CD40 Activation Research Articles

Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

IntroductionSuccess rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction.ResultsDynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms.Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ).Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C).ConclusionThese results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. [Display omitted] DisclosuresKater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

Ibrutinib Treatment in CLL Interrupts CD40 Signaling Capacity and Sensitizes CLL Cells to Venetoclax

Background: For proliferation and survival, chronic lymphocytic leukemia (CLL) cells depend on interactions with cells and soluble factors present in the tumor microenvironment (TME). These interactions also increase expression of B-cell leukemia/lymphoma-2 (Bcl-2) proteins, including Bcl-XL, Mcl-1 and Bfl-1, thereby reducing drug sensitivity.In the VISION HOVON141 clinical trial, patients with relapsed or refractory CLL are treated with 2 cycles of Bruton tyrosine kinase inhibitor ibrutinib (IBR) lead-in followed by 13 cycles of IBR + Bcl-2 inhibitor venetoclax (VEN) combination before randomization. The combination of IBR + VEN may have synergistic anti-tumor effects, since IBR forces CLL cells from lymph node (LN) to the blood (PB) where they become fully dependent on Bcl-2, and succumb to VEN.To support a mechanistic basis for this premise, we investigated changes in expression of Bcl-2 proteins, effects of TME-mimicking signals, and venetoclax sensitivity before/after 2 months IBR treatment.Results: At baseline, lymph node emigrant cells (CXCR4dim/CD5high) have higher Bfl-1 expression, in addition to earlier reported Bcl-XL and Mcl-1 expression than cells immigrating back to the lymph node (CXCR4high/CD5dim)(Haselager et al., 2020). After 2 months of IBR treatment a clear reduction in all four pro-survival proteins was observed (N=17 p<0.001). Despite these changes, VEN sensitivity was not different in unstimulated PB CLL cells obtained at baseline versus at 2 months of IBR treatment (N=8; IC50=0.001µM). In contrast, CD40 stimulated CLL cells obtained at baseline are fully resistant to VEN (IC50>10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value <0.0001; Figure 1A). This suggested reduced CD40 signalling capacity and was accompanied by reduced ability to upregulate Bcl-XL, Mcl-1, and Bfl-1 expression. Indeed, cell surface expression, as well as level of CD40 activation as measured by CD95 induction, was clearly reduced after 2 months IBR. Importantly, these effects occurred in vivo, as IBR did not directly affect CD40 signaling in vitro.These data imply that under IBR treatment, when cells cannot (re-)enter LN sites, a factor is lacking that maintains or induces CD40 expression. In search of stimuli that can augment CD40 signaling capacity, it was found that BCR stimulation had no effect on CD40 expression. In contrast, TLR9 stimulation via CpG led to increased CD40 expression in CLL cells (N=7, p-value <0.0001)(Figure 1B). These findings align well with recent data which indicate a role for TLR9 signalling in vivo by unmethylated mitochondrial DNA in CLL (Kennedy et al., 2021). Taken together, these data provide a mechanistic explanation, by which a triad of signaling pathways not only involving BCR but also CD40 and TLR9 is interrupted by IBR (Figure 1C).Discussion/conclusion: IBR treatment broadly affects Bcl-2 family protein expression and CD40 signaling in vivo. The combined data indicate a novel aspect of IBR efficacy, namely its capacity to interrupt TLR9-induced CD40 upregulation, which normally primes CLL cells in the LN environment for drug resistance. This also suggests that drugs that inhibit TLR9 signaling may synergize with IBR.ReferencesHaselager, M. V., et al (2020). Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood, 136(25), 2918Kennedy, E., et al (2021). TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target. Blood, 137(22), 3064 [Display omitted] DisclosuresLevin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Westerweel: Novartis: Research Funding; Incyte: Consultancy; BMS / Celgene: Consultancy; Pfizer: Consultancy. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Novo Nordisk Foundation: Research Funding; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding.

Role of miR-145-5p/ CD40 in the inflammation and apoptosis of HUVECs induced by PM2.5

Fine particulate matter (PM2.5) exposure can cause the injury of vascular endothelial cells by inflammatory response. CD40 works in inflammation of endothelial cells and it may be regulated by the miRNAs. This study aimed to clarify the role and mechanism of CD40 and miR-145-5p in PM2.5-induced injury of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with different concentrations of PM2.5 exposure (0, 100, 200, 400 μg/mL) for 24 h. The si-RNA was used for CD40 gene silencing (0, 200 μg/mL PM2.5, siRNA-CD40 and siRNA-CD40 + 200 μg/mL PM2.5). Mimics was used for overexpression of miR-145-5p (0, 200 μg/mL PM2.5, mimics and mimics+200 μg/mL PM2.5). The cell viability of HUVECs was detected with Cell Counting Kit8 (CCK8) kit. The level of cell apoptosis was detected by flow cytometry. The inflammation-related factor including interleukin-1β (IL-1β), interleukin-18 (IL-18), tumor necrosis factor α (TNF-α) and C1q complement/tumor necrosis factor (TNF)-associated proteins9 (CTRP9) were tested with enzyme-linked immunosorbent assay (ELISA) kits. The mRNA and protein expression levels of CD40, CD40L, caspase1, NLRP3 (Nod-like receptor family pyrin domain-containing 3) and IKKB were detected with quantitative real-time PCR (qRT-PCR), Western blot and Immunofluorescence. Compared with the control group, the cell viability of HUVECs exposed to PM2.5 was significantly decreased (p < 0.05); the levels of IL-Iβ and TNF-α were significantly increased, while the level of CTRP9 was significantly decreased (p < 0.05). The proportion of apoptotic cells was increased after being treated with PM2.5 (p < 0.05). Besides, the mRNA and protein levels of CD40, CD40L, IKKB, NLRP3 and caspase1 were increased comparing with the control group (p < 0.05). After CD40 silencing, the condition of inflammation and apoptosis in HUVECs exposed to PM2.5 was alleviated, and the expression levels of CD40L, IKKB, NLRP3 and caspase1 were significantly decreased (p < 0.05). Furthermore, miR-145-5p was significantly down-regulated after exposure to 200μg/mL PM2.5 (p < 0.05). After over-expression of miR-145-5p, the expression level of CD40 was decreased (p < 0.05). Taken together, PM2.5 can cause inflammation and apoptosis of HUVECs via the activation of CD40, which can be regulated by miR-145-5p. Over-expression of miR-145-5p can down-regulate CD40, further inhibiting the inflammation and apoptosis of HUVECs induced by PM2.5.

Resveratrol, EGCG and Vitamins Modulate Activated T Lymphocytes.

Vitamins and bioactives, which are constituents of the food chain, modulate T lymphocyte proliferation and differentiation, antibody production, and prevent inflammation and autoimmunity. We investigated the effects of vitamins (vitamin A (VA), D (VD), E (VE)) and bioactives (i.e., resveratrol (Res), epigallocatechin-3-gallate (EGCG)) on the adaptive immune response, as well as their synergistic or antagonistic interactions. Freshly isolated T lymphocytes from healthy individuals were activated with anti-CD3/CD28 antibodies for 4–5 days in the presence of bioactives and were analyzed by cytofluorometry. Interleukins, cytokines, and chemokines were measured by multiple ELISA. Gene expression was measured by quantitative RT-PCR. Res and EGCG increased CD4 surface intensity. EGCG led to an increased proportion of CD8+ lymphocytes. Anti-CD3/CD28 activation induced exuberant secretion of interleukins and cytokines by T lymphocyte subsets. VD strongly enhanced Th2 cytokines (e.g., IL-5, IL-13), whereas Res and EGCG favored secretion of Th1 cytokines (e.g., IL-2, INF-γ). Res and VD mutually influenced cytokine production, but VD dominated the cytokine secretion pattern. The substances changed gene expression of interleukins and cytokines in a similar way as they did secretion. Collectively, VD strongly modulated cytokine and interleukin production and favored Th2 functions. Resveratrol and EGCG promoted the Th1 response. VA and VE had only a marginal effect, but they altered both Th1 and Th2 response. In vivo, bioactives might therefore interact with vitamins and support the outcome and extent of the adaptive immune response.

Abstract MP223: CCR2 + Macrophages And T-cells Infiltrate The Murine Heart Following Immune Regulator Treatment With CD40

Immune checkpoint inhibitors have revolutionized cancer treatment but have been associated with severe adverse cardiac events including myocarditis. CD40 agonist (CD40ag) antibodies are immune regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse cardiac events, C57BL/6J, CCR2-GFP, and CCR2-knockout mice were injected with CD40ag or isotype antibodies for 7 days. Flow cytometry showed a specific increase in CCR2 + macrophages that was independent of monocyte recruitment. There was no change in neutrophil recruitment and only a modest increase in dendritic cells was evident. We also observed a significant expansion of CD4 and CD8 T-cells that displayed an effector memory phenotype. Bulk cardiac tissue gene expression analysis (Figure A) revealed that CD40 activation upregulates multiple inflammatory cytokines (Ifng, Tnf, Il-12β) and chemokines (Ccl3, Ccl5, Ccl7, Cxcl9, Cxcl10), which are regulated by interferon gamma and coordinate the activation of APCs and T-cells. Finally, RNA in situ hybridization (Figure B-C) demonstrated increased levels of Cxcl9 (red) expression in cells consistent with CCR2 + (white) macrophages when comparing isotype control to treatment with CD40ag. These findings reveal that CD40 activation results in a robust expansion of CCR2 + macrophages and activation of T-cells within the heart, initiating a feed forward loop of activation that is mediated by interferon gamma and generates inflammatory cytotoxic mediators that may lead to myocardial injury.

A Novel Nanomedicine Ameliorates Acute Inflammatory Bowel Disease by Regulating Macrophages and T-Cells.

Ramulus mori polysaccharide (RMP), one of the most important active components of R. mori, has been attracting increasing interest because of its potent bioactive properties, including anti-inflammatory, antitumor, and antidiabetic effects. Despite the great therapeutic potential of RMP, its inherent properties of low bioavailability and brief biological half-life have limited its applications to the clinic. Thus, RMP was packaged by poly(lactic-co-glycolic acid) (PLGA) nanoparticles to develop a novel anti-inflammatory nanomedicine (PLGA-RMP) in this study. The nanoparticles were synthesized via a double-emulsion solvent evaporation technique, and the average diameter of PLGA-RMP was about 202 nm. PLGA-RMP nanoparticles reduced the expression of inflammatory cytokines while promoting the production of IL-10, and boosted the phenotypic shift in macrophages in vitro. Furthermore, lipopolysaccharide (LPS)-induced inflammatory bowel disease (IBD) in mouse was used to examine the anti-inflammatory effect of PLGA-RMP in vivo. Oral administration of PLGA-RMP in LPS-induced IBD mice substantially mitigated the intestinal inflammation compared to treatment with LPS alone, as evidenced by attenuation of disease activity index scores and inflammatory damage in the intestine. Meanwhile, PLGA-RMP suppressed the expression and secretion of specific inflammatory cytokines including TNF-α, IL-6, IL-1β, and PGE2 in the inflamed intestine while inhibiting the activation of CD3+CD8+ T-cells and increasing the number of activated Tregs in the intestine. These results indicated that PLGA-RMP deserves further consideration as a potential therapeutic nanomedicine to treat various inflammatory diseases, including IBD.

CD40 Agonist Monoclonal Antibody-Mediated Hepatitis in TNF-Receptor 1 Gene Knockout Mice.

Tumor necrosis factor-alpha (TNF-α) plays an important role in liver inflammation. CD40-CD40 ligand (CD40-CD40L) is a key receptor–ligand signaling pair involved in the adaptive immune response and pathogenesis of autoimmune diseases. In mice, CD40 activation leads to sickness behavior syndrome (SBS) comprising weight loss, sleep disruption and depression, which can be blocked by administration of the TNF-inhibitor etanercept. In the present study, we assessed the extent of hepatic inflammation in mice devoid of the TNF-receptor 1 (TNFR1)-mediated signaling pathway. The TNFR1-depleted (TNFR1−/−) adult mice and their wild type littermates were given a single intra-peritoneal injection of CD40 agonist monoclonal antibody (mAb) or rat IgG2a isotope control. As described previously, TNFR1−/− mice were protected from SBS upon CD40 mAb treatment. Cd40, tnf and tnfr1 mRNA and Tnf-α peptide were increased in the liver of CD40 mAb-stimulated wild type mice. Serum alanine aminotransferase was elevated in both CD40-activated wild type and TNFR1−/− mice. TNFR1−/− mice showed much less intra-parenchymal infiltrates, hepatocellular necrosis, and perivascular clusters upon CD40 mAb activation than their wild type littermates. A gene expression microarray detected increased activity of metabolic and detoxification pathways and decreased activity of inflammatory pathways. We conclude that immune activation and development of liver inflammation in CD40L interactions depend on TNFR1-mediated signaling pathways and are counteracted by alterations in metabolic pathways.

Abstract 1846: DuoBody-CD40×4-1BB (GEN1042) induces dendritic-cell maturation and enhances T-cell activation and effector functions in vitro by conditional CD40 and 4-1BB agonist activity

Abstract The clinical success of immune checkpoint inhibitor monoclonal antibodies (mAbs) has demonstrated potential for re-activation of anti-tumor immunity in cancer treatment. DuoBody®-CD40×4-1BB (GEN1042) is a first-in-class bispecific agonistic antibody that combines targeting of costimulatory molecules CD40 and 4-1BB, resulting in priming and (re-)activation of tumor-specific immunity. We previously showed that DuoBody-CD40×4-1BB induces CD40 and 4-1BB agonist activity that is conditional, i.e., dependent on crosslinking of CD40- and 4-1BB-expressing cells. Here we further elucidate the preclinical mechanism of action of DuoBody-CD40×4-1BB in a broad range of functional immune cell assays. Fixed cell microscopy and live cell imaging of the immune synapse was performed using co-cultures of activated T cells and monocyte-derived dendritic cells (DC). DuoBody-CD40×4-1BB co-localized with LFA-1 at the interface between DC and activated T cells. By binding to both targets, DuoBody-CD40×4-1BB enhanced DC/T cell interactions and increased the contact duration between these cells compared to the combination of monovalent CD40- and 4-1BB-specific antibodies. In mixed lymphocyte reactions (MLR) comprised of naïve CD8+ T cells co-cultured with immature (iDC) or LPS-matured monocyte-derived DCs (mDC), DuoBody-CD40×4-1BB induced DC maturation and increased T cell functionality, shown by a dose-dependent increase in HLA-DR/CD86 expression and production of IFNγ, CXCL10 and granzyme B. DC maturation induced by DuoBody-CD40×4-1BB was also observed in monocultures of iDCs expressing both targets, and was associated with a concurrent dose-dependent increase in TNFα production. Furthermore, in antigen-specific T-cell assays (CLDN6-TCR+CD8+ T cells co-cultured with CLDN6+CD40+ DCs), DuoBody-CD40×4-1BB conditionally enhanced T-cell proliferation and production of IFNγ and TNFα, and induced upregulation of the degranulation marker CD107a and granzyme B. Finally, DuoBody-CD40×4-1BB enhanced clonal expansion of human TILs derived from non-small cell lung and colon cancer patients ex vivo in a dose-dependent manner. In conclusion, by enhancing the formation of the immune synapse between CD40+ DC and 4-1BB+ T cells, DuoBody-CD40×4-1BB induced conditional activation of both cell types, resulting in DC maturation, T-cell activation and effector functions in vitro. Thereby, DuoBody-CD40×4-1BB provides a unique combination of priming and enhancing existing anti-tumor immunity. DuoBody-CD40×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04083599). Citation Format: Alexander Muik, Rachelle Kosoff, Friederike Gieseke, Isil Altintas, Kristina Schödel, Saskia Burm, Eliana Stanganello, Fulvia Vascotto, Aras Toker, Homer Adams, III, Esther Breij, Mustafa Diken, Tahamtan Ahmadi, Kate Sasser, Özlem Türeci, Mark Fereshteh, Ugur Sahin, Maria Jure-Kunkel. DuoBody-CD40×4-1BB (GEN1042) induces dendritic-cell maturation and enhances T-cell activation and effector functions in vitro by conditional CD40 and 4-1BB agonist activity [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 1846.

Abstract 1593: Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy

Abstract Mitazalimab (ADC-1013, JNJ-64457107) is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on antigen-presenting cells (APC), leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. There are several CD40 agonists in clinical development with different characteristics, affecting both efficacy and tolerability. Mitazalimab is an FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and safety. Immunologically “cold” tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, infiltration of effector CD8+ T cells is blocked by the desmoplastic tumor stroma, which surrounds the tumor and hosts immune-suppressive macrophages that dampen the immune response in the tumor microenvironment (TME). By activating and re-directing the immunosuppressive M2 macrophages into tumoricidal M1 macrophages in the TME, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective immune reaction by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy and induced a long-term survival. Further, it was also demonstrated that mitazalimab induced a concentration-dependent activation of tumor-associated macrophages (TAMs), purified from human tumor samples, into a more tumoricidal M1 like phenotype, by upregulation of cell surface markers such as CD83.In conclusion, mitazalimab re-directs human macrophages into a M1 like tumoricidal, less immunosuppressive phenotype, and synergizes effectively with chemotherapy, leading to induction of a long-term survival in a preclinical tumor mouse model. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099) where mitazalimab was well tolerated up to 1200 μg/kg with manageable side effects, support further clinical development of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Adnan Deronic, Mia Thagesson, Anneli Nilsson, Peter Ellmark, Anette Fält, Charlotte Fält, Karin Enell Smith. Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy [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 1593.

Abstract 1852: A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling

Abstract Immunotherapies that target PD1/PDL1 pathway have marked a new era of cancer therapy. Combinatorial treatment of inhibiting PD1/PDL1 signaling and other immunomodulating agents has been widely tested and validated as a better strategy compared to single agent treatment. CD40 (TNFRSF5) is a member of the TNFR super family, a key T cell co-stimulatory signal critical for B-cell maturation and immunoglobulin class switching. Biocytogen developed a novel first in class PD1-CD40 bispecific antibody (YH008) that agonizes CD40 conditionally upon PD1 binding. In vitro study data shows YH008 can block the interaction of PD1 to PDL1 and induced dose dependent NFAT activation. YH008 was able to activate human CD40 on reporter cells in the presence of PD1-expressing jurkat cells in a dose dependent manner without relying on FcγR expressing cells. This feature is beneficial for conditionally activation of CD40+ cells in tumor microenvironment and tumor-draining lymph nodes, where tumor specific PD1+ T cells are enriched. In vivo study result shows that YH008 demonstrated robust anti-tumor efficacy against MC38 and B16F10 tumors in PD1/CD40 double humanized mice (B-hPD1/hCD40). In hCD40 mice which has no human PD1 engagement, YH008 did not show anti-tumor activity against MC38 tumor. Furthermore, YH008 did not elevate liver enzymes of MC38 bearingB-hPD1/hCD40 mice at dose as high as 26 mg/kg, demonstrating improved liver safety profile than selicrelumab. Taken together, these data demonstrated that YH008 is a promising new immunotherapeutic candidate in cancer treatment. Citation Format: Baihong Liu, Li Hui, Chonghui Liu, Zhihong Li, Fangxia Pan, Benny Yang, Qingcong Lin. A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling [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 1852.

Abstract 1733: MP0317, a CD40xFAP targeting multi-specific DARPin® therapeutic, drives immune activation and reverts myeloid-mediated T-cell suppression in vitro and ex vivo

Abstract Therapeutic agonists targeting CD40 have shown encouraging signs of anti-tumor efficacy in patients. Despite their initial promise however, optimal dosing of systemically active agents has been limited by toxicity and their full therapeutic potential not achieved. We have previously demonstrated in vitro the ability of MP0317, a novel multi-specific DARPin® therapeutic, to selectively activate CD40 bearing B-cells, dendritic cells and macrophages in the presence of fibroblast activation protein (FAP). FAP is highly expressed in the stroma of many solid tumors and is found only at lower levels in other tissues. MP0317 utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the presence of FAP and thus avoid systemic CD40 activation classically associated with toxicity. Here we present new data which confirm the unique therapeutic potential of MP0317 in ex vivo model systems, demonstrate modulation of macrophage phenotype and reveal a release of T-cells from macrophage-mediated suppression. Ex vivo functional assays performed with MP0317 on dissociated human tumors demonstrated FAP-dependent activation of CD40-expressing B-cell and myeloid cell populations. Immunohistochemical analysis of tissue micro-arrays demonstrated FAP levels consistent with those required for immune cell activation in a broad range of solid tumor indications. Furthermore, by mining publicly available data, we classified tumor indications based on their immune cell content, pathway activation and MP0317 ex vivo mode of action (T cell/macrophage ratio, FAP and CD40 expression) and proposed patient populations and indications for the upcoming clinical trials. To further investigate the functional impact of MP0317, macrophages were differentiated in vitro using canonical polarising cytokines. Suppressive tumor-associated macrophage (TAM)-like cells, were repolarized by MP0317 to an anti-tumor-like phenotype. Furthermore, we explored whether modulation of macrophage phenotype by MP0317 could relieve their suppressive effect on T-cells. TAM-like macrophages potently supressed anti-CD3/28 driven T-cell activation and indeed the addition of MP0317 was found to restore T-cell response. Together these data further support MP0317's FAP targeted multimodal mechanism of action in human tumors, provide evidence for an improved therapeutic window over existing CD40 agonists and support progression to the clinic with an informed target patient profile. Citation Format: Kyriaki Ioannou, Simone Ragusa, Joanna Roquette, Ana Florescu, Mariam Gachechiladze, Eliane Müller, Julia M. Martinez-Gomez, Sophie Barsin, Sarah Jetzer, Nicolo Rigamonti, Clara Domke, Tamara Lekishvili, Karolin Rommel, Ivana Tosevski, Anne Goubier, Philippe Legenne, Vladimir Kirkin, Mitch Levesque, Hong Ji, Rupert Kenefeck. MP0317, a CD40xFAP targeting multi-specific DARPin® therapeutic, drives immune activation and reverts myeloid-mediated T-cell suppression in vitro and ex vivo [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 1733.

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.

Melanoma reactive TCR-modified T cells generated without activation retain a less differentiated phenotype and mediate a superior in vivo response

Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia.

Simple SummaryPrevious observations have shown that CD40 activation of CLL cells via coculture with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile and sensitization for cell death by aCD20 mAbs. We found that the pro-survival effect of selicrelumab is minimal, while cell death by combined selicrelumab plus anti-CD20 antibody treatment is maintained. Thus, further investigation of applying selicrelumab combined with anti-CD20 mAbs in a therapeutic setting might be considered.Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab.

Human CD3+CD56+NKT-like cells express a range of complement receptors and C3 activation has negative effects on these cell activity and effector function

CD3+CD56+NKT-like cells are a rare population of lymphocytes that serve important roles in various types of immune-related diseases, and particularly in cancer. The complement system regulates inflammatory and immune responses by interacting with complement receptors expressed on a range of immune cells. However, whether CD3+CD56+NKT-like cells are regulated by the complement system has still not been definitively determined. In the present study, the expression of complement receptors and regulators in gated CD3+CD56+NKT-like cells isolated from human peripheral blood was assessed using PCR and flow cytometry. The results showed that human CD3+CD56+NKT-like cells expressed a range of complement receptors and regulators, such as CR3, C3aR, C5aR, C5L2, CD46 and CD55. Furthermore, the presence of complement component 3 (C3), a key component in complement activation in culture supernatant, mitigated the activity, IFN-γ production and killing function of CD3+CD56+NKT-like cells. The present study provides evidences supporting the relationship between complement activation and functional modulation of CD3+CD56+NKT-like cells, expanding our knowledge of the complement regulatory network, and also highlighting a potential target for treatment of numerous immune-related diseases, particularly NKT cell-based tumor adoptive immunotherapy.

IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities.

Adoptive immunotherapy using CAR-T cells is a promising curative treatment strategy for hematological malignancies. Current manufacture of clinical-grade CAR-T cells based on lentiviral/retrovirus transfection of T cells followed by anti-CD3/CD28 activation supplemented with IL-2 has been associated with low transfection efficiency and usually based on the use of terminally differentiated effector T cells. Thus, improving the quality and the quantity of CAR-T cells are essential for optimizing the CAR-T cell preparation. In our study, we focus on the role of IL-21 in the γc cytokine conditions for CAR-T cell preparation. We found for the first time that the addition of IL-21 in the CAR-T preparation improved T cell transfection efficiency through the reduction of IFN-γ expression 24–48 h after T cell activation. We also confirmed that IL-21 enhanced the enrichment and expansion of less differentiated CAR-T cells. Finally, we validated that IL-21 improved the CAR-T cell cytotoxicity, which was related to increased secretion of effector cytokines. Together, these findings can be used to optimize the CAR-T cell preparation.

Transcriptome landscape of double negative T cells by single-cell RNA sequencing

CD4 and CD8 coreceptor double negative TCRαβ+ T (DNT) cells are increasingly being recognized for their critical and diverse roles in the immune system. However, their molecular and functional signatures remain poorly understood and controversial. Moreover, the majority of studies are descriptive because of the relative low frequency of cells and non-standardized definition of this lineage. In this study, we performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of male C57BL/6 mice using strict fluorescence-activated cell sorting. The data was replicated in a subsequent study. Our analysis revealed five transcriptionally distinct naïve DNT cell clusters, which expressed unique sets of genes and primarily performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, in comparison with CD4+, CD8+ T cells and NK cells, Ikzf2 was highly expressed by both naïve and activated cytotoxic DNT cells. In conclusion, we provide a map of the heterogeneity in naïve and active DNT cells, addresses the controversy about DNT cells, and provides potential transcription signatures of DNT cells. The landscape approach herein will eventually become more feasible through newer high throughput methods and will enable clustering data to be fed into a systems analysis approach. Thus the approach should become the “backdrop” of similar studies in the myriad murine models of autoimmunity, potentially highlighting the importance of DNT cells and other minor lineage of cells in immune homeostasis. The clear characterization of functional DNT subsets into helper DNT, cytotoxic DNT and innate DNT will help to better understand the intrinsic roles of different functional DNT subsets in the development and progression of autoimmune diseases and transplant rejection, and thereby may facilitate diagnosis and therapy.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

MiR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Dominant interfering CARD11 variants disrupt JNK signaling in T cells

Abstract Previous studies in mice implicated c-Jun N-terminal kinase 1 and 2 (JNK1/2) as important determinants of helper CD4+ T cell differentiation through their influence on AP-1 and NF-AT-dependent gene transcription. However, mechanisms governing the JNK signaling pathway in human lymphocytes remain nebulous. CARD11 is a critical scaffold protein in T and B cells that connects antigen receptor (AgR) engagement to downstream transcriptional programs responsible for effector cell survival, proliferation and differentiation. Although CARD11 is best known for facilitating AgR-induced NF-κB activation, a handful of studies have shown it is also required for AgR-induced mTORC1 and JNK2 signaling. Using both CARD11-deficient Jurkat T cells and primary human T cells, we found that phosphorylation of both JNK1 and JNK2 in response to T cell receptor (TCR)/CD28 activation is dependent on CARD11. Furthermore, we demonstrate that heterozygous dominant negative (DN) CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease also disrupt TCR-dependent JNK1/2 phosphorylation and c-Jun accumulation with variable potency, closely mirroring the extent of impaired NF-κB activation. Considering that loss of either JNK1 or JNK2 in mice contributes to reduced IFN-γ production and skewed Th2 phenotypes, our findings suggest that defective JNK signaling in CD4+ T cells may contribute to severe atopic disease manifestations noted in most CADINS patients. Ongoing studies will define differences in specific JNK-dependent gene signatures that correlate with specific CARD11 DN variants and clinical phenotypes.