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List of PRMT6-associated proteins

Supplementary Figure 1. Increased expression of PRMT6 in non-small cell lung cancer.

Increased expression of protein arginine methyl transferase 6 (PRMT6) correlates with worse prognosis in lung cancer cases. To interrogate the in vivo functions of PRMT6 in lung cancer, we developed a tamoxifen-inducible lung-targeted PRMT6 gain-of-function mouse model, which mimics PRMT6 amplification events in human lung tumors. Lung-targeted overexpression of PRMT6 accelerated cell proliferation de novo and potentiated chemical carcinogen (urethane)-induced lung tumor growth. To explore the molecular mechanism/s by which PRMT6 promotes lung tumor growth, we used proteomics-based approaches and identified interleukin-enhancer binding protein 2 (ILF2) as a novel PRMT6-associated protein. Furthermore, by using a series of in vitro gain-of-function and loss-of-function experiments, we defined a new role for the PRMT6–ILF2 signaling axis in alternate activation of tumor-associated macrophages (TAM). Interestingly, we have also identified macrophage migration inhibitory factor, which has recently been shown to regulate alternate activation of TAMs, as an important downstream target of PRMT6–ILF2 signaling. Collectively, our findings reveal a previously unidentified noncatalytic role for PRMT6 in potentiating lung tumor progression via the alternate activation of TAMs. Implications: This is the first study to demonstrate an in vivo role for PRMT6 in lung tumor progression via the alternate activation of TAMs.

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. CAR-T cell therapies, however, have limited efficacy in solid tumors and are associated with severe toxicity, highlighting the need for safer and more efficacious novel cell therapies. With their intrinsic tumor killing capacity, few treatment-related toxicities, and the ability to be given to patients off-the shelf, natural killer (NK) cells are an attractive alternative therapy option to CAR-T cells. While most NK cell therapies are produced from healthy donor cells, deriving NK cells from induced pluripotent stem cells (iPSCs) has the unique advantage that a clone with any desired edits can be generated. We aim to leverage our iPSC platform in combination with our proprietary gene editing technologies to create highly differentiated off-the-shelf treatments for solid tumors. Using our proprietary engineered AsCas12a, we generated double knocked-in (DKI) iPSC clones in which a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into the GAPDH locus to increase the effector function and persistence of iNKs. Constitutive surface expression of CD16 and mbIL-15 by the DKI iNKs was demonstrated. DKI iNKs showed significantly increased natural and antibody dependent cellular cytotoxicity when compared to wild type (WT) iNKs in a SKOV3 tumor spheroid assay in vitro. Furthermore, in the absence of exogeneous cytokines, DKI iNKs persistence in vitro was dramatically improved over WT iNKs. The anti-tumor efficacy of the DKI iNKs in vivo was evaluated using a SKOV3 ovarian cancer model. Tumor bearing mice were treated with WT or DKI iNKs intraperitoneally in combination with trastuzumab or treated with trastuzumab alone. No exogenous cytokines were administered. DKI iNKs combined with a single dose or multiple doses of trastuzumab exerted greater tumor control compared to WT iNKs with trastuzumab, or trastuzumab alone. A single dose of DKI iNKs combined with three doses of trastuzumab induced tumor clearance in 6 out of 8 mice and significantly prolonged survival. Importantly, DKI iNKs were detected in the peritoneum of the treated animals for greater than 3 months, demonstrating that the mbIL-15 maintained iNK survival for a prolonged period of time in the absence of exogeneous cytokine support. In summary, knocking-in CD16 and mbIL-15 to the GAPDH locus of iPSCs dramatically increased the persistence of the DKI iNKs which exhibited robust anti-tumor activities in a solid tumor mouse model. These data demonstrate that our platform enables the development of off-the-shelf iNK cell medicines that may be highly effective for treating solid tumors. Citation Format: Alexander G. Allen, Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, Stephen Sherman, Laura Blaha, Mark Shearman, Kate Zhang, Kai-Hsin Chang. AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse 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 562.

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells. Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity. We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing. In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. Disclosures Gerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Although checkpoint immunotherapies have revolutionized the treatment of cancer, not all tumor types have seen substantial benefit. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in which very limited responses to immunotherapy have been observed. Extensive immunosuppressive myeloid cell infiltration in PDAC tissues has been postulated as a major mechanism of resistance to immunotherapy. Strategies concomitantly targeting monocyte or granulocyte trafficking or macrophage survival, in combination with checkpoint immunotherapies, have shown promise in preclinical studies, and these studies have transitioned into ongoing clinical trials for the treatment of pancreatic and other cancer types. However, compensatory actions by untargeted monocytes, granulocytes, and/or tissue resident macrophages may limit the therapeutic efficacy of such strategies. CD11b/CD18 is an integrin molecule that is highly expressed on the cell surface of these myeloid cell subsets and plays an important role in their trafficking and cellular functions in inflamed tissues. Here, we demonstrate that the partial activation of CD11b by a small-molecule agonist (ADH-503) leads to the repolarization of tumor-associated macrophages, reduction in the number of tumor-infiltrating immunosuppressive myeloid cells, and enhanced dendritic cell responses. These actions, in turn, improve antitumor T cell immunity and render checkpoint inhibitors effective in previously unresponsive PDAC models. These data demonstrate that molecular agonism of CD11b reprograms immunosuppressive myeloid cell responses and potentially bypasses the limitations of current clinical strategies to overcome resistance to immunotherapy.

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell–based high–throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high–content screening–based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z′ value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an β1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active β1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS–induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active β1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside–induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte–based screening assays for identifying novel therapeutics for proteinuric kidney diseases.

Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) with unclear etiology and limited treatment options. Immune cell infiltration into the kidneys, a hallmark of LN, triggers tissue damage and proteinuria. CD11b, the α-chain of integrin receptor CD11b/CD18 (also known as αMβ2, Mac-1, and CR3), is highly expressed on the surface of innate immune cells, including macrophages and neutrophils. Genetic variants in the human ITGAM gene, which encodes for CD11b, are strongly associated with susceptibility to SLE, LN, and other complications of SLE. CD11b modulates several key biological functions in innate immune cells, including cell adhesion, migration, and phagocytosis. CD11b also modulates other signaling pathways in these cells, such as the Toll-like receptor signaling pathways, that mediate generation of type I interferons, a key proinflammatory cytokine and circulating biomarker in SLE and LN patients. However, how variants in ITGAM gene contribute to disease pathogenesis has not been completely established. Here, we provide an overview of CD11b modulated mechanisms and the functional consequences of the genetic variants that can drive disease pathogenesis. We also present recent insights from studies after pharmacological activation of CD11b. These studies offer novel mechanisms for development of therapeutics for LN, SLE and other autoimmune diseases.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a−/−) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a−/− animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-β, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Lung cancer is the leading cause of cancer related deaths in the United States. With a 5-year survival rate of 18%, lung cancer patients are in immediate need of new therapeutic options. A major problem facing immunotherapy for cancer is the active immune suppression by the tumor. Tumors show presence of large numbers of tumor associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization to the tumor, and promote tumor survival. Integrin CD11b is highly expressed on TAMs and is essential for their recruitment and biological functions. Reasoning that CD11b activity is important for controlling tumor growth, we developed a novel small molecule agonist called leuhadherin-1 (LA1), which activates CD11b. We found that CD11b activation by LA1 significantly reduced tumor growth in wild type mice bearing Lewis Lung Carcinoma (LLC) tumors as early as one week post treatment. Conversely, LLC tumors grew at a faster rate in CD11b-/- mice, compared to wild type mice, showing that CD11b is important for controlling tumor growth. Importantly, LA1 treatment in tumor bearing CD11b-/- mice did not show any efficacy, demonstrating the specificity of LA1 to CD11b. These data suggest that CD11b activation via LA1 modulates TAMs in tumors and is a novel therapeutic strategy against cancer. Citation Format: Terese Geraghty, Anugraha Rajagopalan, Samia Khan, Judith Varner, Vineet Gupta. Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 137.

Lung cancer kills more people in the United States than any other malignancy. High mortalities are in part due to the diagnosis at an advanced inoperable stage where the 5-year survival is only 4%. Therefore, improving survival will require increased knowledge of genes that drive the complex mechanisms of lung cancer initiation and progression. Emerging data implicate novel roles for protein arginine methylation in lung cancers. In protein arginine methylation, nitrogen/s of arginine can be post-translationally modified via the addition of a methyl group, catalyzed by a class of enzymes, protein arginine methyl transferases (PRMTs). Based on the computational analysis, tissue staining, and immunoblotting we have identified an increased expression of PRMT6 in lung tumors. To test the biologic relevance of PRMT6 in lung tumor development, we employed an inducible lung-targeted PRMT6 gain-of-function (GOF) mouse model. For the first time, we show that lung-targeted overexpression of PRMT6 results in spontaneous lung tumor development. We also observed a robust increase in lung tumors in the PRMT6 GOF mice in response to urethane, a KRas mutagen, and a prototypical model to study lung tumorigenesis. Excitingly, we detected an increase in the number of tumor-associated macrophages (TAMs) in the PRMT6 GOF mice. Further characterization of the TAMs from the PRMT6 GOF mice revealed that they were M2 polarized with immunosuppressive, angiogenesis-enhancing, and tumor-promoting activities. Taken together, these results demonstrate a novel role for PRMT6 in lung tumor development by way of macrophage M2 polarization. Therefore, defining the complete mechanism of PRMT6-mediated macrophage M2 polarization could lead to the development of novel therapeutic strategies to treat lung cancer. Citation Format: Sreedevi Avasarala, Pei-Ying Wu, Samia Q. Khan, Michelle Van Scoyk, Yanlin Su, Odile David, Vineet Gupta, Mark T. Bedford, Robert A. Winn, Rama Kamesh Bikkavilli. PRMT6 promotes lung tumor growth via the modulation of macrophage M2 polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 138.

Genetic variations in the ITGAM gene (coding for CD11b) produce defective CD11b and associate with a risk for systemic lupus erythematosus (SLE, lupus). Elevated level of IFN I in circulation plays a pathogenic role and is a heritable risk factor for SLE. Whether variations in ITGAM are linked to high IFN I and whether CD11b activation could be a therapeutic strategy is unknown and is explored here. We measured serum IFN I activity in 171 SLE patients and report that three ITGAM variants significantly associate with elevated levels of IFN I in lupus, suggesting a direct link between reduced CD11b activity and elevated inflammation in patients. Given that ITGAM SNPs result in functionally deficient CD11b, we tested whether partial CD11b activation with small molecule agonist, leukadherin-1 (LA1) reduces IFN I responses and determined the underlying mechanistic pathways. CD11b activation with LA1 reduced TLR-dependent pro-inflammatory signaling in leukocytes and suppressed IFN I signaling via an AKT-FOXO3-IRF7 pathway. TLR-stimulated macrophages from CD11b SNP carriers showed increased basal expression of IRF7 and IFNB, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1. To test the efficacy of LA1 in vivo, we used the MRL/lpr mice that develop IFN I-dependent multi-organ lupus. LA1 treatment reduced IFN I responses and protected lupus-prone MRL/lpr mice from kidney injury. LA1-treated mice had reduced proteinuria, IgG renal immune complex deposition, and glomerular damage as compared to controls. LA1 suppresses TLR-stimulated overproduction of cytokines in vivo, which drives lupus pathogenesis. These findings suggest that pharmacological CD11b activation is a promising therapeutic strategy in human SLE.

Kidney allograft rejection is associated with infiltration of inflammatory CD11b+ leukocytes. A CD11b agonist leukadherin-1 (LA1) increases leukocyte adhesion, preventing their transmigration and tissue recruitment in vivo. Here, we test the extent to which LA1-mediated activation of CD11b/CD18 enhances kidney allograft survival in a mouse model of fully MHC-mismatched orthotopic kidney transplantation, where C57BL/6J (H-2(b)) recipients received kidney allografts from Balb/c mice (H-2(d)). Isograft control recipients received a kidney from a littermate. Control isograft and allograft recipients were treated daily with cyclosporine (CsA) for 2 weeks, while the test group received CsA therapy and daily LA1 injections during week 1 and alternate days during weeks 2-8. LA1 treatment reduced interstitial leukocyte infiltration in the allograft, reduced neointimal hyperplasia and glomerular damage, and prolonged graft survival from 48.5% (CsA only) to 100% (CsA and LA1) on day 60. Serum creatinine levels showed significantly improved kidney function in LA1-treated mice compared to CsA-treated allograft controls [0.52 ± 0.18 mg/dL vs 0.24 ± 0.07 mg/dL (n = 5), respectively]. Furthermore, combination therapy reduced macrophage infiltration and increased the frequency of FoxP3 + Tregs in the allograft. These findings indicate a crucial role for CD11b/CD18 in the control of leukocyte migration to the transplanted kidney and identify integrin agonist LA1 as a novel potential therapeutic agent for kidney transplantation.

Tumors associated macrophages (TAMs) mediate neovascularization and promote tumor growth. Integrin CD11b/CD18 is highly expressed on cells of myeloid and granulocytic lineage and mediates their adhesion, migration and tissue recruitment functions. CD11b+ TAMs are present at high numbers in several types of human cancers, including breast and non-small cell lung cancer, and are associated with reduced T-cell mediated anti-tumor immunity. This suggests that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. Recently, we have developed a novel small molecule CD11b agonist termed leukadherin-1 (LA1) that binds to and allosterically activates integrin CD11b on leukocytes. We also reported that CD11b activation is a more effective strategy for reducing CD11b+ cell tissue infiltration, as compared to integrin antagonists. LA1 binding transiently increases leukocyte adhesion to the vascular endothelium and reduces their transmigration in vivo. Here, we show that LA1 mediated CD11b activation reduced the number of CD11b+ TAMs in two different murine tumor models that was accompanied with diminished primary tumor growth. LA1 treated animals also had significantly reduced tumor angiogenesis and prolonged survival. These improved aspects were accompanied with reduction in vessel density as indicated by reduced intratumoral expression levels of CD31 and αSMA and increased numbers of CD8+ T cells indicating an anti-tumor immune profile response fostering tumor suppression. Our data provide a rationale for using allosteric CD11b agonists to target TAMs as a novel therapeutic approach for treating cancer. Citation Format: Samia Q. Khan, Mohd. H. Faridi, Shehryar J. Khaliqdina, Antonio J. Barbosa, Judith A. Varner, Vineet Gupta. Integrin agonists reduce infiltration of tumor-associated macrophages to promote T-cell mediated anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B03.

TNF superfamily member 15 (TL1A) is the ligand for TNFR superfamily (TNFRSF)25. We previously reported that TNFRSF25 stimulation with an agonist Ab, 4C12, expands pre-existing CD4+Foxp3+ regulatory T cells (Tregs) in vivo. To determine how the physiological ligand differs from the Ab, we generated a soluble mouse TL1A-Ig fusion protein that forms a dimer of TL1A trimers in solution with an apparent molecular mass of 516 kDa. In vitro, TL1A-Ig mediated rapid proliferation of Foxp3+ Tregs and a population of CD4+Foxp3− conventional T cells. TL1A-Ig also blocked de novo biogenesis of inducible Tregs and it attenuated the suppressive function of Tregs. TNFRSF25 stimulation by TL1A-Ig in vivo induced expansion of Tregs such that they increased to 30–35% of all CD4+ T cells in the peripheral blood within 5 d of treatment. Treg proliferation in vivo was dependent on TCR engagement with MHC class II. Elevated Treg levels can be maintained for at least 20 d with daily injections of TL1A-Ig. TL1A-Ig–expanded Tregs expressed high levels of activation/memory markers KLRG1 and CD103 and were highly suppressive ex vivo. TL1A-Ig–mediated Treg expansion in vivo was protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of conventional T cells to Tregs in the lung and blocking eosinophil exudation into the bronchoalveolar fluid. Thus, TL1A-Ig fusion proteins are highly active and tightly controllable agents to stimulate Treg proliferation in vivo, and they are uniquely able to maintain high levels of expanded Tregs by repeated administration.

Breast cancer remains the second highest cancer-related cause of death in women today. Neoadjuvant therapies reduce tumor size in patients but long-term survival remains poor. Tumor microenvironment shows a significant infiltration of CD11b+ tumor associated macrophages (TAMs) that promote tumor growth and metastases, while suppressing T-cell mediated anti-tumor immunity, suggesting that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. We recently developed a novel small molecule that targets CD11b+ cells and reduces their migration and tissue recruitment. Unlike previously described strategies, our methodology involves allosteric activation of CD11b, which induces transient increase in adhesion of CD11b+ cells to the vascular endothelium, thereby reducing the ability of the myeloid cells to transmigrate out of circulation and suppressing their recruitment into tumor and inflamed tissues. However, it is currently not known as to whether small molecule mediated CD11b activation can reduce TAM infiltration in tumors and whether that will have a therapeutic benefit. Here, we present results from studies with our lead compound leukadherin1 (LA1) in murine models of breast cancer. Our studies show that treatment of established syngeneic Cl66 orthotopic breast tumors in Balb/c mice with LA1 significantly reduces tumor growth and increases survival. LA1 treatment significantly reduced infiltration of CD11b+ cells into the tumor stroma, without affecting the levels of cells in circulation. LA1 treatment also significantly reduced expression of molecules that promote angiogenesis and tumor growth including MMP9, S100A8 and ARG1 that coincided with decreased CD31 and α-SMA, suggesting tumor vasculature disruption. Immune cell profiling revealed that LA1 treatment further promotes anti-tumor immunity by increasing the influx of cytotoxic CD8+ T cells into tumors. These data provide a rationale for using allosteric CD11b agonists to target TAM recruitment as a novel therapeutic approach for breast cancer. Citation Format: Samia Q. Khan, Mohd H. Faridi, Brian Ruffell, Ana M. S. Lopez, Shehryar Jehangir, Antonio J. Barbosa, Marta Torroella-Kouri, Lisa M. Coussens, Judith Varner, Vineet Gupta. An integrin agonist suppresses breast cancer by reducing CD11b+ leukocytes, promoting anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A17.

We demonstrated that the TNF receptor superfamily member 25 (TNFRSF25) agonistic antibody 4C12 drives the in vivo proliferation of preexisting CD4+FoxP3+ cells (Tregs) but not that of conventional CD4 cells (Tconvs) (1). TNFRSF25-mediated Treg proliferation depends upon MHC II loaded with self-antigen and is blocked by calcineurin inhibitors. If antigen is provided to Tconvs in vitro or in vivo, TNFRSF25 signals also costimulate proliferation and effector cell activity (2–5). Thus, Treg proliferation to TNFRSF25 signals is due to the continuous presence of self-antigenic stimulation of the TCR in vivo, rather than to the level of TNFRSF25 expression. TNFRSF25-mediated Treg expansion is not unique to the agonistic 4C12 antibody, as a TL1A-Ig fusion protein mediates similar Treg expansion in vivo (unpublished observations). In support of this conclusion, the subsequent reports by Al-Shamkhani’s group and Siegel’s group demonstrated that in transgenic mouse models overexpressing TL1A, Tregs proliferate at high frequencies throughout the secondary lymphoid organs and have increased frequencies of Tconvs with an effector/memory phenotype systemically, resulting in mice that are prone to spontaneous inflammatory bowel disease (6, 7). Meylan et al. also demonstrate that TL1A inhibits the de novo induction of Tregs from Tconvs in vitro (7). Both of these recent reports agree that TNFRSF25 attenuates the suppressive capacity of Tregs. In a letter to the JCI, Al-Shamkhani and colleagues provide evidence that the level of expression of TNFRSF25 appears much more similar between Tregs and Tconvs than we originally reported. In recent studies, we have further explored the binding of additional anti-TNFRSF25 antibodies and TL1A-Ig fusion proteins generated in our laboratory to Tregs and Tconvs by flow cytometry. We have found clone-specific variations in the intensity of TNFRSF25 staining between Tconvs and Tregs, which suggests that, as indicated by Al-Shamkhani, the absolute expression of TNFRSF25 between Tconvs and Tregs is not a key determinant in the proliferative specificity of TNFRSF25 agonists or TL1A to Tregs. Instead, as both our and Siegel’s group concluded, the determining factor of TNFRSF25 action in vivo is the availability of cognate antigen, with self-antigen continuously present for Treg expansion. Together, these three reports agree that TNFRSF25 is a regulator of Treg proliferative responses, both with the natural ligand (TL1A) and with receptor agonistic reagents (antibodies and fusion proteins). In the transgenic systems used by Taraban and colleagues and Meylan et al., the continuous availability of TL1A stimulates both the proliferation of Tregs and activation of antigen-experienced effector/memory phenotype Tconvs systemically (6, 7). The finding by Meylan et al. with regard to inhibition of in vitro iTreg generation by TL1A is consistent with the possibility of a role for TL1A in inflammatory bowel disease, in which tolerance to endogenous microbial antigens may have been disrupted by transgenic overexpression of TL1A (7). The balance of TNFRSF25 triggering regulatory or effector immunity is likely dependent upon the availability of cognate antigen to Tregs or Tconvs and not on absolute differences in the expression of the receptor between each subset.