishing CD39 and CD73 expression, and decreasing levels of co-inhibitory receptors including PD-1 and TIGIT on tumor-reactive lymphocytes. The effects of RORγ agonists were not observed in RORγ-/- T cells, underscoring the selective on-target activity of the compounds. In vitro treatment of tumor-specific T cells with RORγ agonists, followed by adoptive transfer to tumor-bearing mice is highly effective at controlling tumor growth while improving T cell survival and maintaining enhanced IL-17A and reduced PD-1 in vivo. The in vitro effects of RORγ agonists translate into single agent, immune system-dependent, antitumor efficacy when compounds are administered orally in syngeneic tumor models. RORγ agonists integrate multiple antitumor mechanisms into a single therapeutic that both increases immune activation and decreases immune suppression resulting in robust inhibition of tumor growth. Thus, RORγ agonists represent a novel immunotherapy approach for cancer.

Whole-molecule antibody engineering: generation of a high-affinity anti-IL-6 antibody with extended pharmacokinetics.

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential 'next-generation' antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.

Role of Ets-1 phosphorylation in the effector function of Th cells.

The transcription factor Ets-1 critically regulates differentiation and function of T helper (Th) cells. In vitro studies have demonstrated that DNA binding and transcriptional activity of Ets-1 are regulated by phosphorylation. Depending on the site of phosphorylation, Ets-1 function can either be increased or inhibited. In addition, a splice variant lacking several inhibitory phosphorylation sites has been identified, raising the possibility that this splice variant may function differently from the full-length Ets-1. However, it is unclear how the activating and inhibitory phosphorylation events of Ets-1 are coordinated during Th cell activation. Furthermore, the biological consequences of Ets-1 phosphorylation and alternative splicing in regulating the function of Th cells are unknown. We report here that both activating and inhibitory phosphorylation events of Ets-1 occur simultaneously and independently of each other during Th cell activation. We further demonstrate that the effect of Ets-1 phosphorylation is very modest and that full-length Ets-1 and its splice variant are functionally interchangeable in the regulation of cytokine production in Th cells.

Ets-1 is a negative regulator of Th17 differentiation.

IL-17 is a proinflammatory cytokine that plays a role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. IL-17 is produced mainly by a newly characterized subset of T helper (Th) cells termed Th17. Although the role of Th17 cells in the pathology of autoimmune diseases is well established, the transcription factors regulating the differentiation of Th17 cells remain poorly characterized. We report that Ets-1-deficient Th cells differentiated more efficiently to Th17 cells than wild-type cells. This was attributed to both low IL-2 production and increased resistance to the inhibitory effect of IL-2 on Th17 differentiation. The resistance to IL-2 suppression was caused by a defect downstream of STAT5 phosphorylation, but was not caused by a difference in the level of RORgamma t. Furthermore, Ets-1-deficient mice contained an abnormally high level of IL-17 transcripts in their lungs and exhibited increased mucus production by airway epithelial cells in an IL-17-dependent manner. Based on these observations, we report that Ets-1 is a negative regulator of Th17 differentiation.

Distinct role of MAPKAPK-2 in the regulation of TNF gene expression by Toll-like receptor 7 and 9 ligands.

Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3' untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.

Chronic asthma-induced airway remodeling is prevented by toll-like receptor-7/8 ligand S28463.

RATIONALE: Allergic asthma is a heterogeneous disease, the pathology of which is a result of improper immune responses to innocuous antigens. We and others have previously shown that one of the Toll-like receptor (TLR)-7/8 ligands, the synthetic compound S28463 (resiquimod, R-848), is able to inhibit acute allergic asthma in mice. OBJECTIVES: Given that the efficiency of this pharmacologic compound against the smooth muscle mass increase and goblet cell hyperplasia that are characteristic of chronic allergic asthma has not been previously assessed, we investigated the ability of this compound to prevent these aspects of chronic airway remodeling. METHODS: The impact of S28463 treatment was assessed in a Brown Norway rat model of chronic asthma by histologic, morphometric, and molecular techniques. MEASUREMENTS AND MAIN RESULTS: We demonstrate that treatment with S28463 is able to prevent the development of goblet cell hyperplasia and increases in airway smooth muscle mass, and that this effect is at least partially mediated by inhibiting proliferation of goblet and smooth muscle cells, respectively. Furthermore, we show that the abrogation of airway remodeling is preceded by inhibition of the inflammatory reaction normally occurring in response to allergen challenge in sensitized animals. This inhibition was associated with a reduction of both helper T cell type 1 and type 2 cytokine protein expression in the lungs, demonstrating the potent antiinflammatory effect of this pharmaceutical compound in the context of allergic reactions. CONCLUSIONS: Taken together, our results indicate great potential for the use of S28463 as an antiinflammatory therapeutic agent for the management of chronic asthma.

Role of SLC11A1 (formerly NRAMP1) in regulation of signal transduction induced by Toll-like receptor 7 ligands.

Modulation of immune responses using Toll-like receptor (TLR) ligands is fast becoming one of the main new approaches for the treatment of infectious and allergic diseases. Characterizing the role of genetic factors in modulating responses to these ligands will be crucial in determining the efficacy of a particular treatment. Our previous findings have shown that treatment of Mycobacterium bovis BCG infection with a synthetic TLR7 ligand resulted in a reduction of the splenic bacterial load only in mice carrying a wild-type allele of Nramp1. To understand further how natural resistance-associated macrophage protein 1 (NRAMP1) modulates responses to TLR7 ligands, we have analysed various important TLR7 signal transduction events in macrophage cell lines derived from B10.ANramp1r and B10.ANramp1-/- mice. The Nramp1 genotype did not affect TLR7 receptor expression, ligand uptake or intracellular processing. Following TLR7 ligand stimulation, p38 mitogen-activated protein kinase (MAPK) activation was significantly reduced in B10A.Nramp1-/- macrophages compared with B10A.Nramp1r cells. Interestingly, levels of protein kinase C zeta (PKCzeta) activation were also found to be lower in B10A.Nramp1-/- macrophages and inhibition of this kinase in B10A.Nramp1r cells led to a reduction in cytokine production. Taken together, the data demonstrate a role for NRAMP1 in modulating p38 MAPK and PKCzeta activity, which leads to reduced cytokine induction by TLR7 ligands.

Delayed cutaneous wound healing in mice lacking solute carrier 11a1 (formerly Nramp1): correlation with decreased expression of secretory leukocyte protease inhibitor.

Control of macrophage functions by natural resistance-associated macrophage protein 1 (NRAMP1) has proven to be important for murine resistance to several intracellular pathogens, including Mycobacterium bovis BCG and Salmonella typhimurium, although the exact molecular mechanism of its action remains unknown. We identified secretory leukocyte protease inhibitor (SLPI) as a novel candidate gene whose expression is dependent on Nramp1 gene expression using B10A.Nramp1+/+ and B10A.Nramp1-/- macrophage cell lines in vitro, as well as mice bearing the resistance alleles (wild type (WT)) of the Nramp1 and mice with an ablated Nramp1 gene (knockout (KO)). We established that B10A.Nramp1+/+ cells spontaneously expressed a 10-fold higher level of SLPI messenger RNA (mRNA) compared to B10A.Nramp1-/- expression. Similarly, protein secretion was detected only in supernatants from B10A.Nramp1+/+ macrophages. Induction of SLPI in excisional cutaneous wounds and, most importantly, in macrophages infiltrating these wounds was significantly higher in Nramp1 WT mice compared to KO mice. These differences in SLPI expression in vivo correlated with a significant delay in the kinetics of wound healing in Nramp1 KO mice compared to WT controls. Taken together, these results suggest for the first time that Nramp1 controls macrophage SLPI mRNA and protein expression, and can also have an important effect on the kinetics of wound healing.

Recombinant congenic strains of mice: a new tool for the genetic dissection of efficacy and toxicity of adjuvants.

The efficacy of immunization of a vaccine depends on an antigen, an adjuvant, and on the expression of multiple genes in the host. The responsiveness of various strains of mice to adjuvants is therefore dependent on their genetic background present as a complex, multigenic trait similarly as in man. We have recently developed a gene-discovery platform, termed recombinant congenic strains (RCS), that greatly facilitates the dissection, localization and characterization of genes that mediate complex traits such as responsiveness to adjuvants. These recombinant congenic mice, which were constructed from two progenitor strains (A/J and C57BL/6) that are phenotypically different for several spontaneous or infectious diseases, were generated such that they carry 13.5% of the one genome in 85% of the other genome. The use of these RCS mice therefore enables a more efficient identification of genes that mediate the responsiveness of the adjuvant.