Agonistic mAb Research Articles

Human Hepatic CD56bright NK Cells Display a Tissue-Resident Transcriptional Profile and Enhanced Ability to Kill Allogenic CD8+ T Cells.

Liver-resident CD56brightCD16- natural killer (NK) cells are enriched in the human liver and are phenotypically distinct from their blood counterparts. Although these cells are capable of rapid cytotoxic effector activity, their functional role remains unclear. We hypothesise that they may contribute to immune tolerance in the liver during transplantation. RNA sequencing was carried out on FACS sorted NK cell subpopulations from liver perfusates (n=5) and healthy blood controls (n=5). Liver-resident CD56brightCD16+/- NK cells upregulate genes associated with tissue residency. They also upregulate expression of CD160 and LY9, both of which encode immune receptors capable of activating NK cells. Co-expression of CD160 and Ly9 on liver-resident NK cells was validated using flow cytometry. Hepatic NK cell cytotoxicity against allogenic T cells was tested using an in vitro co-culture system of liver perfusate-derived NK cells and blood T cells (n=10-13). In co-culture experiments, hepatic NK cells but not blood NK cells induced significant allogenic T cell death (p=0.0306). Allogenic CD8+ T cells were more susceptible to hepatic NK cytotoxicity than CD4+ T cells (p<0.0001). Stimulation of hepatic CD56bright NK cells with an anti-CD160 agonist mAb enhanced this cytotoxic response (p=0.0382). Our results highlight a role for donor liver NK cells in regulating allogenic CD8+ T cell activation, which may be important in controlling recipient CD8+ T cell-mediated rejection post liver-transplant.

Macrophage-inducible C-type lectin activates B cells to promote T cell reconstitution in heart allograft recipients

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage‐inducible C‐type lectin (Mincle, Clec4e) expression is up‐regulated in B cells from heart allograft recipients treated with murine anti‐thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro‐inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non‐transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell‐depleted, but not in Mincle‐deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle‐dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage‐associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation.

Abstract 605: MoA-based potency bioassays for immunotherapy programs targeting the TIGIT/CD112R/CD226 axis

Abstract Immune checkpoint (IC) receptors play critical roles in maintaining immune homeostasis and have emerged as proven drug targets for the treatment of various cancers and autoimmune disorders. Monoclonal antibodies (mAb) designed to block immune inhibitory receptors PD-1 and CTLA-4 have been approved and show unprecedented efficacy in cancer treatment. This led to rapid expansion of drug development to many more IC receptors including members of the T cell immunoreceptor with Ig and ITIM domains (TIGIT) - CD112R (PVRIG) - CD226 Axis. TIGIT binds to its ligands CD155 (PVR) and CD112 (PVRL2) while CD112R binds to CD112. Binding of TIGIT or CD112R to CD155/CD112 inhibits the co-stimulatory activity of the paired receptor CD226. Thus, therapeutic targeting of this axis can modulate T cell and NK cell activation and unleash anti-cancer immunity. One of the bottlenecks of immunotherapy drug development is the lack of quantitative and reproducible functional assays for mAb potency determination. Existing methods rely heavily on primary immune cells which is labor intensive and highly variable. To address this need, we developed a panel of cell-based reporter bioassays that can quantitatively measure the potencies of mAbs targeting TIGIT/CD155, TIGIT/CD112, and CD112R/CD112. Owing to positive clinical trial results of TIGIT blockade in combination with PD-1/PD-L1 blockade, we further developed a potency bioassay for bispecific antibodies simultaneously targeting TIGIT and PD-1/PD-L1. These bioassays each consist of an engineered T effector cell line that expresses T cell receptors and IC receptors of interest, and an engineered artificial antigen presenting cells (aAPC) expressing the corresponding ligands for the IC receptors. The T effector cell lines also express a luciferase reporter driven by a promoter specifically responding to TCR activation which can be inhibited by signaling from the IC receptors. Additionally, we developed bioassays that can measure potencies of agonist or antagonist mAbs targeting CD226. These bioassays are designed to reflect the mechanisms of action for the drug candidates designed for each IC receptor and the assay signals are specific. Most are prequalified according to ICH guidelines and show the precision, accuracy and linearity required for relative potency determination and product stability study under cGMP environments. Therefore, these MoA-based bioassays can serve as valuable tools for early drug discovery, potency determination and stability studies throughout the development of mAbs targeting the TIGIT/CD112R/CD226 axis. Citation Format: Jamison Grailer, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-Jie Jey Cheng. MoA-based potency bioassays for immunotherapy programs targeting the TIGIT/CD112R/CD226 axis [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 605.

Abstract 628: Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Abstract Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [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 628.

Safety and Tolerability of MEDI0562, an OX40 Agonist mAb, in Combination with Durvalumab or Tremelimumab in Adult Patients with Advanced Solid Tumors.

Combination therapies targeting immunologic checkpoints have shown promise in treating multiple tumor types. We report safety and tolerability of MEDI0562, a humanized IgG1K OX40 mAb, in combination with durvalumab (anti-PD-L1), or tremelimumab (anti-CTLA-4), in adult patients with previously treated advanced solid tumors. In this phase I, multicenter, open-label study, patients received escalating doses of MEDI0562 (2.25, 7.5, or 22.5 mg) every 2 weeks in combination with durvalumab (1,500 mg) or tremelimumab (75 or 225 mg) every 4 weeks, intravenously, until unacceptable toxicity or progressive disease. Tumor assessments were performed every 8 weeks. The primary objective was to evaluate safety and tolerability. Among the 27 and 31 patients who received MEDI0562 + durvalumab or MEDI0562 + tremelimumab, 74.1% and 67.7% reported a treatment-related adverse event (AE), and 22.2% and 19.4% experienced a treatment-emergent AE that led to discontinuation, respectively. The MTD of MEDI0562 + durvalumab was 7.5 mg MEDI0562 + 1,500 mg durvalumab; the maximum administered dose of MEDI0562 + tremelimumab was 22.5 mg MEDI0562 + 225 mg tremelimumab. Three patients in the MEDI0562 + durvalumab arm had a partial response. The mean percentage of Ki67+CD4+ and Ki67+CD8+ memory T cells increased by >100% following the first dose of MEDI0562 + durvalumab or tremelimumab in all dose cohorts. A decrease in OX40+FOXP3 regulatory T cells was observed in a subset of patients with available paired biopsies. Following dose escalation, moderate toxicity was observed in both treatment arms, with no clear efficacy signals demonstrated.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

BackgroundOn the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane...

PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8+ T cells in breast cancer

Myeloid-derived suppressor cells (MDSCs) are an immature innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. Conversely, efficient cytotoxic T cell priming is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to CD8+ T cells, a process that requires a specific subtype of dendritic cells (DCs) called conventional DC1 (cDC1) which are often dysfunctional in cancer. One way to activate cDC1 is ligation of CD40 which is abundantly expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when combined with agonistic CD40, may result in long-lasting protective immunity. In this study, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation, and recruitment to the tumor microenvironment. Our findings demonstrate that PKC agonists decreased MDSC expansion from hematopoietic progenitors and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers via activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmacytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive activity and enhanced MDSC cross-priming capacity both in vitro and in vivo. Finally, combination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth with a significant increase in intratumoral activated CD8+ T cells and tissue-resident memory CD8+ T cells in a syngeneic breast cancer mouse model. In sum, this work proposes a novel promising strategy to simultaneously target MDSCs and promote APC function that may have highly impactful clinical relevance in cancer patients.

Impact of IFN-y and CD40 signalling on Toxoplasma gondii cyst formation in differentiated Neuro-2a neuroblastoma cells.

Signalling by IFN-y and CD40 is known to trigger anti-microbial activity in macrophages infected with Toxoplasma gondii, but their effects on infected neurons are less well known. Here, we compared how stimulation with IFN-y and an agonistic anti-CD40 mAb impacts infection and cyst formation in the mouse neuroblastoma cell line Neuro-2a relative to bone marrow-derived macrophages. Both IFN-y and CD40 mAb decreased cyst emergence in Neuro-2a cells. In macrophages, these stimuli decreased infection, but had no impact on infection in the neuroblastoma cell line. Resistance to killing in Neuro-2a cells may explain why neurons preferentially harbour parasites during chronic infection in the brain.

Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

IntroductionVγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials.Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity.Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study).Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown.Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1.Material and methods5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays.We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14.We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts.DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR.ResultsWe showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells.We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells.DiscussionOur results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function.These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. DisclosuresDe Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure:Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

Increased Expression of CD95 in CD4+ Effector Memory T Cells Promotes Th17 Response in Patients with Myasthenia Gravis.

Emerging data have revealed that CD95 can evoke non-apoptotic signals, thereby promoting pro-inflammatory functions that link to the severity of autoimmune disorders. Here, we reported that the expression of CD95 in CD4+ effector memory T (CD4+ TEM) cells was increased in myasthenia gravis (MG) patients. We also found increased expression of CD95 in CD4+ TEM cells from MG patients correlated positively with clinical severity scores (QMGs), serum IL-17 levels and plasma cells (PCs) frequencies. Conventional treatment, such as glucocorticoid, could down-regulate the expression of CD95 in CD4+ TEM cells, QMGs, serum IL-17 levels and PCs frequencies from MG patients. In vitro, low-dose of agonistic anti-CD95 mAb could promote Th17 cell development. This effect was reversed by CD95 siRNA. Moverover, CD95 stimulation induced the phosphorylation of p38 and Erk1/2 and Th17 cell differentiation, and p38 specific inhibitor SB203580 or Erk1/2 specific inhibitor PD98059 could induce opposite changes. However, SB203580 or PD98059 do not abrogate the increase of CCR6+IL-17A+ cells, ROR-γt and IL-17 expression induced by CD95 triggering relatively to each corresponding control. This suggests that p38 and Erk1/2 MAPK pathway plays a role in expression of CCR6+IL-17A+ cells, ROR-γt and IL-17, but not in their increase induced by CD95 triggering. Taken together, this study revealed that increased expression of CD95 in CD4+ TEM cells promotes Th17 response under the microenvironment of MG.

Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2.

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab’)2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties.

CD40 is a potent activating receptor within the TNFR family expressed on APCs of the immune system, and it regulates many aspects of B and T cell immunity via interaction with CD40 ligand (CD40L; CD154) expressed on the surface of activated T cells. Soluble CD40L and agonistic mAbs directed to CD40 are being explored as adjuvants in therapeutic or vaccination settings. Some anti-CD40 Abs can synergize with soluble monomeric CD40L. We show that direct fusion of CD40L to certain agonistic anti-CD40 Abs confers superagonist properties, reducing the dose required for efficacy, notably greatly increasing total cytokine secretion by human dendritic cells. The tetravalent configuration of anti-CD40-CD40L Abs promotes CD40 cell surface clustering and internalization and is the likely mechanism of increased receptor activation. CD40L fused to either the L or H chain C termini, with or without flexible linkers, were all superagonists with greater potency than CD40L trimer. The increased anti-CD40-CD40L Ab potency was independent of higher order aggregation. Moreover, the anti-CD40-CD40L Ab showed higher potency in vivo in human CD40 transgenic mice compared with the parental anti-CD40 Ab. To broaden the concept of fusing agonistic Ab to natural ligand, we fused OX40L to an agonistic OX40 Ab, and this resulted in dramatically increased efficacy for proliferation and cytokine production of activated human CD4+ T cells as well as releasing the Ab from dependency on cross-linking. This work shows that directly fusing antireceptor Abs to ligand is a useful strategy to dramatically increase agonist potency.

TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pretransplant T cell phenotypes of patients who reject versus those who remain stable on belatacept identified three potential "risky" memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4+ CD28+ TEM , CD8+ CD28null , and CD4+ CD57+ PD1- subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Coculture of human memory CD4+ and CD8+ T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3+ Treg, suggesting that agonism of the TIGIT pathway increases FOXP3+ Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.

Remodeling the Tumor Myeloid Landscape to Enhance Antitumor Antibody Immunotherapies.

Simple SummaryIn addition to cancer cells themselves, tumors consist of several cell types that either function to suppress or promote tumor growth and disease progression. Macrophages are amongst the most abundant of these cell types and possess contrasting abilities to either enhance tumor growth and spread or to destroy cancer cells. Anti-cancer therapeutics such as Rituximab, Herceptin and Cetuximab, are cornerstones of current treatment for cancer patients. These therapeutics are antibodies that directly bind to cancer cells and aid macrophages in detecting and destroying these cells, through a process known as antibody dependent cellular phagocytosis (ADCP). Although the use of anti-cancer antibodies to treat large established tumors has improved survival rates, significant numbers of patients do not respond to this type of therapy. Thus, recently there has been an intense focus on designing new therapies which alter macrophages inside tumors, so that they can more effectively eliminate cancer cells through ADCP. Several molecules on the surface of macrophages can be targeted with antibodies to improve their ability to recognize and eliminate cancer cells. Here we review the most promising of these new targets, which offer the potential to circumvent resistance to therapy in cancer patients. Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called ‘phagocytosis checkpoints’. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of ‘don’t eat me signals’ or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance.

A Novel CD16xCD33 Bispecific Killer Cell Engager (BiKE) Mediates a Double Hit For Natural Killer (NK) Cells To Target CD33+ Myelodysplastic Syndrome (MDS) Cells and Myeloid Derived Suppressor Cells (MDSCs) At All Disease Stages

MDS is a clonal heterogeneous stem cell disorder identified by pancytopenia that frequently progresses to acute myeloid leukemia (AML). The only curative treatment is hematopoietic cell transplantation (HCT), but many patients are ineligible due to advanced age and comorbidities. New therapeutic strategies are urgently needed to reduce disease burden and improve overall survival. Studies of NK cell function from MDS patients have shown diminished natural cytotoxicity against K562 targets. These NK cell defects have been ascribed to abnormalities in expression and function of the NK cell activating receptors NKG2D, NKp30, NKp46 and DNAM-1. However, the properties of the potent activating receptor CD16 (FcgRIII) and its potential for mediating targeted immunotherapy remain unexplored in MDS. Frozen pre-transplant samples from 67 MDS patients (age 19-74 years, Median=47) receiving unrelated HCT were obtained from the research sample repository of the National Marrow Donor Program and were compared to normal volunteers. NK cell degranulation, cytotoxicity and cytokine production were evaluated using published methods. We focused on CD16-induced NK cell function using a bispecific killer cell engager generated to target CD16 along with the myeloid differentiation antigen CD33 (CD16xCD33 BiKE). CD16 expression on total CD56+ MDS-NK cells was significantly lower compared to normal donors (MDS vs. normal: 63 ± 3% (n=67) vs. 89 ± 2% (n=20); p < 0.0001). However, reverse-antibody dependent cell-mediated cytotoxicity (R-ADCC) assays revealed no significant differences in CD107a (46 ± 5% (n=16) vs. 46 ± 3% (n=12)), IFN-g (6 ± 1% vs. 10 ± 2%) or TNF-a (9 ± 2% vs. 6% ± 1%) production when resting MDS-NK cells were triggered with an agonistic CD16 mAb, indicating MDS-NK cell function triggered through CD16 is intact. To evaluate the therapeutic potential of targeting CD16 in MDS patients, resting MDS-mononuclear cells were coated with 10mg/mL of the CD16xCD33 BiKE and co-cultured for 4 hours with the CD33+ target cell line HL-60. MDS-NK cell CD107a (CD16xCD33 BiKE vs. no reagent: 42 ± 2% (n=62) vs. 22 ± 1% (n=20); p < 0.0001), IFN-g (15 ± 1% vs. 3 ± 1%; p < 0.0001) and TNF-a (13 ± 1% vs. 3 ± 1%; p < 0.0001) production was significantly increased compared to the no reagent and scFv CD16 controls. Moreover, in the absence of HL-60 targets, MDS-NK cells treated with the CD16xCD33 BiKE targeted endogenous CD33+ targets, significantly enhancing degranulation (37 ± 2% vs. 17 ± 1%; p < 0.0001), IFN-g (4 ± 1% vs. 2% ± 1%; p < 0.01) and TNF-a (5 ± 1% vs. 2 ± 1%; p < 0.01) production. Recent data have shown an increase in the frequency of MDSCs, phenotypically defined as CD33+/CD11b+/CD14-/HLA-DRlo/-, during oncogenesis and in the presence of inflammatory cytokines. Indeed, in our MDS patient cohort, the frequency of MDSCs of total viable mononuclear cells was significantly higher compared to normal donors (6 ± 1% (n=64) vs. 0.1 ± 0.02% (n=18), respectively; p < 0.0001) (figure). Furthermore, CD16xCD33 BiKE-treated resting NK cells from normal donors co-cultured with cytokine-induced MDSCs generated from normal PBMCs in a 51Cr-release cytotoxicity assay successfully targeted and lysed MDSCs (CD16xCD33 BiKE vs. no reagent (E:T = 20:1): 63 ± 5% vs. 7 ± 2% [p < 0.0001, n=4]; (E:T = 6.6:1): 35 ± 3% vs. 4 ± 1% [p < 0.0001]). CD107a (48 ± 2% (n=6) vs. 4 ± 1% (n=3); p < 0.0001), IFN-g (8 ± 1% vs. 1 ± 1%; p < 0.0001) and TNF-a (8 ± 1% vs. 1 ± 1%; p < 0.0001) production was also significantly increased against MDSC targets in the presence of the CD16xCD33 BiKE, demonstrating its ability to facilitate killing of both the CD33+ MDS clone and an immune suppressor cell population. Lastly, analysis of MDS patient karyotypes in correlation with CD16xCD33 BiKE-induced MDS-NK cell CD107a, IFN-g and TNF-a production did not reveal any significant differences between favorable, intermediate or poor international prognostic scoring system (IPSS) cytogenetic scores, further demonstrating the therapeutic potential of the CD16xCD33 BiKE to induce optimal MDS-NK cell function against CD33+ targets despite disease heterogeneity. In summary, our data suggest that the CD16xCD33 BiKE may function not only against CD33+ MDS targets themselves, but also against MDSCs. This double hit approach may protect against MDS progression/transformation to AML through immune targeting and may improve blood counts by targeting MDS-MDSCs that contribute to ineffective hematopoiesis. [Display omitted] Disclosures:No relesvant conflicts of interest to declare.

Abstract CT005: T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial

Abstract Deploying CD40 activation to stimulate T cell responses upstream of immune checkpoint molecules is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological treatment effects especially treatment-related modulation of the tumor microenvironment (TME), remain poorly understood. Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy (gemcitabine and nab-paclitaxel) to 16 resectable patients with pancreatic ductal adenocarcinoma (PDAC) prior to surgery followed by adjuvant chemotherapy and CD40 mAb. The toxicity profile was acceptable, including only grade 1 or 2 cytokine release syndrome and expected toxicities from chemotherapy. Disease-free survival was 13.8 months (95% CI 2.9 - 24.8 months) and median overall survival was 23.4 months (95% CI 18.0 - 28.8), with 8 patients alive at a median of 20.0 months after surgery (follow-up range 12.2 to 34.8 months). Neoadjuvant selicrelumab induced major pharmacodynamic differences in the TME, as revealed by a multiplex imaging platform auditing the immune ecosystem, compared to resection specimens from PDAC patient previously untreated or given neoadjuvant chemotherapy/chemoradiotherapy only. For tumors resected after selicrelumab, 82% (9/11) were T-cell enriched, compared to 37% (38/104) (p=0.004) of untreated tumors and 23% (93/13) of chemotherapy/chemoradiation-treated tumors (p=0.012). Moreover, for selicrelumab tumors, tumor-associated fibrosis was less, “M2” macrophages were fewer, dendritic cells were more mature, and T cells were activated and proliferative, compared to the non-selicrelumab groups. In the periphery, CD8+ and CD4+ T cells were more activated and proliferative, and serum inflammatory cytokines CXCL10 and CCL22 increased after treatment. This study provides proof-of-concept in patients that agonistic CD40 mAb alters the TME, enhances T-cell infiltration, and modulates systemic inflammatory responses. These findings inform design of next-generation CD40 clinical trials. Citation Format: Katelyn T. Byrne, Courtney B. Betts, Rosemarie Mick, Shamilene Sivagnanam, David L. Bajor, Daniel A. Laheru, E. Gabriela Chiorean, Mark H. O'Hara, Shannon M. Liudahl, Craig Newcomb, Cécile Alanio, Ana P. Ferreira, Byung S. Park, Takuya Ohtani, Austin P. Huffman, Sara A. Väyrynen, Andressa Dias Costa, Judith C. Kaiser, Andreanne M. Lacroix, Colleen Redlinger, Martin Stern, Jonathan A. Nowak, E. John Wherry, Martin A. Cheever, Brian M. Wolpin, Emma E. Furth, Elizabeth M. Jaffee, Lisa M. Coussens, Robert H. Vonderheide. T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT005.

Abstract 1766: Triggering the resolution of inflammation with agonistic anti-ChemR23 antibody dampens inflammation-driven carcinogenesis

Abstract Chronic inflammation is associated with abnormal non-phlogistic clearance (efferocytosis) of apoptotic cells by macrophages and a defect of the resolution of inflammation pathways. The resolution of inflammation is an active immunological process mediated by specialized pro-resolving mediator (SPM) which target specific resolutive G-protein coupled receptors expressed by different immune cells and participate to the tissue homeostasis return after an injury. Defects in the clearance of (chemotherapy-induced) apoptotic tumor cell debris strengthens inflammation and has been associated with exacerbated tumor growth in several preclinical models. Proresolutive therapeutic approaches, such as using exogenous resolvin E1 (RvE1), the natural lipidic proresolutive ligand of the GPCR ChemR23, have been shown to dampen tumor-associated inflammation and to reduce tumor growth. Using single-cell RNA sequencing analysis, we found that ChemR23 is mainly expressed by tumor-associated macrophages in melanoma and lung cancers. Moreover, ChemR23 expression was barely expressed in non-inflamed tissues indicating a preferential expression on the inflamed or tumor site. We screened and identified an anti-ChemR23 mAb which induces RvE1-like Akt and ERK signaling in mouse and human macrophages and favors macrophage polarization towards a pro-resolutive phenotype. In vitro, the agonist antibody significantly increased the phagocytosis of apoptotic tumor primary mesothelioma cells by human tumor-polarized macrophages. In vivo, the agonist ChemR23 mAb accelerates the resolution of inflammation in an acute inflammatory model, as illustrated by a significant decrease of inflammatory macrophages and neutrophils infiltrates as compared to isotype control groups both in mice and non-human primate models. Similarly, ChemR23 triggering with the anti-ChemR23 mAb improved weight recovery, reduced diarrhea and decreased the development of colon neoplastic foci in a chronic colitis model coupled with the injection of a carcinogen agent. Furthermore, the administration of the anti-ChemR23 mAb as a monotherapy displayed some partial and complete antitumor responses in immunocompetent mouse models of subcutaneous colon carcinoma (MC38 and CT26) as well as orthotopic mesothelioma (AK7). Finally, using an orthotopic triple-negative breast cancer model (4T1), while we observed a limited impact on primary tumor growth, spontaneous metastasis development in the lung was significantly inhibited by the ChemR23 agonist mAb monotherapy. Our study reveals for the first time the therapeutic potential of triggering the proresolutive pathways using an anti-ChemR23 agonistic mAb to limit chronic inflammation in the tumor microenvironment and inhibit metastasis development. Citation Format: Vanessa Gauttier, Margot Lavy, Charlène Trilleaud, Kévin Biteau, Isabelle Girault, Lyssia Belarif, Géraldine Teppaz, Caroline Mary, Virginie Thépénier, Christophe Blanquart, Sophie Barillé-Nion, Nicolas Poirier. Triggering the resolution of inflammation with agonistic anti-ChemR23 antibody dampens inflammation-driven carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1766.

Abstract 2816: Immuno-PET monitoring of CD8+ T cell infiltration post anti-ICOS agonist antibody treatment alone and in combination with PD-1 blocking antibody using a 89Zr anti-CD8+ mouse minibody in EMT 6 syngeneic tumor mouse

Abstract Introduction Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor that is important for promoting immune activation and function. Despite reported clinical activity and a wide range of non-clinical studies supporting a role for ICOS in lymphocyte activation, proliferation and pro-inflammatory cytokine secretion, little is known regarding the potential of monoclonal agonist antibody-mediated ICOS signaling to drive cytotoxic T cell infiltration into tumors. Feladilimab (GSK3359609) is a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials. Here, we used PET/CT imaging to evaluate CD8+ T cell infiltration following treatment with a rodent surrogate of feladilimab (7E.17G9 mouse [m] IgG1) alone or in combination with anti-PD-1 mAb (RMP-14 rat IgG2a) in a syngeneic model of breast cancer (EMT6). Method Female BALB/c mice with established tumors (~150 mm3) received 10µg, IP of either IgG control mAbs, ICOS mAb, or ICOS + PD-1 mAbs on day 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 &amp; 48 hrs post IV dose of 89Zr labeled CD8 minibody (IAB42M1-14, ImaginAb, CA) on day 0, 3, 5, 9, or 14. In addition to the CD8 minibody uptake in tumor &amp; tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images. Top ranked features were used for hierarchical clustering to identify treatment effects. Results Tumor size regressed in all treated groups relative to IgG control, with a number of mice clearing tumors (ICOS: 4 mice, ICOS + PD-1: 9 mice). The in vivo uptake of CD8 minibody in TDLN was significantly higher in the ICOS + PD-1 group on day 4, 6, &amp; 7 relative to IgG control P&amp;lt;0.05. The CD8 minibody uptake in tumor was significantly higher in the ICOS group on day 6, 11, &amp; 16, and in the ICOS + PD-1 group on day 11 compared to IgG control P&amp;lt;0.05. Top ranked CT radiomic features were predictive for treatment effects at earlier days (day 3 - 5), while PET features were predictive at later days (6 - 10). Texture features in TDLN were consistently selected at earlier days and shape features in tumor were consistently selected in later days. Conclusions Herein, we demonstrated for the first time that treatment of tumor-bearing mice with ICOS agonist mAb alone or in combination with PD-1 blockade can increase CD8+ T cell infiltration into tumors &amp; TDLN, and is correlated with reduced tumor burden. Notably, radiomics features predicted an effect of treatment on CD8+ T cell infiltration earlier than the detection of absolute changes in the CD8 minibody uptake in tumor &amp; TDLN. Overall, these data support the ongoing pivotal investigation of feladilimab. Moreover, this translational imaging method may be a useful tool to non-invasively monitor CD8+ T cell in response to immunotherapies and understand the temporal relationship between CD8+ T cell flux in tumor and in TDLN. Citation Format: Hasan Alsaid, Shih-Hsun Cheng, Meixia Bi, Mary V. Rambo, Tinamarie Skedzielewski, Bao Hoang, Sunish Mohanan, Andrew Gehman, Chih-Yang Hsu, Minh Doan, Fang Xie, M. Reid Groseclose, Christopher Hopson, Sara Brett, Ian A. Wilson, Andrew Nicholls, Marc Ballas, Jeremy D. Waight, Beat M. Jucker, Axel Hoos. Immuno-PET monitoring of CD8+ T cell infiltration post anti-ICOS agonist antibody treatment alone and in combination with PD-1 blocking antibody using a 89Zr anti-CD8+ mouse minibody in EMT 6 syngeneic tumor mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2816.

TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity

Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.