Increased Production Of IFN-g Research Articles

Abstract P260: Development and validation of a novel T-cell modulating, microbiome-based peptide for combination with immunotherapy

Abstract The gut microbiome is an important determinant for the success of anti-tumor therapies including chemotherapeutics and anti-checkpoint inhibitors. In this study we wanted to leverage Second Genome’s large and curated microbiome database coupled with its proprietary bioinformatics and machine learning tools to discover bioactive peptides from Bifidobacteria that have the potential to drive response to immunotherapy. The genome of Bifidobacterium (B.) breve and B. longum were analyzed for proteins which were potentially secreted, and had unknown functions. 50 peptides were chemically synthesized and then screened in cell based assays for T cell activation and cytokine secretion. In the present study we describe one such novel B. breve-derived 42-aa peptide (SG-3-0020). The peptide stimulated secretion of effector cytokines by in vitro-cultured T cells (IFNg, TNF-a, IL-10 and IL-2) and increased the expression of PD-1 on both CD4+ and CD8+ T cells when stimulated with low-dose anti-CD3 antibody. To identify the binding partners and mechanism of action of the peptide, Mass Spec and Single-cell RNA-seq was used. Mass spec analysis showed that SG-3-05308, a variant of SG-3-0020 binds to a transmembrane glycoprotein of the immunoglobulin superfamily. Silencing this gene via CRISPR-Cas significantly decreased PD1 levels, cell proliferation and IFNg production in human pan T cells. Single cell RNA-seq data showed that SG-3-0020 activates NF-kB signaling and modulates calcium signaling in T cells. The potency of SG-3-0020 was further optimized for binding to activated T cells by using alanine scanning, saturation, and combinatorial mutation libraries using phage display. The results of protein engineering demonstrated that the 13 amino acids from the C-terminus of SG-3-0020 were not critical for binding and a 29-aa long (lacking the C-term) peptide stimulated increased IFNg production in human T cells across multiple human donors in a dose dependent manner. The peptide with the highest potency SG-3-05429 was selected for further understanding of how it interacts with the identified glycoprotein target and activates downstream T cell signaling pathways . Collectively, these data suggest that SG-3-0020’s ability to up-regulate key co-stimulatory and checkpoint molecules on T cells provides a strong rationale for its potential future use in combination with IO. These results validate the capability of the Second Genome drug discovery platform to identify novel microbial peptides/proteins of potential therapeutic relevance in IO and demonstrate a unique approach that can identify microbial derived bioactive molecules involved in modulating immune cell effector functions and/or immune cell differentiation. Citation Format: Michelle Lin, Archana S. Nagaraja, Dhwani Haria, Yuliya Katlinskaya, Divya Ravichandar, Preston Williams, Roberta Hannibal, Todd Z. DeSantis, Bum-Yeol Hwang, Michi Wilcoxon, Toshi Takeuchi, Karim Dabbagh, Preeti Lal, Helena Kiefel. Development and validation of a novel T-cell modulating, microbiome-based peptide for combination with immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P260.

Role of Extracellular Vesicles in human uveitis

Abstract Non-infectious uveitis (NIU) is a significant cause of blindness. It is an immune mediated disease however the exact pathogenesis remains elusive. Considerable evidence exists on the immunomodulatory effects of Extracellular Vesicles (EVs) in various diseases; but little is known on the role of EVs in uveitis patients. We have previously shown that cytokine stimulated ARPE19 cells, release EVs; these EVs are capable of inhibiting T cell proliferation, inducing proinflammatory cytokine production from monocytes and monocyte cell death, in PBMCs from uveitis patients. The purpose of this study was to isolate EVs from uveitis patients and to determine their immune-modulatory potential in-vitro. EVs were isolated from the serum of NIU patients using Size Exclusion Chromatography (SEC) and set up in culture with PBMCs or ARPE19 cells; after stimulation cells were stained to determine activation and cytokine production. A higher number of EVs were isolated from patients, as compared to healthy controls; but EVs from both, were of the appropriate size, had a low protein content and were positive for the exosome associated markers CD9, CD63 and CD81. EVs from patients expressed increased levels of CD8, CD29, CD44, and HLA-DR/DP/DQ, indicating their potential to activate cells in-vivo as compared to controls. An upregulation of CD69 and CD25 on T cells and increased IFNg production was observed when the cells were cultured with EVs from patients. Incubating ARPE19 cells with the EVs from patients, increased the expression of MCP1 and IL8 in the cells. Preliminary data indicates that EVs isolated from uveitis patients can induce a robust activation of cells in-vitro. Current experiments are underway to further substantiate these results.

Lupus susceptibility gene Esrrg regulates Treg development and suppressive function through mitochondrial metabolism

Abstract Estrogen-Related Receptor Gamma (Esrrg) is the candidate gene for the Sle1c2 susceptibility locus, which encodes the orphan nuclear receptor ERR-g, in the NZM2410 mouse model of Systemic Lupus Erythematosus (SLE). Congenic C57BL/6 (B6) mice expressing this locus have an increased IFN-g production and dysregulated metabolism in their CD4 T cells, but the role of ERR-g in CD4 T cells is still unclear. In muscle, heart, iPSCs, pancreatic β cells and neurons, Esrrg has been identified as a major regulator of mitochondrial metabolism. We generated Treg conditional Esrrg knockout mice Foxp3cre Esrrgf/f (Esrrg-KO) and compared CD4+ T cells between Esrrg-KO and B6 mice. Tregs from Esrrg-KO mice showed a reduced expression of Foxp3 and CD25, and other markers associated with Treg suppressive activity. Esrrg-KO mice demonstrated non-Treg global CD4 T cell activation, including the increased frequency and number of CD69 and T effector memory cells, as well as increased spontaneous T follicular helper (Tfh) cells and decreased T follicular regulatory (Tfr) cell frequencies. In the T-dependent NP-KLH immunization, Esrrg-KO mice produced increased Tfh and germinal center B cells and less Tfr, which led to high affinity anti-NP IgG. Esrrg-deficiency in Tregs significantly altered the expression of genes and metabolites, ATP, NAD+ and ROS, involved in mitochondria function. Esrrg-KO mice also exhibited a high expression of mTORC1 and Hif1-a, a pathway that is known to destabilize Foxp3 expression. Finally, Esrrg-deficiency decreased both respiration and glycolysis in Tregs. These results demonstrate that Esrrg-deficiency results in defective mitochondrial function in Tregs, which leads to defective Tregs and global CD4 T cell hyperactivation.

Intratumoral Administration of the Immunotherapeutic Combination Anti-ctla4, Anti-cd137 and Anti-ox40: Comparison to Systemic Administration, Peri-Draining Lymph Node Injection, and Cellular Vaccine in a Mouse Lymphoma Model

Immunotherapy has come of age and is truly a breakthrough in the treatment of cancer. Immune checkpoint inhibition is now an established and effective modality for treatment of multiple tumor types. Combination immunotherapy creates even more potential for efficacy by targeting synergistic immune pathways. However, systemically administered immunomodulators can activate T-cells non-specifically resulting in off-target toxicity, which is dose-limiting and potentially lethal in combination. Intratumoral administration of immunotherapeutic agents has several advantages including 1) higher concentrations of agents in close proximity to target antigens and tumor-infiltrating lymphocytes 2) lower doses overall with less systemic exposure and toxicity 3) potentially a novel mechanism of action such as depletion of intratumoral Tregs 4) potential for an abscopal effect, acting as an in-situ cancer vaccine.Here we report a novel combination of immunomodulators, anti-CTLA4, CD137 and OX40 administered by intratumoral route in a mouse lymphoma model (A20) as well as a colon cancer model (MC38). CTLA4 is an immune checkpoint, and CD137 and OX40 are members of the TNF receptor superfamily - all agents in clinical practice or clinical trials.For both the A20 and MC38 cell lines, a dual tumor model was used in which tumor cells were inoculated bilaterally on the flanks. BALBC mice were used for the A20 model and C57BL/6 mice for MC38. Treatment was started approximately day 8 when there were visible tumors between 5-7mm in diameter. Intratumoral injection was to the left tumor only.Injection of the triple combination at doses about 1/10 that of usual systemic doses (30 ug each) resulted in significant tumor growth inhibition/regression and a significant survival advantage for both the A20 and MC38 models. For the A20 model, at doses of approximately 1/40 systemic doses (10ug each) the triple combination resulted in tumor clearance in 70-100% of mice, and significantly prolonged survival. There was no toxicity observed. The majority of the mice remained tumor free, and did not grow tumor when rechallenged with A20 cells. The response was shown to be CD8 T-cell dependent, and in vivo CD8 depletion, but not CD4 depletion led to complete abrogation of the treatment effect. A memory t-cell response was confirmed in vitro, with CD8CD44(hi) T-Cells demonstrating increased IFN-g production when incubated with A20 cells, but not control cells (4T1 breast ca). Intratumoral administration at low doses was more effective than 2 systemic routes - intraperitoneal (IP) and subcutaneous (SC), supporting a local in situ vaccine effect. Interestingly, in single tumor model, SC injection in proximity to the tumor draining lymph node (DLN) was significantly more effective than SC injection close to a non-tumor DLN, suggesting that the tumor DLN may be equally involved as the tumor microenvironment in establishing an in situ vaccine effect. Finally, in a single tumor model, a cell based vaccine of irradiated A20 cells co-injected subcutaneously with the low dose triple combination at a site distant from the tumor and tumor DLN had anti-tumor effects greater than the triplet alone SC at the same location, but less than IT or when injected SC in proximity to the tumor DLN.Thus we demonstrate that this novel combination of immunomodulators delivered intratumorally induces a dramatic anti-tumor effect. At low doses, the triple combination is more effective when delivered IT than by systemic routes and the anti-tumor immune response is completely dependent on CD8 T-cells, supporting an in situ vaccine effect. Importantly, it appears both the tumor microenvironment and DLN are important to the anti-tumor immune response. Injecting in the region of the tumor DLN may be a viable and effective option clinically when an injectable tumor site is not easily accessible. The A20 cellular vaccine delivered together with the low dose triple combo at a site distant from the tumor was more effective than triple combo alone, but less effective than intratumoral, suggesting that elements of the tumor microenvironment and/or tumor DLN are important for full effect.Overall these results demonstrate potent anti-tumor effects of the triple combination anti-CTLA4, -CD137 and -OX40, all agents in clinical practice or clinical trials, and support the intratumoral route as safe and highly efficacious route of administration. DisclosuresHebb:Alligator Biosciences: Research Funding. Ellmark:Alligator Biosciences: Employment. Norlen:Alligator Biosciences: Employment. Felsher:Alligator Biosciences: Research Funding.

Abstract 1486: Pharmacodynamic response to HER2/CD3 bispecific antibody (HER2-TDB): evidence of T-cell recruitment and further rationale for combination treatment with anti-PD-L1

Abstract Based on the recent clinical success of tumor immunotherapies that block immune suppressive mechanisms to restore T cell function, there is a profound interest in the clinical development of T cell targeted therapies. We have produced a trastuzumab-based HER2 T cell dependent bispecific antibody (HER2/CD3; HER2-TDB; Junttila et al Cancer Res 2014, 74:5561) that conditionally activates T cells resulting in lysis of HER2 expressing cancer cells at low picomolar concentrations. In vivo, HER2/CD3 can inhibit growth of established mammary tumors in mice at doses as low as 0,01 mg/kg. Due to its unique mechanism of action, which is unrelated to HER2 signaling or sensitivity to chemotherapeutic agents, HER2/CD3 can eliminate cells refractory to currently approved HER2 therapies and inhibit growth of Kadcyla insensitive mammary tumor models. Treatment with HER2-TDBs also results in tumor regression as a second line treatment for Kadcyla relapsed mammary tumors in mice. Spontaneous mammary tumors arising in MMTV-huHER2 transgenic animals are poorly immunogenic or “immunological deserts” due to their low mutational load and low T cell infiltration. Despite these attributes, HER2/CD3 shows remarkable activity in this model characterized by a robust increase in the number of infiltrating lymphocytes and CD8+ T cells as well as a significant enhancement of tumor CD8+ cytotoxic effector function as measured by increased IFNg production. In addition to inducing T cell proliferation, we further show that HER2/CD3 strongly recruits new T cells from the periphery and this leads to significantly increased T cell numbers in the mammary tumor tissue. Another notable feature of the response to HER2/CD3 treatment is a significant upregulation of PD-1 expression on tumor infiltrating CD8+ T cells. This observation adds a strong rationale for combining HER2/CD3 with anti-PDL1 treatment. Our in vitro experiments further demonstrate that PD-1/PD-L1 signaling can inhibit killing mediated by CD3 bispecific antibodies and that combining two immune therapies - direct polyclonal recruitment of T cell activity together with inhibiting the T cell suppressive PD-1/PD-L1 axis results in enhanced and durable long term responses in vivo. Citation Format: Ji Li, Maria Hristopoulos, Robyn Clark, Jennifer Johnston, Dion Slaga, Bu-Er Wang, Rafael Cubas, Klara Totpal, Melissa R. Junttila, Teemu T. Junttila. Pharmacodynamic response to HER2/CD3 bispecific antibody (HER2-TDB): evidence of T-cell recruitment and further rationale for combination treatment with anti-PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1486.

Tuning Of mTOR In Therapeutic T Cells As A Strategy To Manufacture Effector and Memory T Cells

Retroviral gene transfer of T cell receptors (TCRs) or chimeric-antigen receptors has been successfully used to redirect T cell specificity to tumor antigens and has shown promising results in clinical trials for patients with melanoma and B cell malignancies. Successful therapy relies not only on the generation of an efficient effector response but also in the ability of T cells to engraft and persist during and after cancer rejection. Yet, both aspects can be compromised. On the one hand, effector T cells can be disarmed in vivo by the immunosuppressive environment of the tumor, where the depletion of key nutrients (glucose, arginine, etc.) and the predominance of inhibitory signals (TGF-β, PD-L1, etc.) dampen their functions. On the other hand, current retroviral gene transfer protocols require T cells to be activated and expanded ex vivo,driving terminal effector differentiation at the expense of the capacity for self-renewal (“stemness”). This may prevent the generation of long-term protective memory and compromise therapeutic success. The mechanistic target of rapamycin (mTOR) pathway has recently emerged as a driver of effector differentiation in CD8 T cells by acting as a signalling bridge between extracellular stimuli and effector differentiation. Whilst high doses of rapamycin are known to be immunosuppressive, a low dose regimen increases memory output after a viral challenge without impairing viral clearance. These and other studies suggest that mTOR can be used as a rheostat to regulate T cell differentiation to clinical advantage. Using a mouse model of TCR gene therapy we aimed to produce T cells in which the mTOR pathway is either hyperactive, in order to generate ‘super-effectors’ that can function in the tumor microenvironment, or hypoactive in order to preserve “stemness”, increase engraftment and produce better recall immunity. To this purpose we transferred genes into therapeutic T cells encoding either (1) an mTOR activator, RHEB (Ras homolog enriched in brain) or (2) an mTOR inhibitor, PRAS40 (proline-rich Akt substrate 40 kDa). Upon stimulation in vitro,the phosphorylation of ribosomal protein S6, an mTOR downstream target, was increased in RHEB-expressing cells and decreased in PRAS40-expressing cells. RHEB T cells demonstrated a greater propensity than controls for blast formation, lower expression of L-selectin, increased IFN-g production and resistance to TGF-b. In contrast, PRAS40 T cells were smaller in size, expressed higher surface levels of L-selectin and had reduced but not ablated production of IFN-g, IL-2 and TNF-a production. In vivo, RHEB T cells co-transduced with a tumor-specific TCR showed increased initial expansion that was associated with increased tumor protection. However, this was followed by a steep decrease in T cell numbers compared to control cells, with preliminary experiments indicating that RHEB cells produced a less robust re-call response upon antigenic re-challenge. In contrast, PRAS40 cells failed to expand in vivo or to infiltrate tumors, resulting in complete lack of protection. Yet, the few PRAS40 cells remaining in circulation were predominately high for L-selectin, IL-7Ra and Sca-1 (markers associated with ‘stemness’). These results demonstrated that constitutive suppression of mTOR was of no therapeutic advantage. To circumvent this, we developed a doxycycline (DOX)-inducible PRAS40 vector in which mTOR suppression in T cells can be temporally controlled in vivo. Unlike the constitutive vector used previously, with the DOX-ON system the level of mTOR suppression was lower and furthermore, could be applied only during the period of antigen exposure. Thus, when DOX was added during tumor challenge, PRAS40-expressing T cells could reject tumor as well as controls. However, after DOX was withdrawn and transgene expression turned off, increased numbers of therapeutic T cells were found in peripheral blood. When re-challenged, these T cells produced a stronger recall response relative to cells in which mTOR had not been suppressed initially. Thus, our results show that genetic manipulation of mTOR can be used to alter the intrinsic properties of T cells and direct either their effector or memory differentiation. This approach maybe useful in designing better therapeutic immunotherapeutic strategies according to the type of T cell response required in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 4842: p300 targeting impairs treg function and promotes host anti-tumor immunity

Abstract The long-held view that tumors were not sufficiently immunogenic to promote host immune responses has now been revised to the assessment that at least for some tumors, Foxp3+ T-regulatory (Treg) cells actively suppress anti-tumor immunity. Seeking to build on our ongoing assessment of the regulation of Foxp3+ Treg biology by HDAC and HAT enzymes, we have begun to use combined genetic and pharmacologic approaches to identify and modulate key mechanism within Tregs that limit host immunity. Microarray studies of gene expression in WT, p300-/- and CBP-/- Tregs showed that p300 or CBP deletion led to down-regulation of Foxp3 expression, as well as that of a number of signature Treg genes. Cells co-transfected with Foxp3 and increasing amounts of p300 expression vectors showed that Foxp3 acetylation was enhanced by increasing levels of p300 levels, whereas a p300 small molecule inhibitor, C646 (p300i), impaired Foxp3 acetylation and inhibited Treg function using in vitro suppression assays. In vivo use of C646 showed negligible effects on CD4, CD8 or Treg cell numbers, but qPCR studies showed C646 decreased Treg expression of CTLA-4, GITR, IL-10 and TGF-b, in vitro assays again showed impaired Treg suppression. Additional studies showed, critically, that use of the p300i impaired Treg but not T effector cells in allograft recipients. Thus, parent-to-F1 assays involving alloactivation and proliferation of CD4 an dCD8 T cells were unaffected by C646, and this p300i restored allograft rejection in Treg-dependent models of allograft survival. Turning to tumor models, TC1 lung cancer cells were injected in the flanks of WT B6 mice, B6 mice in which p300 was deleted in CD4+ T cells (Treg plus Teff using CD4-Cre) or just in Tregs (Foxp3-Cre) (n=10/grp), p300 was inhibited using C646 (Alzet pumps, 14 d, 0.9 mg/kg/d) from day 6 post-tumor injection; in both genetic and pharmacologic approaches, p300 targeting suppressed tumor cell growth. qPCR analysis of tumors in C646-treated mice showed increased CD4 and granzyme B mRNA but decreased Foxp3; these data were confirmed by immunohistology, along with increased IFN-g production by purified CD8 cells (ELISPOT), along with reductions in tumor volumes and weights in these C646-treated mice. C646 use did not impair tumor growth in immunodeficient (RAG-/-) mice, in which tumors were exposed to drug in RAG-/- mice as seen by decreased acetylation of histone 3 in tumor extracts (Western blot); no effects on acetylation of certain p300-independent targets within tumor extracts was seen (e.g. acetylation of alpha-tubulin was unimpaired). The p300i also had no direct effect on tumor cell proliferation in vitro. We conclude that our comprehensive genetic and pharmacologic studies show that p300 is an important target for modulation of host Foxp3+ Treg functions, such that Treg suppression can be incrementally reduced and host anti-tumor responses promoted without inducing concomitant autoimmunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4842. doi:1538-7445.AM2012-4842

Positional effect of amino acid replacement on peptide antigens for the enhancement of IFNg production

Rationale Since certain amino acid replacement on peptide antigens induce increased IFNg production from CD4T cell clones, we investigated if specific position of peptide is prone to induce such a change. Methods Human Th0 clones BC20, BC33, and BC42 express distinct TCRa and TCRb but recognize the same TCR ligand; i.e., the same framework of peptide antigen BCGa84-100 in the context of DRB1 ∗1405. We stimulated these T cells with various analogue peptide with one residue replacement, and determined their proliferation and IFNg production. Results Increased IFNg productin was observed, if position 2 of BCGa84-100 was replaced by more hydrophobic residues. Such a change was associated with the structure of CDR3 of TCRa. Conclusions Thus structure-function relationship is apparent in increased IFNg production induced by analogue peptide.