anti-CD40 mAb Research Articles

A novel anti-CD47 antibody with therapeutic potential for NK/T-cell lymphoma

ABSTRACT NK/T-cell lymphoma (NKTCL) is a rare type of non-Hodgkin lymphoma (NHL). Although L-asparaginase-based chemotherapy has significantly improved survival in early-stage patients, the prognosis is poor in advanced and relapsed or refractory patients. CD47 is a promising target for cancer immunotherapy. However, the expression of CD47 in NKTCL and the antitumor effect and mechanism of the anti-CD47 monoclonal antibody (mAb) AK117 in NKTCL remain unclear. Firstly, the expression level of CD47 protein in NKTCL cells was detected by immunoblot and flow cytometry. Secondly, in order to validate the role of CD47 downregulation in the proliferation, apoptosis, and cell cycle of NKTCL cells, we used shRNA transfection to knock down CD47 expression. We determined the effect of knocking down CD47 and the novel anti‐CD47 antibody AK117 on the phagocytosis of NKYS and YTS cells by M2 macrophages in vitro. Finally, we assessed the ability of AK117 to inhibit tumor growth in an NKTCL xenograft model in which YTS cells were engrafted in SCID mice. The results showed that CD47 is relatively highly expressed in NKTCL cells. CD47 knockdown in NKTCL promoted phagocytosis by M2 macrophages in an in vitro coculture assay. The study also demonstrated that anti-CD47 mAb AK117 promoted phagocytosis of NKTCL cells by M2 macrophages. In addition, in vivo experiments showed that the anti-CD47 mAb AK117 significantly inhibited the growth of subcutaneous xenograft tumors in SCID mice compared to the control antibody IgG. Our results indicate that targeting CD47 monoclonal antibodies is a potential therapeutic strategy for NKTCL.

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

Evaluation of a Combinatorial Immunotherapy Regimen That Can Cure Mice Bearing MYCN-Driven High-Risk Neuroblastoma That Resists Current Clinical Therapy.

Background: Incorporating GD2-targeting monoclonal antibody into post-consolidation maintenance therapy has improved survival for children with high-risk neuroblastoma. However, ~50% of patients do not respond to, or relapse following, initial treatment. Here, we evaluated additional anti-GD2-based immunotherapy to better treat high-risk neuroblastoma in mice to develop a regimen for patients with therapy-resistant neuroblastoma. Methods: We determined the components of a combined regimen needed to cure mice of established MYCN-amplified, GD2-expressing, murine 9464D-GD2 neuroblastomas. Results: First, we demonstrate that 9464D-GD2 is nonresponsive to a preferred salvage regimen: anti-GD2 with temozolomide and irinotecan. Second, we have previously shown that adding agonist anti-CD40 mAb and CpG to a regimen of radiotherapy, anti-GD2/IL2 immunocytokine and anti-CTLA-4, cured a substantial fraction of mice bearing small 9464D-GD2 tumors; here, we further characterize this regimen by showing that radiotherapy and hu14.18-IL2 are necessary components, while anti-CTLA-4, anti-CD40, or CpG can individually be removed, and CpG and anti-CTLA-4 can be removed together, while maintaining efficacy. Conclusions: We have developed and characterized a regimen that can cure mice of a high-risk neuroblastoma that is refractory to the current clinical regimen for relapsed/refractory disease. Ongoing preclinical work is directed towards ways to potentially translate these findings to a regimen appropriate for clinical testing.

Abstract 2693: MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy

Abstract Background: CD163 positive tumor associated macrophages (TAM), have been associated with poor response to immunochemotherapy in DLBCL. Targeting these TAMs to reverse immunosuppression is one approach for improving treatment response rates. MicroRNAs have the potential to modify hundreds of genes by interacting with multiple mRNA targets, thereby altering the global transcriptional profile of cells. Manipulation of microRNAs may prove an effective means of switching the transcriptomic profiles of TAMs to more activated, anti-tumor states resulting in the reversal of immune resistance and enhancement of treatment efficacy. Methods: MicroRNAs differentially expressed in DLBCL and associated with poor response were identified using whole gene correlated network analysis of DLBCL datasets. Peripheral blood mononuclear cells (PBMCs) were obtained from normal blood donors and differentiated into macrophages following cell culture with MCSF. Macrophages were polarized using immunosuppressive (IL4 + IL13) or immunostimulating (LPS + IFNγ) cytokines and microRNA expression measured using RT-qPCR. To assess antibody dependent cellular phagocytosis (ADCP), macrophages were generated and polarized as above and transfected with microRNA inhibitors (Thermofisher) prior to co-incubation with target cells. Human Chronic Lymphocytic Leukemia (CLL) cells were labelled with CSFE and used as target cells with Rituximab as the ADCP-inducing monoclonal antibody (mAb) and Herceptin as an isotype control. The percentage of Fc gamma receptor IIIA (FcγRIIIA)-expressing macrophages taking up target cells was determined using flow cytometry and analyzed using Flowjo software. Diagnostic biopsies from patients were then analyzed for microRNA 142-5p and CD206 expression using miRNAScope assays and immunohistochemistry. Results: There was significant upregulation of microRNA 142-5p in macrophages polarized with IL4 plus IL13 compared to macrophages polarized with LPS plus IFNγ and non-polarized macrophages. Inhibition of microRNA 142-5p in IL4 plus IL13 polarized macrophages led to a significant increase in FcγRIIIA, CD40 and CD47 expression resulting in enhanced phagocytosis of CLL cells opsonized with Rituximab plus anti-CD47 mAb when compared with controls. High microRNA 142-5p expression in combination with high CD206 expression predicted poor response to immunochemotherapy in 38 treatment naïve human DLBCL samples (median PFS 12 months v NR p=0.0096). Discussion: Transfection of a microRNA 142-5p inhibitor can repolarize immunosuppressive macrophages to a more anti-tumor phenotype leading to enhanced ADCP. CD206 positive TAM infiltration in combination with high microRNA 142-5p expression predicted poor outcome following treatment with immunochemotherapy in DLBCL. It may be possible to use microRNA directed therapies targeting the tumor microenvironment to enhance treatment efficacy. Citation Format: Jemma Longley, Russell Foxall, Steven Thirdborough, Steve Beers, Mark Cragg. MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2693.

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5244.

CD99 tumor associated antigen is a potential target for antibody therapy of T-cell acute lymphoblastic leukemia.

Monoclonal antibodies (mAbs) are an effective drug for targeted immunotherapy in several cancer types. However, so far, no antibody has been successfully developed for certain types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is an aggressive hematologic malignancy. T-ALL patients who are treated with chemotherapeutic drugs frequently relapse and become drug resistant. Therefore, antibody-based therapy is promising for T-ALL treatment. To successfully develop an antibody-based therapy for T-ALL, antibodies that induce death in malignant T cells but not in nonmalignant T cells are required to avoid the induction of secondary T-cell immunodeficiency. In this review, CD99 tumor associated antigen, which is highly expressed on malignant T cells and lowly expressed on nonmalignant T cells, is proposed to be a potential target for antibody therapy of T-ALL. Since certain clones of anti-CD99 mAbs induce apoptosis only in malignant T cells, these anti-CD99 mAbs might be a promising antibody drug for the treatment of T-ALL with high efficiency and low adverse effects. Moreover, over the past 25 years, many clones of anti-CD99 mAbs have been studied for their direct effects on T-ALL. These outcomes are gathered here.

KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes.

Develop a novel therapeutic strategy for patients with subtypes of mature T-cell and NK-cell neoplasms. Primary specimens, cell lines, patient-derived xenograft models, commercially available, and proprietary anti-KLRG1 antibodies were used for screening, target, and functional validation. Here we demonstrate that surface KLRG1 is highly expressed on tumor cells in subsets of patients with extranodal NK/T-cell lymphoma (ENKTCL), T-prolymphocytic leukemia (T-PLL), and gamma/delta T-cell lymphoma (G/D TCL). The majority of the CD8+/CD57+ or CD3-/CD56+ leukemic cells derived from patients with T- and NK-large granular lymphocytic leukemia (T-LGLL and NK-LGLL), respectively, expressed surface KLRG1. The humanized afucosylated anti-KLRG1 monoclonal antibody (mAb208) optimized for mouse in vivo use depleted KLRG1+ TCL cells by mechanisms of ADCC, ADCP, and CDC rather than apoptosis. mAb208 induced ADCC and ADCP of T-LGLL patient-derived CD8+/CD57+ cells ex vivo. mAb208 effected ADCC of subsets of healthy donor-derived KLRG1+ NK, CD4+, CD8+ Tem, and TemRA cells while sparing KLRG1- naïve and CD8+ Tcm cells. Treatment of cell line and TCL patient-derived xenografts with mAb208 or anti-CD47 mAb alone and in combination with the PI3K-δ/γ inhibitor duvelisib extended survival. The depletion of macrophages in vivo antagonized mAb208 efficacy. Our findings suggest the potential benefit of a broader treatment strategy combining therapeutic antibodies with PI3Ki for the treatment of patients with mature T-cell and NK-cell neoplasms. See related commentary by Varma and Diefenbach, p. 2300.

The CD47-Blocking Immune Checkpoint Inhibitor Maplirpacept Does Not Interfere with Blood Transfusion Compatibility Testing

CD47 is a transmembrane glycoprotein that delivers an anti-phagocytic (“do-not-eat”) signal by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Many tumors express high levels of CD47 as a means to escape macrophage-mediated immune surveillance and blockade of the CD47-SIRPα pathway has emerged as a novel therapeutic strategy with demonstrated anti-tumor activity both in vitro and in vivo (Petrova et al. 2017; Weiskopf 2017). Maplirpacept (PF-07901801, TTI-622) is a soluble human SIRPαFc fusion protein with an IgG4 domain which acts as an innate immune checkpoint inhibitor that prevents CD47-SIRPα binding, engages activating Fcγ receptors on macrophages, and promotes phagocytosis of malignant cells (Lin et al. AACR 2018). Maplirpacept is currently in clinical development to treat patients with ovarian cancer, multiple myeloma, acute myeloid leukemia and lymphoma. Additionally, CD47 is expressed on red blood cells (RBCs) and plays a role in the natural clearance of aged RBCs by splenic macrophages in vivo (Oldenborg et al. 2000). CD47 expression on RBCs was shown to interfere with pre-transfusion laboratory testing in patients treated with the anti-CD47 mAb, Hu5F9-G4 (Velliquette et al. 2019). Unlike anti-CD47 mAbs, minimal binding to human RBCs is observed with Maplirpacept (Lin et al. AACR 2018). In this study we investigated whether the minimal binding of Maplirpacept to human RBCs precludes interference with routine pre-transfusion laboratory tests. RBC suspensions from healthy donors representing all 8 ABO-Rh blood groups were spiked with Maplirpacept at 0.6, 1.2 or 2 mg/ml or anti-CD47 (clone Hu5F9). Blood typing was performed according to the manufacture's protocol (Seraclone, Biorad) and observed for the presence or absence of agglutination. For cross matching experiments, plasma was prepared from blood of healthy donors and spiked with Maplirpacept or anti-CD47 at the same concentrations used for the blood typing experiments. RBCs from two healthy donors were mixed with plasma and an indirect antiglobulin test (IAT) was performed according to the manufacture's protocol (Coombs Anti-IgG, Biorad). Using RBCs from healthy donors representing the 8 major ABO-Rh blood groups, we now show that the addition of Maplirpacept does not cause agglutination of test RBCs and produces the expected blood typing result in a standard forward blood typing test. No interference was observed at the highest concentration tested. However, addition of an anti-CD47 mAb caused pan-agglutination as well as false positive blood typing results (Table 1). In a standard IAT that mimics the in vitro compatibility cross matching test performed prior to a blood transfusion, the anti-CD47 mAb gave false positive results while Maplirpacept produced results identical to the control serum condition (Table 2). This data is consistent with a study demonstrating interference by Hu5F9 in routine pre-transfusion blood testing (Velliquette et al. 2019). Additionally, no interference was observed when Maplirpacept was added to conditions which produced agglutination in a standard IAT across a range of blood types. Interference with RBC panel testing has important consequences in transfusion medicine as the presence of irregular antibodies can complicate the selection of suitable RBC units for treated patients. Here we demonstrate that interference with blood compatibility testing is dependent upon the type of CD47-blocking agent. Decoy receptors such as Maplirpacept, unlike anti-CD47 mAb, do not interfere with blood group serologic testing, which may confer a significant clinical advantage by negating the need for additional approaches to prevent interference.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

Single-Cell Characterization of TP53-Mutated AML Patients Treated with Frontline Azacitidine, Venetoclax, and Magrolimab Reveals Mechanisms of Response and Resistance

Background: TP53 mutations, found in around 20% of AML cases, often lead to therapy resistance and unfavorable outcomes. Recent studies hint at the potential efficacy of CD47, a ‘don't eat me’ signal, in treating TP53-mutated AML. The ongoing clinical trial (NCT04435691) is assessing the efficacy of a triple therapy combo-magrolimab (anti-CD47 mAb), azacitidine, and venetoclax (AVM)-with promising initial results in TP53-mutated AML patients. However, understanding resistance and relapse remains crucial. Methods: We performed paired 5' scRNA and scTCR profiling on 3 healthy donors and 27 samples from 11 treatment-naive TP53-mutated AML patients (median age 63, range 59-73 years; 8 responders (CR/CRi); 3 non-responders (NR)), both pre-AVM (n=11) and post-AVM-point of remission (n=11), relapse (n=2), or primary resistance (n=3). Results: 42,910 AML cells and 137,169 bone marrow microenvironment cells passed quality checks. AML cell identification was confirmed via correlation with clinical flow cytometry, inferred copy number alterations, and immunohistochemistry markers. We leveraged Symphony to phenotypically infer AML cell types based on their transcriptional similarity to normal hematopoietic cells. Overall, samples displayed heterogeneous distribution of cell types at time of diagnosis. However, the 3 samples from the 3 primary resistant patients demonstrated a marked and significant expansion of AML cell proportion exhibiting an erythroid differentiation phenotype post-AVM (Fig. A). Also, the 2 patients who relapsed 4.8- and 7.1-months following AVM demonstrated expansion of erythroid differentiation proportion (Fig. A) and higher LSC17 at time of relapse. This suggests a selection favoring erythroid clones as a mechanism of primary and acquired resistance following AVM therapy. We conducted a pathway enrichment analysis using Hallmark gene sets to identify significantly altered AML-intrinsic biological pathways between responders and non-responders. CR/CRi patient-derived AML cells exhibited an enriched inflammatory response, specifically in TNFα and IFNγ signaling pathways-known CD47 expression regulators-and increased CD47 expression (Fig. B). In contrast, AML cells from non-responsive patients showed an enrichment of oxidative phosphorylation (Fig. B). Of note, phagocytic activity of myeloid cells was high in non-responders suggesting that despite the likely activity of magrolimab, other mechanisms of primary resistance were at play. As anti-CD47-mediated phagocytosis can stimulate antigen presentation and trigger T cell activation, we delved into the T cell subtypes and their corresponding cellular responses. There were not significant differences in the distribution of CD4 and CD8 pretreatment in CR/CRi versus NR patients. However, CD8 dysfunction score was significantly higher in non-responders at baseline. Following therapy, the CD8 dysfunction score was also significantly higher at time of relapse among responders. Further, effector CD8 T cells of NR patients had enrichment for cytotoxicity and TCR signaling pathways, and higher expression of exhaustion-related genes. Conclusions: Through single cell transcriptomic profiling of TP53-mutated AML patients treated with the triplet combination of AVM on protocol we identified erythroid differentiation and oxidative phosphorylation as possible intrinsic resistance mechanisms. Additionally, non-responders exhibited a dysfunctional T cell state prior to treatment, while relapses were associated with development of CD8 dysfunction. Overall, this data provides valuable insight into the mechanisms of response and resistance to magrolimab-based therapy. TCR repertoire and AML-immune cell interaction analyses are ongoing.

IMMU-07. TACEDINALINE (CI994), A CLASS I HDAC INHIBITOR, TARGETS INTRINSIC TUMOR GROWTH AND LEPTOMENINGEAL DISSEMINATION IN MYC-DRIVEN MEDULLOBLASTOMA WHILE MAKING THEM SUSCEPTIBLE TO ANTI-CD47 INDUCED MACROPHAGE PHAGOCYTOSIS VIA NF-KB-TGM2 DRIVEN TUMOR INFLAMMATION

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified medulloblastoma, which sensitizes them to macrophage-mediated phagocytosis upon targeting the CD47-SIRPa innate checkpoint pathway. METHODS We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), medulloblastoma (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven medulloblastoma. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven medulloblastoma, with little to no activity in non-MYC-driven medulloblastoma, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting Class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in-vitro phagocytosis assays and in-vivo orthotopic xenograft models. RESULTS CI-994 displayed anti-tumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven medulloblastoma. Furthermore, RNA sequencing revealed NFκB pathway induction as a response to CI-994 treatment, followed by TGM2 expression, which enhanced inflammatory cytokine secretion. We further show interferon-gamma (IFN-g) release and cell surface expression of engulfment (“eat-me”) signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPa phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC-amplified medulloblastoma and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

A phase 1b, open-label, two-part safety, tolerability, and efficacy study of a soluble beta-glucan (odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma whose disease did not progress during first-line (1L) chemotherapy.

TPS4201 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease with a 5-year overall survival (OS) rate of 3%. Standard of care treatment is continuous cytotoxic chemotherapy which produces modest improvements in OS but is also associated with significant toxicity raising the need for novel treatment paradigms. Although immunotherapy has yet to produce clinical benefit for most patients with PDAC, preclinical modeling demonstrates its potential and show that combinations of myeloid agonists can trigger robust synergistic anti-tumor activity against PDAC tumors that are otherwise resistant to immunotherapy including immune checkpoint blockade (anti-CTLA4, anti-PD1). In this study, odetiglucan (OD), a novel beta glucan pathogen associated molecular pattern (PAMP), will be combined for the first time with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody in the maintenance setting after an induction phase of cytotoxic chemotherapy. OD and a CD40 agonist directly promote the activation and maturation of antigen presenting cells and shift the suppressive TME to enhance T-cell responses through non-redundant myeloid signaling pathways. We hypothesize that the combination of OD and CD40 agonist therapy has the potential to trigger anti-tumor immunity in PDAC and thus, extend and deepen responses to 1L cytotoxic chemotherapy. Methods: A two-part Phase 1b study of metastatic PDAC pts who have evidence of response or stable disease following 16-24 wks of chemotherapy is being conducted. Part A uses a 3+3 design; pts will receive OD 4 mg/kg QW + CDX-1140 1.5 mg/kg Q3W. Following a dose-limiting toxicity observation period, the dose will be held at 1.5 mg/kg or decreased to 0.72 mg/kg Q3W. Once preliminary safety is established, the cohort will be expanded to 15 pts. Part B pts will receive OD 4mg/kg + CDX-1140 at the dose identified in Part A Q3W. The study will enroll 30 pts. Main eligibility criteria: Metastatic PDAC having durable stable disease or response to 1L chemotherapy; serum ABA ≥20 µg/mL, and no prior anti-CD40 mAb or immunomodulatory treatment exposure. Primary endpoints are MTD, RP2D, and safety. Secondary endpoints include DOR, PFS, ORR, and OS and exploratory endpoints include evaluating immune pharmacodynamic responses in peripheral blood and tumor. Safety parameters will be listed and summarized using descriptive statistics. ORR will be tabulated by overall frequency; DOR, TTF, PFS and OS will be summarized and presented using Kaplan-Meier and waterfall plots. 5 US sites will participate. Clinical trial information: NCT04834778 .

3D culture boosting fullerenol nanoparticles to induce calreticulin exposure on MCF-7 cells for enhanced macrophage-mediated cell removal

Calreticulin (CRT) on the cell surface that acts as an "eat me" signal is vital for macrophage-mediated programmed cell removal. The polyhydroxylated fullerenol nanoparticle (FNP) has appeared as an effective inducer to cause CRT exposure on cancer cell surface, but it failed in treating some cancer cells such as MCF-7 cells based on previous findings. Here, we carried out the 3D culture of MCF-7 cells, and interestingly found that the FNP induced CRT exposure on cells in 3D spheres via re-distributing CRT from endoplasmic reticulum (ER) to cell surface. Phagocytosis experiments in vitro and in vivo illustrated the combination of FNP and anti-CD47 monoclonal antibody (mAb) further enhanced macrophage-mediated phagocytosis to cancer cells. The maximal phagocytic index in vivo was about three times higher than that of the control group. Moreover, in vivo tumorigenesis experiments in mice proved that FNP could regulate the progress of MCF-7 cancer stem-like cells (CSCs). These findings expand the application of FNP in tumor therapy of anti-CD47 mAb and 3D culture can be used as a screening tool for nanomedicine.

DOP67 A transcriptional module associated with neutrophil degranulation is highly conserved across species and is a core molecular pathway of treatment resistance in ulcerative colitis

Abstract Background Advanced therapeutics are changing the treatment landscape in ulcerative colitis (UC), but drug resistance is still common. New strategies are needed to identify the molecular and cellular drivers of treatment resistance and inform the development of novel molecular diagnostics and therapies. In this study we analysed transcriptional modules conserved across species and different colitis models to look for fundamentally important pathways of disease. Methods We surveyed the colonic transcriptome in preclinical UC models, including the T-cell transfer, Il10-/-, DSS, DNBS, anti-CD40 mAb and TRUC (Tbx21-/- x Rag2-/- UC) models and multiple cohorts of patients with UC. Gene expression and pathway level changes were defined. Conserved gene and pathway level changes were defined and their relationship to treatment response with anti-TNF, ustekinumab and vedolizumab assessed in independent UC cohorts, including key registration trials. Results Principal component analysis showed clear separation between control and experimental colitis samples, which clustered according to the specific model and classification, such as chemically induced (DSS and DNBS), T-cell dependent (T-cell transfer model and Il10-/-, and T-cell independent, innate-mediated models (anti-CD40 mAb and TRUC models). Between 1700 and 8200 genes were differentially expressed (FDR < 0.05) between the experimental models and the corresponding controls; 145 genes had expression levels at least doubled up in all cases. Pathway analysis highlighted significant shared effects (FDR<0.05) on known cytokine-cytokine receptor interactions, signalling by interleukins and neutrophil degranulation. Genes expressed more specifically in the T-cell dependent models were enriched in immune processes such as the complement system, FCERI signalling and downstream activation of MAPK and NFKB, while those more specific to chemically-induced models were enriched in extracellular matrix functions like degradation and remodelling. A set of 21 genes involved in neutrophil degranulation, and over-expressed across all models of colitis, was also significantly enriched in the colon of patients with active UC. Moreover, the magnitude of enrichment of this gene list could differentiate between responders and non-responders to infliximab (difference in median -704.58, p=0.01) and ustekinumab (difference in median -771.08, p=2.19e-08) but not vedolizumab (difference in median 2212.71, p=0.37). Conclusion This transcriptional atlas provides a new lens to scrutinize fundamental biological pathways of gut inflammation and new opportunities to understand the core molecular features of the resistome. A set of neutrophil degranulation genes was associated with resistance to anti-cytokine therapies in UC.

Antibody-induced erythrophagocyte reprogramming of Kupffer cells prevents anti-CD40 cancer immunotherapy-associated liver toxicity

BackgroundAgonistic anti-CD40 monoclonal antibodies (mAbs) have emerged as promising immunotherapeutic compounds with impressive antitumor effects in mouse models. However, preclinical and clinical studies faced dose-limiting toxicities mediated by necroinflammatory liver...

Tacedinaline (CI-994), a class I HDAC inhibitor, targets intrinsic tumor growth and leptomeningeal dissemination in MYC-driven medulloblastoma while making them susceptible to anti-CD47-induced macrophage phagocytosis via NF-kB-TGM2 driven tumor inflammation

BackgroundWhile major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant...

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Inhibition of the CD47-SIRPα axis for cancer therapy: A systematic review and meta-analysis of emerging clinical data

CD47-SIRPα interaction acts as a "don't eat me" signal and is exploited by cancer to downregulate innate and adaptive immune surveillance. There has been intense interest to develop a mechanism of blockade, and we aimed to analyze the emerging data from early clinical trials. We performed a systematic review and meta-analysis of relevant databases and conference abstracts including clinical trials using CD47 and/or SIRPα inhibitors in cancer treatment. Nonlinear mixed models were applied for comparison of response and toxicity. We retrieved 317 articles, 24 of which were eligible. These included 771 response-evaluable patients with hematologic (47.1%) and solid tumors (52.9%). Of these, 6.4% experienced complete response, 10.4% partial response, and 26.1% stable disease for a 16.7% objective response rate (ORR), 42.8% disease control rate, and 4.8-month median duration of response. ORR was significantly higher for hematologic cancers (25.3%) than solid cancers (9.1%, p=0.042). Comparing by mechanism, seven CD47 monoclonal antibodies (mAbs) and six selective SIRPα blockers were given alone or combined with checkpoint inhibitors, targeted therapy, and/or chemotherapy. In solid cancers, selective SIRPα blockade showed a higher ORR (16.2%) than anti-CD47 mAbs (2.8%, p=0.079), which was significant for combination therapies (ORR 28.3% vs 3.0%, respectively, p=0.010). Responses were seen in head and neck, colorectal, endometrial, ovarian, hepatocellular, non-small cell lung, and HER2+gastroesophageal cancers. Dose-limiting toxicity (DLT) was seen in 3.3% of patients (5.4% anti-CD47 mAbs, 1.4% selective SIRPα blockers; p=0.01). The frequency of treatment-related adverse events (TRAEs) ≥grade 3 was 18.0%, similar between the two groups (p=0.082), and mostly laboratory abnormalities. For anti-CD47 mAbs, the most common toxicities included grade 1-2 fatigue (27.2%), headache (21.0%), and anemia (20.5%). For selective SIRPα blockers, these included grade 1-2 infusion reaction (23.1%) and fatigue (15.8%). Anti-CD47 mAbs were significantly more likely than selective SIRPα blockers to cause grade 1-2 fever, chills, nausea/vomiting, headache, and anemia. In conclusion, combination therapies using selective SIRPα blockade had higher response rates in solid tumors than anti-CD47 mAb combinations. Hematologic changes were the main TRAEs, and selective SIRPα blockers seemed to have a better grade 1-2 toxicity profile. Treatment was well-tolerated with minimal DLTs.

Conjugation of Listeriolysin O to Anti-CD47 mAb: Harnessing Listeria Toxin to Weaponize Macrophages against Tumor Cells

<h3>Purpose/Objective(s)</h3> CD47 is expressed on various tumor types including breast, prostate, glioblastoma, non-Hodgkin lymphoma, and hepatocellular carcinoma. Interrupting the CD47-SIRP alpha axis by monoclonal antibodies fosters macrophage-mediated phagocytosis of solid and hematopoietic tumor cells. The pore-forming toxin listeriolysin O (LLO) is activated in the acidic vacuoles of bone marrow-derived macrophages. We hypothesize that LLO conjugation to the anti-CD47 mAb enhances the release of autophagic cargo within macrophages thereby augmenting cGAS-STING activation, antigen presentation, and tumor phagocytosis. <h3>Materials/Methods</h3> The anti-CD47 mAb was conjugated to the LLO toxin using a water-soluble SPDP crosslinker and purified by affinity chromatography. The cytotoxicity of anti-CD47-LLO mAb relative to anti-CD47 mAb was tested in the mouse breast cancer cell line E0771, the human breast cancer cell line MDA-MB-468, the mouse lung cancer cell line LLC, and the human leukemia monocytic cell line THP-1. C57B6 mouse bone marrow-derived macrophages (BMDM) co-cultured with tumor cell lines were analyzed in the presence of anti-CD47-LLO mAb and anti-CD47 mAb to determine the efficiency of BMDM tumor cell phagocytosis, STING activation, and antigen presentation. Tumor growth of E0771-bearing wildtype mice was measured following intratumoral injection of anti-CD47 mAb and anti-CD47-LLO mAB. <h3>Results</h3> The anti-CD47-LLO mAb conjugate enhanced BMDM phagocytosis of LLC and E0771 tumor cells in a dose dependent manner that was significantly higher than cells treated with the anti-CD47 mAb alone. The anti-CD47-LLO mAb significantly increased BMDM levels of phosphorylated STING, interferon-stimulated genes, and antigen presentation relative to anti-CD47 mAb treatment. At doses compatible with preserved animal total body weight, red blood cell counts, and lymphocyte counts, intratumoral injection of anti-CD47-LLO mAb significantly suppressed tumor growth of E0771-bearing wildtype mice relative to the anti-CD47 mAb. <h3>Conclusion</h3> Conjugation of the LLO toxin to anti-CD47 mAb enhances macrophage cGAS-STING activation, antigen presentation, and tumor cell phagocytosis. This novel CD47 antibody builds on clinical interest in targeting macrophages for the treatment of malignancy and may be studied as a supplemental therapy for patients with tumors resistant or refractory to checkpoint therapy.

Effect of clinical application of anti-CD38 and anti-CD47 monoclonal antibodies on blood group detection and transfusion therapy and treatment

BackgroundTo investigate the effect of anti-CD38 monoclonal antibodies (mAb) (daratumumab, DARA) and anti-CD47 mAb combined with azacytidine on blood transfusion compatibility tests, transfusion effects in the treatment of multiple myeloma or acute myeloid leukemia and the corresponding management strategy. Materials and methodsAmong the 19 patients who were treated with DARA and anti-CD47 mAb, 4 patients with cross matching incompatibility were selected. The ABO blood group, the Rh blood group, irregular antibody screening and direct antiglobulin test (DAT) and cross matching testing were performed before and after the application of mAbs using serological methods. Then, irregular antibody screening and microcolumn gel cross matching tests were performed with donor and recipient erythrocytes and serum treated with DL-dithiothreitol (DTT) and Immucor kit, respectively. The transfusion effect was monitored. Results21.05% (4/19) patients had mismatched cross-matching results after mAb treatment. The agglutination intensity of irregular antibody screening tests (3 + ∼ 4 +) after anti-CD47 mAb was higher than that (1 + ∼ 2 +) after DARA. In the DARA group, treating RBCs with 0.2 mol L−1 DTT eliminated the DARA interference with antibody screening. In the anti-CD47 mAb group, the antibody screening, cross-matching test and DAT had been strongly interfered, and using Immucor kit eliminated the interference with antibody screening testing. There was no difference in the transfusion effect. ConclusionThe application of mAb drugs led to incompatibility of cross matching tests, and the transfusion effect was not affected.