anti-CD40 mAb Research Articles

Abstract 553: Combined use of human iPSC-derived natural killer cells with macrophages and anti-CD47 blockade to improve killing of acute myeloid leukemia

Abstract Despite many recent advances, treatment of acute myeloid leukemia (AML) remains challenging. To generate an improved cell-based therapy against AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from combination with other therapies. Based on studies showing that targeting the CD47 pathway on macrophages (Mϕ) improves anti-tumor activity in AML, we investigated the combination of iPSC-NK cells with iPSC-Mϕ with and without CD47 blockade for the treatment of AML. To determine if the addition of iPSC-Mϕ can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-Mϕ in standard cytotoxicity assays. While Mϕ alone did not kill AML blasts, the addition of iPSC-Mϕ to iPSC-NK cells significantly improved killing of AML blasts by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of AML blasts by an additional 23% (p<0.01). Addition of the CD47 mAb to iPSC-Mϕ without NK cells did not result in increased killing. We also demonstrated that blockade of SIRPα (receptor for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-Mϕ + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-Mϕ alone (p<0.01). To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased killing via loss of the inhibitory CD47-SIRPα interaction on NK cells and not via another mechanism, we tested the effect of adding anti-CD47 mAb and SIRPα antibodies simultaneously. Compared to addition of either mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. We also found the addition of the SIRPα mAb to iPSC-Mϕ alone did not increase cytotoxicity. We next evaluated if antibody dependent cellular cytotoxicity (ADCC) mediated by anti-CD47 mAb binding AML cells could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity, indicating no role for ADCC. Finally, we showed that direct contact between NK cells and Mϕ is required to increase killing of AML in a transwell assay. Separation of iPSC-Mϕ from iPSC-NK cells by a transwell insert abrogated the increase in anti-AML cytotoxicity seen with addition of iPSC-Mϕ to NK cells without the insert. In vivo studies testing the combination of iPSC-NK cells, iPSC-Mϕ and CD47 mAb against human AML in mouse xenograft-models are ongoing. Together our results indicate that CD47 blockade combined with iPSC-Mϕ leads to increased NK cell-mediated anti-tumor activity. iPSC-NK cells and iPSC-Mϕ provide an important, standardized, allogeneic cell therapy approach. Citation Format: Benjamin Goldenson, Manuel Fierro, Somayeh Pouyanfard, Dan S. Kaufman. Combined use of human iPSC-derived natural killer cells with macrophages and anti-CD47 blockade to improve killing of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 553.

Abstract 5958: CD276/CD47-targeted antibody-drug conjugates to treat triple-negative breast cancers

Abstract Triple-negative breast cancers (TNBCs) are highly aggressive, heterologous and frequently recurrent. The standard radiotherapy and chemotherapy have limited clinical benefits while the combined therapies or antibody-drug conjugates (ADCs) are promising to treat TNBC. The objective of this study was to develop a combined therapy, CD276/CD47-targeted ADCs, for TNBC treatment. Specifically, the CD276 and CD47 surface receptors were identified with immunohistochemistry staining of drug-sensitive and resistant patient tissues and confirmed in other assays. Two innovative monoclonal antibodies (mAbs), i.e. anti-CD276 mAb and anti-CD47 mAb that target the N-glycosylated extracellular domains (Leu29-Pro245 and Gln19-Glu141) of surface receptors, were developed using hybridoma technology. The top mAb clones with high surface binding, drug delivery and affinity were screened, produced in fed-batch culture, purified using liquid chromatography, and conjugated with payload mertansine (DM1). Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the CD276/CD47 ADCs effectively target human and mouse TNBC cells and xenograft models. The in vitro anti-cancer cytotoxicity was confirmed in multiple cell lines (MDA-MB-468, MDA-MB-231, 4T1), and in vivo evaluations using TNBC cell line-derived xenograft models and patient-derived xenograft (PDX) models demonstrated high anti-tumor efficacy and tumoral immunity of the dually targeted ADCs. In addition, whole blood analysis indicated that neither CD276 mAb nor CD47 mAb showed general immune toxicity in immunocompetent model. The maximal tolerated dose (MTD), pharmacokinetics (PK) and biodistribution will be further investigated. This study indicated that the ADCs dually targeting CD276 and CD47 have great potential to treat TNBCs. Citation Format: Yingnan Si, Kai Chen, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. CD276/CD47-targeted antibody-drug conjugates to treat triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5958.

MODL-18. Enhancing anti-CD47 mAb efficacy with radiotherapy for Group 3 paediatric medulloblastoma in preclinical models

Abstract Medulloblastoma is the most common paediatric brain cancer. Standard treatment approaches, including craniospinal irradiation (CSI), can result in severe lifelong side effects and have not changed for decades resulting in a stagnation of survival outcomes for children with aggressive medulloblastoma. Despite successes in other cancers, no immunotherapies have been approved for use in paediatric medulloblastoma. Unlike other solid tumours, medulloblastomas are myeloid dominant, and immunotherapies must be rationally designed with the tumour microenvironment in mind. Anti-CD47 antibody therapy activates macrophages against cancer cells by blocking anti-phagocytic signalling mediated by CD47-SIRPa ligation and has shown preclinical efficacy in brain cancer models. We have developed preclinical CSI protocols that mimic clinical treatment response using a small animal radiotherapy platform. We show that CSI depletes adaptive immune cells in the brain, increasing the proportional abundance of myeloid cells, suggesting an opportunity to combine radiation with myeloid-targeted immunotherapy. We show that anti-CD47 therapy is ineffective as a single agent against a patient-derived xenograft model of Group 3 medulloblastoma (SJ_MB002), and that while the CSI protocol causes temporary tumour regression, the combination of anti-CD47 with CSI results in marked and persistent tumour regression. To enhance our preclinical evaluation of CSI and anti-CD47, we have developed new mouse models that more accurately reflect the developing microenvironment of children and show that immune populations in paediatric brain are distinct from adult mouse brain. Future work will elucidate the mechanisms by which radiotherapy alters the medulloblastoma microenvironment to enhance the anti-tumour activity of myeloid immune cells in the brain. By evaluating this novel combination of immunotherapy with standard medulloblastoma treatments, in age-appropriate models, our research should facilitate the rational selection and rapid translation of optimised treatment combinations for future medulloblastoma clinical trials.

Anti-CD47 immunotherapy in combination with BCL-2 inhibitor to enhance anti-tumor activity in B-cell lymphoma.

CD47 expressed on cancer cells enables macrophage immune evasion. Blocking CD47 using anti-CD47 monoclonal antibodies (mAbs) is a promising strategy. The anti-CD47 mAb TJC4 has anti-tumor activity but lacks hematological toxicity. Venetoclax, a B-cell lymphoma 2 (BCL-2) inhibitor for B-cell malignancy, induces phosphatidylserine (PS) extracellular exposure, representing an "eat-me" signal for macrophages. The present study aimed to explore whether TJC4-Venetoclax combined therapy exerts synergistic anti-cancer properties in B-cell lymphoma. In vitro, flow cytometry and microscopy assessed whether TJC4 monotherapy or combination treatment could promote macrophage-mediated phagocytosis of tumor cells. Induced PS exposure on the cell membrane was measured using flow cytometry with Annexin V-FITC staining. In vivo, Venetoclax and TJC4's synergistic anti-tumor effects were evaluated. B cell lymphoma cell lines express high levels of CD47 and patients with diffuse large B cell lymphoma expressing CD47 have a worse clinical prognosis. TJC4 eliminates tumor cells via macrophage-mediated phagocytosis. In vitro and in vivo, the TJC4-Venetoclax combination increased phagocytosis significantly compared with either agent alone, showing synergistic phagocytosis, and displayed synergistic anti-cancer properties in B-cell lymphoma. Our results support the TJC4-Venetoclax combination as a promising therapy, and suppressing BCL-2 and CD47 simultaneously could represent a novel therapeutic paradigm for B-cell lymphoma.

Targeting Fc Receptor-Mediated Effects and the "Don't Eat Me" Signal with an Oncolytic Virus Expressing an Anti-CD47 Antibody to Treat Metastatic Ovarian Cancer.

mAbs blocking immune checkpoints have emerged as important cancer therapeutics, as exemplified by systemic administration of the IgG1 anti-CD47 mAb that blocks the "don't eat me" pathway. However, this strategy is associated with severe toxicity. To improve therapeutic efficacy while reducing toxicities for ovarian cancer, we engineered an oncolytic herpesvirus (oHSV) to express a full-length, soluble anti-CD47 mAb with a human IgG1 scaffold (OV-αCD47-G1) or IgG4 scaffold (OV-αCD47-G4). Both IgG1 and IgG4 anti-CD47 mAbs secreted by oHSV-infected tumor cells blocked the CD47-SIRPα signal pathway, enhancing macrophage phagocytosis against ovarian tumor cells. OV-αCD47-G1, but not OV-αCD47-G4, activated human NK-cell cytotoxicity and macrophage phagocytosis by binding to the Fc receptors of these cells. In vivo, these multifaceted functions of OV-αCD47-G1 improved mouse survival in xenograft and immunocompetent mouse models of ovarian cancer when compared with OV-αCD47-G4 and a parental oHSV. The murine counterpart of OV-αCD47-G1, OV-αmCD47-G2b, also enhanced mouse NK-cell cytotoxicity and macrophage phagocytosis and prolonged survival of mice bearing ovarian tumors compared with OV-αmCD47-G3. OV-αmCD47-G2b was also superior to αmCD47-G2b and showed a significantly better effect when combined with an antibody against PD-L1 that was upregulated by oHSV infection. Our data demonstrate that an oHSV encoding a full-length human IgG1 anti-CD47 mAb, when used as a single agent or combined with another agent, is a promising approach for improving ovarian cancer treatment via enhancing innate immunity, as well as performing its known oncolytic function and modulation of immune cells.

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

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

IPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML.AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01).To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC.To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, “off-the-shelf” cell therapy approach that can be translated into novel clinical therapies. [Display omitted] DisclosuresKaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of Relapsed Refractory Multiple Myeloma

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT ®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of relapsed/refractory multiple myeloma (rrMM). The CD38 binding arm consists of two bi-paratopic Fabs that strongly bind to CD38 through avidity-induced interactions. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the CD38 Fab arm preferentially drives binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. Hence, ISB 1442 is anticipated to induce minimal unintended effects on red blood cell (RBC) hemagglutination compared to benchmark anti-CD47 monoclonal antibodies (mAb) magrolimab. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). ISB 1442 is the first-in-class with these unique molecular attributes, which are designed to overcome mechanisms of resistance to daratumumab in rrMM patients.In vitro, ISB 1442 exhibited a higher killing potency compared to benchmark daratumumab across a broad range of CD38-expressing tumor cells. Specifically, ISB 1442 showed a high potency to kill CD38 high tumor cells through CDC and a superior potency to kill CD38 low expressing tumor cells through ADCC and ADCP compared to daratumumab. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, an anti-CD47 mAb (IgG4) acting mostly through ADCP. Consistent with its molecular design, which includes a functional Fc, ISB 1442 induced more potent killing of CD38 high and CD38 low tumor cells by CDC and ADCC compared to magrolimab. To characterize the complex mechanisms of action of ISB 1442 in a single system more fully, a multiple mode of action of killing (MMoAK) in vitro assay was established where autologous macrophages and PBMCs from healthy donors were incubated with CD38 low expressing tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs, autologous macrophages, and complement from human serum. In MMoAK, ISB 1442 exhibited prominent tumor cell killing that was twice as high as daratumumab. The presence of soluble CD38 or RBCs, the main source of antigen sinks for CD38 and CD47, did not affect the killing potency of ISB 1442 across in vitro assays using CD38 high or CD38 low expressing tumor cells. These data suggest that the molecular design of ISB 1442 mitigates the potential risk of CD47 and CD38 antigen sink and the related side effects.On-target specificity was evaluated in vitro by measuring binding to human RBCs, induction of hemagglutination and RBC depletion, hemolysis and platelet aggregation. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation in vitro and showed a marked reduction in human RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile.Finally, the potency of ISB 1442 was assessed in vivo in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice which have functional complement, macrophages and NK cells of murine origin. ISB 1442 induced higher tumor growth inhibition relative to daratumumab and comparable tumor regression compared to magrolimab.In summary, we report a novel approach for the treatment for rrMM by co-targeting CD38 and CD47 using a 2+1 biparatopic bispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in rrMM patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance. DisclosuresNo relevant conflicts of interest to declare.

Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity

Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs).Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb.Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing.Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. [Display omitted] DisclosuresKaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

Targeting the cluster of differentiation 47/signal-regulatory protein alpha axis in myeloid malignancies.

The antitumor activity of macrophages is regulated by a balance of prophagocytic and antiphagocytic signals. Cluster of differentiation 47 (CD47), the dominant macrophage immune checkpoint ('do not eat me' signal), interacts with its receptor signal-regulatory protein alpha (SIRPα) to suppress phagocytic activities. This axis plays a pivotal role in immune evasion in myeloid malignancies as well as multiple cancers providing strong rationale for therapeutic exploitation. Preclinical studies have revealed overexpression of CD47 on leukemic stem cells and myeloblasts from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), which contributes to immune surveillance evasion and is associated with poor outcomes. Blockade of CD47 with different approaches has demonstrated proof-of-concept antitumor activities mainly through phagocytic clearance. Early phase clinical trials combining the anti-CD47 mAb magrolimab with the hypomethylating agent azacitidine have showed synergistic activities, deep and durable responses, as well as a tolerable safety profile in these patients, including those with TP53 mutations. Targeting CD47/SIRPα axis, in combination with other therapeutic agents, represents a promising treatment approach for patients with myeloid malignancies, particularly the challenging TP53-mutated subgroup.

CD47 blockade enhances the efficacy of intratumoral STING-targeting therapy by activating phagocytes.

Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.

Anti-CD47 Monoclonal Antibody-Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody–drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC50 in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69+ NK, CD11c+ DC, and CD4+ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.

Targeting the CD47-SIRPα pathway promotes macrophage-mediated trogocytosis of ovarian cancer cells

Targeting the CD47-SIRPα pathway promotes macrophage-mediated trogocytosis of ovarian cancer cells

Targeting CD47 As a Novel Therapeutic Strategy in Multiple Myeloma

Introduction: CD47 is an immune checkpoint coordinating the innate and adaptive immune systems which allows cancer cells to escape the innate immune response by sending inhibitory signals to macrophages and regulates the adaptive immune anti-tumor response. Hence, CD47 has been identified as a novel therapeutic target in cancers, including multiple myeloma (MM) which is localized mainly in the bone marrow (BM). MM cells rely heavily on the interactions with tumor microenvironment (TME) and immune system. Macrophages are enriched in the BM of MM patients compared to healthy controls, and were demonstrated to support MM growth, in opposition to their intrinsic phagocytic properties. We study the new class of immune checkpoint inhibitor such as anti-CD47 monoclonal antibody (mAb) which enhance MM killing. We tested two anti-CD47 mAbs provided; Vx1000R (blocking only) - blocking CD47/SIRPα binding and Vx1004R (dual function) - blocking CD47/SIRPα binding and inducing cell death activity.The objectives of this study were: (1) to test the expression of CD47 (Vx1000R and Vx1004R binding) to MM cells, stromal cells (MSP-1 and HS5) and peripheral blood mononuclear cell (PBMC) sub-populations; (2) to study anti-CD47 mAbs binding in hypoxic conditions; (3) to investigate the effect of anti-CD47 mAbs on MM cell apoptosis in 2D and the 3D tissue engineered BM (3DTEBM; a scaffold incorporating BM plasma from MM patients, MM cells, and accessory cells) to reproduce the TME; (4) to test the cytotoxicity of Vx1004R on PBMC sub-populations in 3D culture; and (5) to examine the effect of Vx1004R on MM cell phagocytosis ex vivo by macrophages.Methods: We performed meta-analysis of CD47 mRNA expression in dataset available online in healthy, MGUS, smoldering MM and MM subjects. We tested the binding of Vx1000R and Vx1004R mAbs on MM.1S, H929, RPMI8226 and U266 cells and stromal cells (MSP-1 and HS5) in normoxia (21% O2) and hypoxia (1% O2), and in the 3DTEBM at 24h and 72h. We also tested Vx1000R binding to PBMC sub-populations (from 12 MM patients) including CD3 (T cells), CD14 (monocytes/macrophages), CD16 (NK cells, eosinophils, and neutrophils), CD19 (B cells), CD123 (pDC and basophils) and CD138 (MM cells).Next, we examined the cytotoxic activity of CD47 mAbs on MM cells alone and in co-culture with the BM negative fraction (CD138-depleted) and stromal cells (MSP-1) in 2D cultures by performing MTT survival assay, and in 3DTEBM cultures by flow cytometry. The cytotoxicity of Vx1004R was studied on PBMCs incorporated into a similar 3D model and treated with Vx1004R; proliferation and apoptosis of different sub-populations of PBMC were assessed via flow cytometry. Lastly, the effect of Vx1004R on MM cell phagocytosis by macrophages (CD14+; green) was tested after 2h of co-culture with MM cells (pre-labeled with DiD+; red) in the presence or absence of Vx1004R. Phagocytosis was determined as double positive (green+ red+) cells by flow cytometry.Results: The meta-analysis results demonstrated that CD47 mRNA expression increases with tumor progression (compared to early stage disease and healthy subjects). We demonstrated that MM.1S, H929, U266 and RPMI8226 cells and stromal cells (MSP-1 and HS5) express high levels of CD47, which does not alter in hypoxia, and that MM cells increase CD47 expression in 3DTEBM at 72h. CD47 (Vx1000R) is expressed in different sub-populations of PBMCs with the highest expression in MM cells. In addition, we have shown that anti-CD47 dual function mAb inhibits proliferation of MM cells, especially when co-cultured with negative fraction and MSP-1 stroma in 3DTEBM cultures. Moreover, we showed that anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages.Conclusions: CD47 is a promising target candidate for MM treatment, since CD47 mRNA increases with MM progression, and CD47 protein increases in MM cells in the presence of TME in the 3DTEBM, suggesting involvement in pathophysiology of MM. Moreover, anti-CD47 mAb enhances killing of MM cells when co-cultured with TME; however, it does not affect apoptosis of PBMC sub-populations in the 3DTEBM. Finally, anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages. These results suggest that anti-CD47 mAbs are applicable for MM treatment. Further studies are warranted to examine the effect of anti-CD47 mAbs as a novel strategy to inhibit the progression and dissemination of MM in vivo, especially when combined with chemotherapy. DisclosuresVij:Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Konypharma: Honoraria. Capoccia:Tioma: Employment. Manning:Tioma: Employment. Azab:Abbvie: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Cleave Bioscience: Research Funding; Cellatrix LLC: Other: Founder and owner; Glycomimetics: Research Funding; Tioma: Research Funding.

The Interaction between Integrin Mac-1 (αMβ2, CD11b/CD18) and Sirpα Mediates Macrophage Fusion

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. While a number of membrane proteins have been implicated in mediating cell-cell attachement during fusion, their binding partners are not yet clear. Recently, we have demonstrated that IL-4-induced fusion of mouse peritoneal macrophages depends on integrin Mac-1 (αMβ2, CD11b/CD18). Surprisingly, the genetic deficiency of ICAM-1, an established ligand of Mac-1, resulted in no decrease of macrophage fusion, suggesting the involvement of other counter-receptors for Mac-1. In the present study, we demonstrated that SIRPα, which like ICAM-1 belongs to the Ig superfamily and was previously implicated in fusion, interacts with Mac-1. Expression of Mac-1 on the surface of HEK293 cells resulted in their adhesion to recombinant ectodomain of SIRPα and this process was mediated through the αMI-domain of Mac-1. In addition, adhesion of RAW264.7 mouse macrophages to SIRPα was dependent on Mac-1. Direct interaction between the αMI-domain and SIRPα was shown by biolayer interferometry. Analyses of the interaction of αMI-domain with a peptide library spanning the ectodomain of SIRPα showed that recognition of SIRPa conforms to general principles that govern binding by Mac-1 of many of its ligands. SIRPα reportedly interacts with CD47; however, adhesion of Mac-1-expressing HEK293 cells, which express CD47, to SIRPα was not dependent on CD47 and an anti-CD47 function-blocking mAb produced only a limited inhibition of adhesion of RAW264.7 cells. Co-immunoprecipitation experiments revealed that Mac-1 forms a complex with CD47 on the surface of RAW264.7 and Mac-1-expressing HEK293 cells. Finally, co-culturing of SIPRα- and Mac-1-expressing HEK293 cells resulted in cell fusion. Collectively, these data identify SIRPα as a novel counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion. DisclosuresNo relevant conflicts of interest to declare.

MO244CD47 BLOCKADE AMELIORATES AUTOIMMUNE VASCULITIS VIA THE EFFEROCYTOSIS OF NEUTROPHIL EXTRACELLULAR TRAPS*

Abstract Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by systemic necrotizing vasculitis in small vessels. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs) which represent a form of lytic cell death. The persistent NETs serve as autoantigens against ANCAs and cause organ damage in a vicious cycle. Considering dead cells are essentially cleared by phagocytic cells as a process of efferocytosis, why the NETs persist in tissue remains unclear. During efferocytosis, macrophages engulf apoptotic cells to prevent the leakage of intracellular components including toxic enzyme into the surrounding cells and these processes are regulated by the expression of CD47 as a “don’t eat me” signal. In this study, we hypothesized that ANCA-mediated NETs in AAV escape from efferocytosis via the up-regulation of CD47 and the persistent NETs amplify the disease. Method Human data: Human kidney biopsy specimens from patients with AAV and minor glomerular abnormality (MGA, as a case control) were subjected to immunohistochemistry (IHC) staining for CD47. In vitro: The expression of CD47 on neutrophils was evaluated by flow cytometry (FCM). Human neutrophils from healthy donor were treated with ANCA-IgGs from MPO-AAV patients or control IgGs. For the efferocytosis assay, macrophages were co-incubated with unstimulated, apoptotic, and ANCA-IgGs treated neutrophils in the presence of anti-CD47 monoclonal antibody (mAb) or a control antibody. The neutrophils were labeled with CFMDA cell tracker (fluorescent probe) and the efferocytosis was evaluated as neutrophil engulfed (CFMDA positive) macrophages using fluorescent microscopy. In vivo: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice (8-week-old age) were treated with intraperitoneal injection of anti-CD47 mAb or a control antibody every 5 days for two weeks. The severity of glomerulonephritis was assessed by the levels of serum creatinine, haematuria, mRNA expression of pro-inflammatory genes, and histopathological findings. To address the immune response against the CD47 blockade, the titre of MPO-ANCA and the number of splenic cell subset was assessed by ELISA and FCM analysis, respectively. Results Human data: The IHC analysis of human renal specimens revealed that the positive area of CD47 of AAV was greater than that of MGA. In particular, the CD47-overexpressed cells were seen in glomeruli with necrotic crescent formation. In vitro: Mean fluorescence intensity (MFI) of CD47 in ANCA-IgGs treated neutrophils (NETs) was significantly higher than that in control IgGs treated neutrophils (ANCA-IgG; 442±21.4 a.u. vs control IgG; 402±10 a.u., p&amp;lt;0.05). In efferocytosis assay, apoptotic neutrophils were engulfed by macrophages (efferocytosis rate/ apoptotic neutrophil; 20.5±3.8%, live neutrophils; 0.9±0.5%). The efferocytosis rate of ANCA-induced NETs significantly decreased compared to apoptotic neutrophil, but anti-CD47 mAb improved the efferocytosis of ANCA-NETs (efferocytosis rate/ anti-CD47 mAb; 19.1±4.2%, control antibody; 7.7±2.2%, p&amp;lt;0.05). In vivo: the renal histopathological severity score, serum creatinine level of AAV mice treated with anti-CD47 mAb decreased compared to that of AAV mice treated with a control antibody (anti-CD47 mAb; 0.96±0.30 vs control antibody; 0.61±0.32 mg/dL). Although there was no significant difference in the number of splenic cells between anti-CD47 and control antibody treated mice, CD47 blockade therapy significantly reduced serum MPO-ANCA titre (28.5±10.4 vs 45.2±14.5 μg/mL) and renal mRNA expression (IFNα, IFNγ, MCP-1 and perforin) of AAV mice. Conclusion ANCA-mediated NETs might escape from efferocytosis through up-regulation of CD47 and provoke necrotizing vasculitis. CD47 blockade could be a potential novel therapeutic strategy for AAV.

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis.

CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated in Escherichia coli support the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with Plasmodium infection

CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen.