Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

Proinflammatory polarization strongly reduces human macrophage in vitro phagocytosis of tumor cells in response to CD47 blockade.

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.

Development and evaluation of a human CD47/HER2 bispecific antibody for Trastuzumab-resistant breast cancer immunotherapy.

The treatment for trastuzumab-resistant breast cancer (BC) remains a challenge in clinical settings. It was known that CD47 is preferentially upregulated in HER2+ BC cells, which is correlated with drug resistance to trastuzumab. Here, we developed a novel anti-CD47/HER2 bispecific antibody (BsAb) against trastuzumab-resistant BC, named IMM2902. IMM2902 demonstrated high binding affinity, blocking activity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and internalization degradation effects against both trastuzumab-sensitive and trastuzumab-resistant BC cells in vitro. The in vivo experimental data indicated that IMM2902 was more effective than their respective controls in inhibiting tumor growth in a trastuzumab-sensitive BT474 mouse model, a trastuzumab-resistant HCC1954 mouse model, two trastuzumab-resistant patient-derived xenograft (PDX) mouse models and a cord blood (CB)-humanized HCC1954 mouse model. Through spatial transcriptome assays, multiplex immunofluorescence (mIFC) and in vitro assays, our findings provided evidence that IMM2902 effectively stimulates macrophages to generate C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, thereby facilitating the recruitment of T cells and NK cells to the tumor site. Moreover, IMM2902 demonstrated a high safety profile regarding anemia and non-specific cytokines release. Collectively, our results highlighted a novel therapeutic approach for the treatment of HER2+ BCs and this approach exhibits significant anti-tumor efficacy without causing off-target toxicity in trastuzumab-resistant BC cells.

Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation.

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.

TLR3 agonism augments CD47 inhibition in acute myeloid leukemia.

CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

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.

Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance.

Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome.

Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a "don't eat me" signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic "eat me" signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.

Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.

Macrophage LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Is Required for the Effect of CD47 Blockade on Efferocytosis and Atherogenesis-Brief Report.

OBJECTIVE: Antibody blockade of the "do not eat me" signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE-/-), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE-/- mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls (P<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. CONCLUSIONS: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE-/- model of atherosclerosis.

Combined strategies for effective cancer immunotherapy with a novel anti-CD47 monoclonal antibody.

CD47 is a widely expressed cell-surface protein that regulates phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, signal regulatory protein (SIRP)-α, which in turn inhibits phagocytosis. Several targeted CD47 therapeutic antibodies have been investigated clinically; however, how to improve its therapeutic efficacy remains unclear. Herein, we developed a CD47 blocking antibody, named IBI188, that could specifically block the CD47-SIRP-α axis, which transduces the "don't eat me" signal to macrophages. In vitro phagocytosis assays demonstrated the pro-phagocytosis ability of IBI188. Furthermore, several in vivo models were chosen to evaluate the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment.

Phase 1 study of anti-CD47 monoclonal antibody CC-90002 in patients with relapsed/refractory acute myeloid leukemia and high-risk myelodysplastic syndromes.

CC-90002 is an anti-CD47 antibody that inhibits CD47-SIRPα interaction and enables macrophage-mediated killing of tumor cells in hematological cancer cell lines. In this first clinical, phase 1, dose-escalation and -expansion study (CC-90002-AML-001; NCT02641002), we evaluated CC-90002 in patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS). CC-90002 was administered in escalating doses of 0.1-4.0 mg/kg, using a modified 3 + 3 design. Primary endpoints included dose-limiting toxicities (DLTs), non-tolerated dose (NTD), maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary endpoints included preliminary efficacy, pharmacokinetics, and presence/frequency of anti-drug antibodies (ADAs). Between March 2016 and July 2018, 28 patients were enrolled (24 with AML and 4 with MDS) at 6 sites across the USA. As of July 18, 2018, all patients had discontinued, mainly due to death or progressive disease. The most common treatment-emergent adverse events were diarrhea (46.4%), thrombocytopenia (39.3%), febrile neutropenia (35.7%), and aspartate aminotransferase increase (35.7%). Four patients experienced DLTs (1 patient had grade 4 disseminated intravascular coagulation and grade 5 cerebral hemorrhage, 1 had grade 3 purpura, 1 had grade 4 congestive cardiac failure and grade 5 acute respiratory failure, and another had grade 5 sepsis). The NTD and MTD were not reached. No objective responses occurred. CC-90002 serum exposure was dose-dependent. ADAs were present across all doses, and the proportion of ADA-positive patients in cycle 1 increased over time. Despite no unexpected safety findings, the CC-90002-AML-001 study was discontinued in dose escalation for lack of monotherapy activity and evidence of ADAs. However, as other anti-CD47 agents in clinical trials are showing promising early results for AML and MDS, understanding preclinical and clinical differences between individual agents in this class will be of high importance.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

CD47 blockade enhances therapeutic efficacy of cisplatin against lung carcinoma in a murine model.

Cisplatin (CDDP) is the first generation of platinum-based drug and is widely used to treat many cancers due to its potency. The present study aims to explore the effects of CDDP on lung carcinoma and its relationship with macrophage phagocytosis. In in vitro study, murine and human lung cancer cell lines were applied and treated with CDDP, CD47 antibody (aCD47), or CDDP plus aCD47. In in vivo study, a tumor xenograft animal model was treated with CDDP, aCD47, or CDDP plus aCD47. Real-time PCR was applied to determine the mRNA expressions. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and Immunofluorescent staining were applied to determine the protein expressions. Flow cytometry was applied to analyze cell apoptosis, phagocytosis, and specific cell populations. CDDP enhanced the expressions of CD47 in lung cancer cells. Interestingly, the blockage of CD47 enhanced the macrophages' phagocytic activity on the CDDP-treated tumor cells. The treatment of CDDP and aCD47 exhibited anti-tumor effects and prolonged the LLC tumor-bearing mice survival time. Mechanistic studies revealed that the treatment of CDDP and aCD47 regulated the phagocytic activity of macrophage, percentage of CD8+ T cells, and cytokines (tumor growth factor (TGF)-ß, interleukin (IL)12p70, and interferon (IFN)-γ) in the tumor-bearing model. CD47 blockade enhanced therapeutic efficacy of cisplatin against lung carcinoma in vivo and in vitro.

A bispecific antibody targeting GPC3 and CD47 induced enhanced antitumor efficacy against dual antigen-expressing HCC.

Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both in vitro and in vivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells in vitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.

Novel fully human anti-CD47 antibodies stimulate phagocytosis and promote elimination of AML cells.

Although most patients with acute myeloid leukemia (AML) enter remission after induction chemotherapy, the risk of relapse remains considerable. Therefore, some novel therapeutic strategies are still required. This study found that the overexpression of CD47 on AML cells was at least twofold more than that on normal bone marrow (NBM) cells in 81% (17/21) of the investigated patients; no patients had lower expression level of CD47 compared with healthy donors. The study also demonstrated that blocking the CD47/SIRPα (signal regulatory protein α) signal with the established novel fully human anti-CD47 monoclonal antibodies increased the phagocytosis of AML cells by macrophages in vitro. Furthermore, in vivo experiments showed that the novel fully human anti-CD47 monoclonal antibodies could significantly prolong the survival time of mice. Overall, the novel fully human anti-CD47 antibodies could block CD47/SIRPα interaction, increase macrophage-mediated phagocytosis, and enhance the elimination of AML cells.

CD47 blocking antibody accelerates hematoma clearance and alleviates hydrocephalus after experimental intraventricular hemorrhage.

Background CD47, a glycoprotein on red blood cell membranes, inhibits phagocytosis via interaction with signal regulatory protein α on phagocytes. Our previous research has demonstrated that blocking CD47 accelerates hematoma clearance and reduces brain injury after intracerebral hemorrhage. The current study investigated whether phagocytosis or erythrocyte CD47 impacts hematoma resolution and hydrocephalus development after intraventricular hemorrhage (IVH). Methods Adult (3-month-old) male Fischer 344 rats were intraventricularly injected with 200 µl autologous blood, mixed with either CD47 blocking antibody or isotype IgG, or 200 µl saline as control. In subgroups of CD47 blocking antibody treated rats, clodronate liposomes (to deplete microglia/monocyte-derived macrophages) or control liposomes were co-injected. Magnetic resonance imaging (MRI) was used to evaluate ventricular volume and intraventricular T2* lesion volume (estimating hematoma volume). The brains were harvested after 4 or 72 h for histology to evaluate phagocytosis. Results In adult male rats, CD47 blocking antibody alleviated hydrocephalus development by day 3. In addition, the CD47 blocking antibody reduced intraventricular T2* lesion and T2* non-hypointense lesion size after IVH through day 1 to day 3. Erythrophagocytosis was observed as soon as 4 h after IVH and was enhanced on day 3. Furthermore, intra-hematoma infiltration of CD68, heme oxygenase-1 and ferritin positive phagocytes were upregulated by CD47 blockade by day 3. Clodronate liposomes co-injection caused more severe hydrocephalus and weight loss. Conclusion Blocking CD47 in the hematoma accelerated hematoma clearance and alleviated hemolysis and hydrocephalus development after IVH, suggesting CD47 might be valuable in the future treatment for IVH.

CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma.

BACKGROUND: The Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin's lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically. METHODS: We conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin's lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose. RESULTS: A total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing. CONCLUSIONS: The macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; ClinicalTrials.gov number, NCT02953509 .).

Tumor-selective blockade of CD47 signaling with a CD47/PD-L1 bispecific antibody for enhanced anti-tumor activity and limited toxicity.

CD47, an immune checkpoint receptor frequently unregulated in various blood and solid tumors, interacts with ligand SIPRα on innate immune cells, and conveys a "do not eat me" signal to inhibit macrophage-mediated tumor phagocytosis. This makes CD47 a valuable target for cancer immunotherapy. However, the therapeutic utility of CD47-SIRPα blockade monoclonal antibodies is largely compromised due to significant red blood cell (RBCs) toxicities and fast target-mediated clearance as a result of extensive expression of CD47 on normal cells. To overcome these limitations and further improve therapeutic efficacy, we designed IBI322, a CD47/PD-L1 bispecific antibody which attenuated CD47 activity in monovalent binding and blocked PD-L1 activity in bivalent binding. IBI322 selectively bound to CD47+PD-L1+ tumor cells, effectively inhibited CD47-SIRPα signal and triggered strong tumor cell phagocytosis in vitro, but only with minimal impact on CD47 single positive cells such as human RBCs. In addition, as a dual blocker of innate and adaptive immune checkpoints, IBI322 effectively accumulated in PD-L1-positive tumors and demonstrated synergistic activity in inducing complete tumor regression in vivo. Furthermore, IBI322 showed only marginal RBCs depletion and was well tolerated in non-human primates (NHP) after repeated weekly injections, suggesting a sufficient therapeutic window in future clinical development of IBI322 for cancer treatment.

The effects of cell surface CD47 downregulation on ischaemia-reperfusion injury during pig liver transplantation.

This study aimed to investigate the effect of cell surface CD47 downregulation on ischaemia-reperfusion injury (IRI) during pig liver transplantation. Blood samples were collected from healthy miniature Bama pigs randomly and equally divided into CD47 antagonism (group A), without CD47 antagonism (group B) and a sham group (group C). Blood samples were collected from groups A and B at 0, 8 and 48 hours after establishment of the new liver through an indwelling tube in the right internal jugular vein. Blood samples were collected at the same time points after liver dissociation from group C. The expression of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA); CD47 expression was detected by Western blot; and liver function indices, including alanine aminotransferase (ALT) and aspartic transaminase (AST), were directly read by an automatic biochemical analyzer. The concentrations of both receptors in group A were significantly lower than groups B and C, at 8 and 48 hours after establishment of blood flow. At 8 and 48 hours in the new liver stage, the values of CD47 levels, ALT and AST in group A were significantly reduced compared to groups B and C (P < 0.05). The levels of CD47 in the three groups were consistent with the trends of the aforementioned observation indicators. The liver functions of the recipients were significantly improved by reducing the release of the inflammatory mediators. This study provides new ideas and intervention approaches for the treatment of IRI in liver transplantation.