Abstract LBA022: IMM20324, a first-in-class, anti-interleukin-38 monoclonal antibody, rescues myeloid cell activation in vitro and induces robust anti-tumor responses in vivo

2021 ◽  
Author(s):  
John P Dowling ◽  
Pavel A Nikitin ◽  
Fang Shen ◽  
James P Finn ◽  
Nirja Patel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Activation ◽  
Myeloid Cell

scholarly journals Membrane-associated and secreted forms of the Rhesus macaque rhadinovirus-encoded CD200 homologue and cellular CD200 demonstrate differential effects on Rhesus Macaque CD200 Receptor signaling and regulation of myeloid cell activation

2020 ◽  
pp. JVI.01654-20
Author(s):  
Ryan D. Estep ◽  
Aparna N. Govindan ◽  
Kristin Fitzpatrick ◽  
Tiffany C. Blair ◽  
S.A. Rahim Rezaee ◽  
...  
Keyword(s):  
Immune Responses ◽  
Rhesus Macaque ◽  
Cell Activation ◽  
Myeloid Cell ◽  
Functional Protein ◽  
Dna Viruses ◽  
Cd200 Receptor ◽  
First Time

The CD200-CD200R pathway is involved in inhibition of immune responses, and the importance of this pathway to infectious disease is highlighted by the fact that viral CD200 (vCD200) molecules have been found to be encoded by several DNA viruses, including the human gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV), and the closely related rhesus macaque rhadinovirus (RRV). KSHV vCD200 is the most extensively studied vCD200 molecule, however, the only herpesvirus vCD200 molecule to be examined in vivo is that encoded by RRV. Our prior studies have demonstrated that RRV vCD200 is a functional CD200 homologue that is capable of affecting immune responses in vivo, and further, that RRV can express a secreted form of vCD200 (vCD200-Sec) during infection. Despite this information, RRV vCD200 has not been examined specifically for effects on RM CD200R signaling, and the functionality of vCD200-Sec has not been examined in any context. Thus, we developed an in vitro model system in which B cells expressing vCD200 were utilized to assess the effects of this molecule on the regulation of myeloid cells expressing RM CD200R, mimicking interactions that are predicted to occur in vivo. Our findings suggest that RRV vCD200 can bind and induce functional signals through RM CD200R, while vCD200-Sec represents a non-functional protein incapable of affecting CD200R signaling. We also provide the first demonstration of the function of RM CD200, which appears to possess more robust signaling capabilities than RRV vCD200, and also show that KSHV vCD200 does not efficiently induce signaling via RM CD200R.IMPORTANCE Viral CD200 homologues are encoded by KSHV and the closely related RRV. Though RRV vCD200 has been examined, questions still exist in regard to the ability of this molecule to induce signaling via rhesus macaque CD200R, as well as the potential function of a secreted form of vCD200. Further, all previous in vitro studies of RRV vCD200 have utilized an Fc fusion protein to examine functionality, which does not replicate the structural properties of the membrane-associated form of vCD200 that is naturally produced during RRV infection. In this study, we demonstrate for the first time that membrane-expressed RRV vCD200 is capable of inducing signal transduction via RM CD200R, while the secreted form of vCD200 appears to be non-functional. Further, we also demonstrate that RM CD200 induces signaling via RM CD200R, and is more robust than RRV vCD200, while KSHV vCD200 does not appear to induce efficient signaling via RM CD200R.

550 An Axl-targeting monoclonal antibody that inhibits Axl activity and potently stimulates the innate immune response

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A587-A587
Author(s):  
Diego Alvarado ◽  
Laura Vitale ◽  
Mike Murphy ◽  
Thomas O’Neill ◽  
Edward Natoli ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Human Cancer ◽  
Cell Activity ◽  
Negative Regulator

BackgroundAxl is a member of the TAM (Tyro3/Axl/MerTK) family of receptor tyrosine kinases and a negative regulator of innate immunity. Activation of Axl through its ligand Gas6 leads to suppression of myeloid cell activity, while its activation in tumor cells drives tumor growth, metastasis, and is associated with acquired resistance to targeted therapies, radiotherapy and chemotherapy.MethodsPurified monoclonal antibodies and variants thereof were tested in human cancer lines and primary human myeloid cells for effects on Axl signaling and immune activation, respectively.ResultsWe describe a humanized IgG1 Axl-targeting monoclonal antibody (mAb), CDX-0168, that binds to the ligand-binding domain of Axl with sub-nanomolar affinity and potently inhibits Gas6 binding. In tumor cells, CDX-0168 inhibits Gas6-dependent Axl phosphorylation and signaling and elicits tumor cell killing via ADCC in vitro and in vivo. In primary human immune cells, CDX-0168 treatment induces potent release of pro-inflammatory cytokines and chemokines from dendritic cells, monocytes and macrophages through an Fc receptor-dependent mechanism and enhanced T cell activation in mixed lymphocyte reactions. Axl inhibition may further enhance antitumor activity associated with PD-(L)1 blockade. To this end, we generated a tetravalent bispecific Axl x PD-L1 antibody combining CDX-0168 with a potent anti-PD-L1 mAb (9H9) using an IgG-scFv format. The bispecific antibody elicits greater cytokine release and T cell activation in vitro than the combination of the parental antibodies, while maintaining robust Axl and PD-L1 blockade.ConclusionsAdditional studies investigating simultaneous blockade of the Axl and PD-L1 pathways with other agents may further exploit the potential for this novel anti-cancer therapeutic approach.

scholarly journals VISTA Is a Novel Regulator of Macrophage Biology

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2320-2320
Author(s):  
Parth Shah ◽  
Mohamed Eltanbouly ◽  
Nicole Smits ◽  
Aurelien Sarde ◽  
Randolph Noelle ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Biology ◽  
Time Course ◽  
Cell Activation ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Mouse Bone Marrow ◽  
Rna Seq

Background:V-domain Ig suppressor of T cell activation (VISTA) is an immune checkpoint receptor with an established role as an inhibitory receptor on T cells. However, its role in myeloid cell biology remains unclear. Owing to the much higher expression of VISTA on monocytes and macrophages, we investigated its role in regulating myeloid biology. Methods: Mouse bone marrow-derived macrophages (BMDMs) were differentiated and treated with agonistic anti-VISTA mAb under steady-state and conditions of M1 inflammatory polarization (LPS + IFN-𝛾). Human CD14+ monocytes were isolated from peripheral blood and treated with anti-VISTA in vitro. Phosphoarrays were used to assess signaling downstream of VISTA engagement and Luminex analysis was used to evaluate cytokine production. Time-course total RNA-seq was performed on BMDMs (at 1, 2, 4, 8 and 16 hours of anti-VISTA treatment). To evaluate the effect of anti-VISTA in vivo, single-cell RNA-seq (scRNA-seq) was performed on FACS sorted CD11b+ myeloid populations 12 hours after mice were treated with intravenous anti-VISTA. Results: Anti-VISTA treatment of BMDMs and human monocytes in vitro induced rapid phosphorylation events including robust phosphorylation of Raf-1 kinase at the suppressive serine residue 43.Next, we generated a comprehensive timestamp of VISTA activation in BMDMs in vitro by time-course deep RNA-seq analysis of agonistic anti-VISTA treated cells under rigorous M1 inflammatory polarization. This revealed a unique activation state marked by the upregulation of inhibitory cytokines IL-10 and IL1RA as well as ablation of IL-12 family cytokines, suggestive of an M2-like signature. The presence of an M2-like immunoregulatory signature defined by upregulation of the IL-10 was also seen in macrophages identified post clustering of in vivo scRNA-seq data on myeloid cells. Conclusion: Using novel agonistic anti-VISTA antibodies, we reveal that VISTA triggering on myeloid cells can induce an alternative anti-inflammatory cell-state, even under conditions of strong inflammatory polarization. This introduces VISTA as an intrinsic checkpoint of macrophage tolerance. Disclosures Noelle: Immunext: Employment, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals 585 LIGHT (TNFSF14) co-stimulation enhances myeloid cell activation and anti-tumor immunity in the setting of PD-1 and TIGIT checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A615-A615
Author(s):  
George Fromm ◽  
Kyung Jin Yoo ◽  
Kellsey Johannes ◽  
Casey Shuptrine ◽  
Zach Opheim ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Acquired Resistance ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Preclinical Model ◽  
And Function ◽  
Anti Tumor Activity

BackgroundCo-inhibition of TIGIT and PD-1/L1 improves response rates compared to monotherapy PD-1/L1 blockade in checkpoint naïve NSCLC with PD-L1 expression >50%. TIGIT mAbs with an effector competent Fc can induce myeloid cell activation, and some have also demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT antibody blockade translates to anti-tumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Further, DNAM-1 is downregulated on TIL in advanced and CPI resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or CPI resistant tumors, may be enhanced by immune co-stimulation that independently operates from PD-1/L1 inhibition.MethodsMouse and human TIGIT-Fc-LIGHT molecules were generated and assessed using Octet, MSD, and cell binding assays, and function was evaluated using in vitro/in vivo activation and anti-tumor efficacy experiments; including a preclinical model engineered to mimic human CPI acquired resistance.ResultsTIGIT-Fc-LIGHT was nominated using in vitro and genomic screening assays designed to identify TNF costimulatory receptors widely expressed on TIL, T stem cell memory (Tscm), and NK cells; relative to DNAM-1 expression. HVEM was prioritized, and its ligand TNFSF14 (LIGHT) also directly activates myeloid cells through binding to a second receptor, LTβR. TIGIT-Fc-LIGHT simultaneously engaged TIGIT and LIGHT receptors at low nanomolar affinities (~3.5–6.5 nM), without the requirement for an effector competent Fc. HVEM signaling overlaps with DNAM-1, and TIGIT-Fc-LIGHT activated canonical and non-canonical NFκB pathways, leading to increased tumor infiltration of antigen-specific CD8+ T and NK cells. Importantly, anti-tumor efficacy induced by monotherapy TIGIT-Fc-LIGHT was maintained in aggressive anti-PD-1 acquired resistant tumors, a model where combined PD-1 and TIGIT antibody blockade was inactive. Because HVEM lacks cytoplasmic domain homology to DNAM-1, HVEM signaling is unlikely to be regulated by PD-1. Indeed, while anti-tumor activity of TIGIT-Fc-LIGHT was enhanced by PD-1/L1 blockade, it was not dependent upon combination. TIGIT-Fc-LIGHT also directly activated myeloid cells and increased the expression of CXCL10 and CXCL11, and stimulated proinflammatory cytokines, including CCL2, CCL4, and CXCL13.ConclusionsTIGIT-Fc-LIGHT was designed to overcome the limitations of TIGIT blocking antibodies through: 1) preserved costimulation in advanced tumors, 2) direct myeloid cell activation, 3) blockade of all known TIGIT ligands, and with 4) no risk of depleting effector lymphocytes since TIGIT-Fc-LIGHT activity does not require Fc function. Pre-clinical data indicate that these goals were achieved, and further development is warranted.

scholarly journals Immunometabolism orchestrates training of innate immunity in atherosclerosis

2019 ◽  
Vol 115 (9) ◽  
pp. 1416-1424 ◽  
Author(s):  
Julia van Tuijl ◽  
Leo A B Joosten ◽  
Mihai G Netea ◽  
Siroon Bekkering ◽  
Niels P Riksen
Keyword(s):  
Cell Activation ◽  
Cholesterol Metabolism ◽  
Myeloid Cell ◽  
Metabolic Reprogramming ◽  
Immune Memory ◽  
Low Grade ◽  
Trained Immunity ◽  
Monocytes And Macrophages

Abstract Atherosclerosis is characterized by a persistent, low-grade inflammation of the arterial wall. Monocytes and monocyte-derived macrophages play a pivotal role in the various stages of atherosclerosis. In the past few years, metabolic reprogramming has been identified as an important controller of myeloid cell activation status. In addition, metabolic and epigenetic reprogramming are key regulatory mechanisms of trained immunity, which denotes the non-specific innate immune memory that can develop after brief stimulation of monocytes with microbial or non-microbial stimuli. In this review, we build the case that metabolic reprogramming of monocytes and macrophages, and trained immunity in particular, contribute to the pathophysiology of atherosclerosis. We discuss the specific metabolic adaptations, including changes in glycolysis, oxidative phosphorylation, and cholesterol metabolism, that have been reported in atherogenic milieus in vitro and in vivo. In addition, we will focus on the role of these metabolic pathways in the development of trained immunity.

scholarly journals Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

Blood ◽  
2009 ◽  
Vol 114 (26) ◽  
pp. 5322-5330 ◽  
Author(s):  
Siao-Yi Wang ◽  
Suresh Veeramani ◽  
Emilian Racila ◽  
Jeffrey Cagley ◽  
David C. Fritzinger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Antibody Therapy ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Target Cells

Abstract Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)–cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

scholarly journals 782 PD-L1-targeted ISAC combines myeloid cell activation, immune-checkpoint inhibition and ADCP to improve anti-tumor efficacy over anti-PD-L1 antibodies in preclinical models

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A817-A817
Author(s):  
Justin Kenkel ◽  
Rishali Gadkari ◽  
Karla Henning ◽  
Romas Kudirka ◽  
William Mallet ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Myeloid Cell ◽  
Checkpoint Inhibition ◽  
Immune Checkpoint Inhibition ◽  
Tumor Models

BackgroundPD-L1 is an immune checkpoint that regulates anti-tumor T cell responses and is expressed on tumor cells as well as tumor-infiltrating immune cells across many tumor types. Immune-stimulating antibody conjugates (ISACs) consist of tumor-targeting antibodies conjugated to immune stimulants and are designed to activate the innate and adaptive immune systems against tumor cells following systemic administration. Here we show that PD-L1-targeted TLR7/8 ISACs elicit robust myeloid cell activation which leads to improved anti-tumor responses compared to anti-PD-L1 treatment in pre-clinical tumor models.MethodsA panel of proprietary anti-PD-L1 antibodies was identified through a phage display screen and subsequently tested for PD-L1 binding affinity and specificity, PD-L1/PD-1 blocking, antibody-dependent cellular phagocytosis (ADCP) by myeloid cells, and anti-tumor efficacy. Lead antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting PD-L1 ISACs were evaluated for in vitro myeloid cell activation and in vivo efficacy against syngeneic and xenograft tumors.ResultsAnti-PD-L1 antibodies induced robust ADCP by myeloid effector cells and medium to strong PD-L1/PD-1 blockade in vitro. Selected antibodies inhibited the growth of syngeneic MC38-hPD-L1 tumors in vivo, confirming efficient immune-checkpoint blockade. The conjugated PD-L1 ISACs induced robust, target-dependent activation of myeloid cells when co-cultured with PD-L1-expressing tumor cells, as measured by increased secretion of such cytokines as IL-12p70, IFN-alpha, and TNF-alpha. Importantly, myeloid activation was observed following co-culture with tumor cells having various levels of endogenous PD-L1 expression that was within the range of PD-L1 expression observed in human tumors. Systemically administered surrogate PD-L1 ISACs were well tolerated in mice and showed improved anti-tumor efficacy over anti-PD-L1 antibodies, with significant tumor growth delay or complete responses frequently observed in syngeneic (e.g. MB49, MC38-hPD-L1) as well as xenograft (e.g. HCC1954-hPD-L1) tumor models.ConclusionsThese data demonstrate the potential of a PD-L1-targeted ISAC as a multifunctional therapeutic that may improve efficacy of PD-L1/PD-1 inhibition by combining three mechanisms of action into a single molecule: TLR-mediated myeloid cell activation, T cell activation through immune-checkpoint inhibition as well as ADCP.Ethics ApprovalAll animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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