Induction Of Phagocytosis Research Articles

Microfluidic-Generated Injectable Bulking Agents with Biocompatible Surfaces and Their Mid-term Outcomes in a Rat Model with Anal Sphincter Injury.

Bulking agents have gained attention as new, minimally invasive treatments for fecal incontinence. Various materials and surface treatment techniques have been extensively studied to ensure good biocompatibility and long-term stability. Despite significant improvements in biocompatibility, the nonuniform particle size of existing materials has led to other challenges, such as the induction of phagocytosis or reduction of injectability during in vivo tests. This study aimed to conduct a preclinical test of the midterm stability of bulking agents with newly formulated particles with uniform size. To this end, the particles were fabricated using microfluidics, resulting in a narrow size dispersity of less than 5% as the coefficient of variation, which is essentially distinct from conventional bulking agents. The microfluidic fabrication resulted in uniformly sized particles larger than the in vivo migratory limit of 80 μm and in a reduction in maximum injection pressure. Histological staining and microscopic observations confirmed proper positioning of the filler materials in vivo and a negligible immune response for up to 6 months, indicating successful midterm stability.

Minimal Bacterial Cell JCVI-syn3B as a Chassis to Investigate Interactions between Bacteria and Mammalian Cells.

Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides (Mmc) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes (MMSYN1-0179 to MMSYN1-0186, dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.

Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile’s innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile’s innate immune system.

Correlation of Serum Vitamin D Levels with the Severity of Diabetic Foot Ulcers in Dr. Mohammad Hoesin General Hospital, Palembang, Indonesia

Background: Vitamin D has a role in diabetes mellitus. Low levels of vitamin D are associated with the development of diabetic foot infections. Effects of vitamin D supplementation on wound healing, insulin resistance, inflammatory biomarkers, and oxidative stress are mediated by effects on induction of phagocytosis and bacterial killing by macrophages, suppression of interferon-gamma mediating macrophage activation, activation of insulin receptor expression, and downregulation from cytokines. This study aimed to determine the correlation between serum vitamin D levels and the severity of diabetic foot ulcers in Dr. Mohammad Hoesin General Hospital, Palembang, Indonesia.
 Methods: This study was an observational analytics study, where as many as 30 research subjects participated in this study. The study observed vitamin D levels and the severity of diabetic foot ulcers as assessed by the classification of the wound, ischemia, and foot infection (WIfI). Univariate and bivariate data analysis was carried out using SPSS.
 Results: The results of Sommer's correlation statistical test obtained a value of p = 0.001 (p <0.05) with a correlation strength of r = 0.564, which means sufficient strength (r = 0.40-0.70). This means that the higher the deficiency in serum Vitamin D levels, the higher the degree of WIfI classification experienced.
 Conclusion: There is a correlation between serum vitamin D levels and the severity of diabetic foot ulcers in Dr. Mohammad Hoesin General Hospital, Palembang, Indonesia.

Circadian metabolism regulates the macrophage inflammatory response

Abstract Macrophages are an integral part of the innate immune system and coordinate host defense to microbial infections, as well as shaping the remodeling response after tissue injury. Metabolism is now appreciated to be a powerful and pervasive regulator of the identity and function of macrophages. Upon exposure to microbial ligands, macrophage inflammatory activation and the associated induction of phagocytosis, inflammatory responses, and other host defense activities are supported by dynamic changes to cellular metabolism. Of note, metabolic activity is robustly regulated in a circadian fashion, with many metabolic processes displaying peak activity in one phase of the circadian cycle and trough activity in an antiphase manner. Here, we review recent findings suggesting that circadian metabolism influences macrophage activities and particularly the inflammatory response. First, we summarize macrophage activities known to display time-of-day–dependent variation and their mechanistic basis. Second, we review metabolic processes that have been shown to be rhythmically regulated in macrophages and discuss how such circadian metabolism affects or is likely to affect macrophage activities. Third, we discuss the concept of entrainment of the macrophage clock, and consider how loss of rhythmic regulation of macrophage activities may contribute to pathophysiological conditions like shift work, obesity, and aging. Finally, we propose that circadian metabolism can be used to understand the rationale and mechanistic basis of dynamic regulation of inflammatory responses during infection.

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of hematologic malignancies. 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 designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes. 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 effects on red blood cells (RBC) compared to benchmark anti-CD47 monoclonal antibodies (mAb), such as 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). Preclinical activity of ISB 1442 for the treatment of relapsed/refractory multiple myeloma (rrMM), including increased killing potency relative to daratumumab and magrolimab benchmarks as well as more favorable off tumor/on target specificity, was reported previously (Sammicheli et al. ASH 2021). ISB 1442 is currently in a Phase 1 clinical trial for rrMM. Beyond MM, CD38 and CD47 are expressed in other hematologic cancers, including acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL). In AML, unleashing tumor cell phagocytosis using magrolimab shows clinical benefit in combination with demethylating agents and BCL-2 inhibitors. T-ALL is characterized with high CD38 expression, and daratumumab in combination with chemotherapy is under investigation (NCT03384654) for this indication. ISB 1442 has the potential to treat AML and T-ALL patients with its first-in-class molecular attributes of co-targeting CD38 and CD47 by a single antibody with enhanced Fc effector functions. ISB 1442 induced potent phagocytosis of both AML and T-ALL cell lines in vitro. The induction of phagocytosis was more prominent in cell lines expressing high level of CD38. ISB 1442 also induced higher phagocytosis of both AML and T-ALL tumor cells as compared to daratumumab and anti-CD47 (5F9) in cell lines expressing low level of CD38 and CD47. In addition to phagocytosis, ISB 1442 induced potent ADCC of both AML and T-ALL cell lines. To characterize the complex mechanisms of action of ISB 1442 in a single assay, a multiple mode of action of killing (MMoAK) in vitro assay was established where macrophages and PBMCs are incubated with AML or T-ALL tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs (ADCC Mechanism of Action (MOA)), macrophages (ADCP MOA), and complement from human serum (CDC MOA). ISB 1442 induced higher killing of AML and T-ALL cell lines in MMoAK assays as compared to daratumumab or anti-CD47 (5F9). ISB 1442 induced potent killing of primary tumor samples from both AML and T-ALL patients in a heterologous assay using macrophages from healthy donors as effector cells, suggesting that target cancer cells do not have intrinsic mechanisms protecting them from the immune cell-mediated MOA of ISB 1442 killing . In patients' samples having detectable levels of myeloid or NK cells in bone marrow aspirates, ISB 1442 induced killing of tumor cells in the autologous setting in a direct killing assay ex vivo. In summary, these data support the clinical development of 1442 in AML and T-ALL. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to have antitumor activity in AML and T-ALL patients in a single antibody relative to anti-CD38 or anti-CD47 monoclonal antibodies as well as their combination.

SeV C Protein Plays a Role in Restricting Macrophage Phagocytosis by Limiting the Generation of Intracellular Double-Stranded RNA.

Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-β stimulation and neutralizing antibodies against IFN-β suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-β-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

Porcine iucA+ but rmpA- Klebsiella pneumoniae strains proliferate in blood of young piglets but are killed by IgM and complement dependent opsonophagocytosis when these piglets get older

Klebsiella (K.) pneumoniae causes different diseases in humans and animals including the life-threatening liver abscess syndrome and septicemia, respectively. However, host-pathogen interactions of K. pneumoniae in porcine blood have not been studied. We investigated the working hypothesis that only distinct K. pneumoniae strains have the capacity to survive in porcine blood and that this feature is associated with specific molecular markers such as sequence type, profile of siderophore genes and the regulator of the mucoid phenotype (rmp). Furthermore, we characterize the immune response in growing piglets leading to killing of an invasive K. pneumoniae strain.The veterinary isolates showed great diversity in sequence types and profile of siderophore genes. Porcine isolates were mainly positive for the aerobactin gene iucA but did not carry rmpA and this genotype was associated with proliferation in blood of 4-week-old piglets. Supernatants of an iucA+ but not an iucA- strain boosted growth in porcine serum. Between four and eight weeks of age, piglets showed a prominent increase of IgM binding to K. pneumoniae. Immunglobulin M and complement were crucial for killing of a serum-resistant iucA+ porcine K. pneumoniae strain at eight weeks of age. Flow cytometry analysis confirmed induction of phagocytosis and oxidative burst mediated by serum samples of 8-week-old piglets. Based on our in vitro findings we propose that many porcine iucA+ rmpA- K. pneumoniae strains have the ability to cause bacteremia in young piglets in association with aerobactin-mediated iron acquisition and that this phenotype is lost as specific IgM increases after weaning.

Mechanism of action and clinical results of immunotherapy for multiple myeloma

Treatment outcomes for multiple myeloma (MM) have improved due to the introduction of autologous stem cell transplantation and novel drugs. However, many patients develop resistance to existing therapies; hence, novel treatment strategies for these patients must be established. Therapeutic antibodies, including daratumumab and isatuximab targeting CD38 and elotuzumab targeting SLAMF7, have been introduced as immunotherapies for MM. These antibodies exert cytotoxic effects on myeloma cells through the activation of effectors such as natural killer cells and complement, and induction of phagocytosis by macrophages. Suppressed anti-tumor immunity may be related to acquisition of drug resistance by myeloma cells in patients with MM. It has been reported that the effect of therapeutic antibodies is through the stimulation of anti-tumor immunity. Thus, as each therapeutic antibody displays its own mechanism of action, therapy based on this mechanism of action should be introduced. Furthermore, chimeric antigen receptor (CAR) T-cell therapy, antibody drug conjugates (ADC), and bispecific antibodies (BsAbs) are gradually being introduced as novel immunotherapies for MM. CAR T-cells with high proliferation levels and persistence in recipients to improve the duration of therapeutic response are currently being developed.

HMBD004, a Novel Anti-CD47xCD33 Bispecific Antibody Displays Potent Anti-Tumor Effects in Pre-Clinical Models of AML

CD47 is the “don't-eat-me” signal and is ubiquitously expressed on normal cells where binding to SIRPalpha on macrophages inhibits phagocytosis. CD47 is commonly over-expressed in tumors where it correlates with immune evasion and poor prognosis. Blocking CD47-SIRPalpha interaction restores macrophage phagocytosis of tumor cells and anti-CD47 mAbs have shown anti-tumor efficacy in mouse models of solid tumors and hematological malignancies. Nevertheless, the widespread expression of CD47 decreases the bioavailability of anti-CD47 mAbs at the tumor due to antigen sink effects, and also risks off-target toxicities, most significantly from cross-linking of erythrocytes that show high expression of CD47. Therefore, increasing the specificity for binding CD47, and inhibiting the interaction with SIRPalpha, to tumor cells alone should increase the efficacy and safety of anti-CD47 therapy.CD33 is expressed on myeloid cells, but is highly over-expressed in 90% of AML patients, where it marks leukemic blast cells. The safety of CD33 as an antibody target for directing effector molecules to tumor cells has been convincingly demonstrated by the efficacy of gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate (ADC) that binds CD33 with high affinity, in the treatment of relapsed AML. However, ADCs are notable for their significant risk of toxicities, and gemtuzumab ozogmicin was withdrawn for this reason in 2010. Still, other companies continue development on next generation anti-CD33 ADCs, e.g. vadastuximab talirine.Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against CD47. Computational sequence and structural analyses predicted a highly specific surface epitope, with strong antigenicity and good safety profile, where antibody binding would inhibit binding to SIRPalpha. Subsequently, an in-house mouse immunization and screening strategy efficiently yielded a monoclonal antibody (mAb, 11a1) that bound with low nM (KD 5 nM) affinity and high specificity to the desired epitope on human CD47. In vitro, 11a1 demonstrated 100% inhibition of SIRPalpha binding at 1 nM, and potently induced phagocytosis of Raji cells by isolated macrophages.11a1 was subsequently humanized by CDR grafting and back-mutation, and the resulting humanized mAb maintained high affinity binding to CD47. Hummingbird Bioscience then constructed a bispecific anti-CD47xCD33 antibody (bsAb), based on a standard 1+1 IgG format, with a humanized anti-CD47 variable domain as an effector arm and the gemtuzumab variable domain as an anti-CD33 specificity arm. This bsAb (HMBD004) maintained the potent inhibition of SIRPalpha binding and induction of phagocytosis but, unlike many CD47 mAbs, caused negligible hemagglutination (i.e. cross-linking) of erythrocytes in vitro . Furthermore, HMBD004 showed preferential sorting of CD47+ CD33+ cells during FACS of mixed populations of CD47+ cells. Finally, in vivo CDX mouse models of AML were established by engrafting NCr nude mice with the CD47+ CD33+ AML cell line, HL-60, via subcutaneous implantation. Treatment of these mice with HMBD004 showed a significant decrease in tumor burden and increased PFS.Together, these data highlight the benefit of using CD33 binding to enhance the specificity, efficacy and safety of anti-CD47 antibody therapy in AML. Hummingbird Bioscience is currently advancing HMBD004 towards the clinic in 2018. DisclosuresNo relevant conflicts of interest to declare.

Potential therapeutic applications of phytoconstituents as immunomodulators: Pre-clinical and clinical evidences.

Autoimmune and infectious diseases are the major public health issues and have gained great attention in the last few years for the search of new agents with therapeutic benefits on the host immune functions. In recent years, natural products (NPs) have been studied broadly for their multi-targeted activities under pathological conditions. Interestingly, several attempts have been made to outline the immunomodulatory properties of NPs. Research on in-vitro and in-vivo models have shown the immunomodulatory activity of NPs, is due to their antiinflammatory property, induction of phagocytosis and immune cells stimulation activity. Moreover, studies on humans have suggested that phytomedicines reduce inflammation and could provide appropriate benefits either in single form or complex combinations with other agents preventing disease progression, subsequently enhancing the efficacy of treatment to combat multiple malignancies. However, the exact mechanism of immunomodulation is far from clear, warranting more detailed investigations on their effectiveness. Nevertheless, the reduction of inflammatory cascades is considered as a prime protective mechanism in a number of inflammation regulated autoimmune diseases. Altogether, this review will discuss the biological activities of plant-derived secondary metabolites, such as polyphenols, alkaloids, saponins, polysaccharides and so forth, against various diseases and their potential use as an immunomodulatory agent under pathological conditions.

The IFN-γ-IDO1-kynureine pathway-induced autophagy in cervical cancer cell promotes phagocytosis of macrophage.

Background: Cervical cancer is a common malignant disease in female patients accompanied by activation of autophagy in tumor cells. However, the exact regulatory factors of autophagy and its effects on the immune response remain unknown.Methods: The induction of autophagy in HeLa and SiHa cells treated with IFN-γ, tryptophan depletion, kynurenine and epacadostat was detected by western blot analysis and by an autophagy detection kit. Following co-culture with pre-treated HeLa and SiHa cells, U937 cells were analyzed by flow cytometry to detect CD80, CD86, CD163 and CD206 expression and the induction of phagocytosis.Results: IFN-γ caused a significant increase in the autophagy levels of HeLa and SiHa cells by promoting indoleamine-2,3-dioxygenase-1 (IDO1) expression. The induction of phagocytosis in HeLa and SiHa cells and the expression levels of CD80 and CD86 in U937 cells were increased significantly following treatment with recombinant human IFN-γ. This effect was associated with the induction of tumor cell autophagy. IFN-γ treatment and IDO1 overexpression promoted tryptophan depletion and kynurenine accumulation in cervical cancer cells. The latter was more potent in inducing autophagy of cervical cancer cells and promoting phagocytosis of macrophages. In vivo, IDO1 overexpression restricted tumor growth in C57 mice and enhanced the induction of phagocytosis in macrophages.Conclusions: IFN-γ promoted induction of autophagy and macrophage phagocytosis in cervical cancer cells possibly via IDO1 expression and kynurenine metabolism.

Current status and future prospects of immunotherapy for multiple myeloma

The introduction of autologous stem cell transplantation, proteasome inhibitors, and immunomodulatory drugs (IMiDs) has improved the treatment outcome for multiple myeloma (MM). However, many patients develop resistance to existing therapies, and novel treatment strategies for these patients must be established. Therapeutic antibodies including daratumumab targeting CD38 and elotuzumab targeting SLAMF7 have been introduced in the clinic as immunotherapies for MM. These antibodies exert cytotoxic effects on myeloma cells through the activation of effector cells such as natural killer cells and induction of phagocytosis by macrophages. Suppressed anti-tumor immunity may be related to acquisition of drug resistance by myeloma cells in patients with MM. It has been reported that IMiDs such as lenalidomide and pomalidomide enhance the effect of therapeutic antibodies through the stimulation of anti-tumor immunity. This stimulation of anti-tumor immunity is also observed in the effects of anti-CD38 antibodies, such as daratumumab and isatuximab. Therefore, it is expected that combination therapy with anti-CD38 antibodies and IMiDs may enhance anti-tumor immunity. Furthermore, chimeric antigen receptor (CAR) T cell therapy, antibody drug conjugates (ADC), and bispecific antibodies (BsAbs) are in the process of their introduction to the clinic as novel immunotherapies for MM.

The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host Immune Response.

Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.

Cell invasion by intracellular parasites - the many roads to infection.

Intracellular parasites from the genera Toxoplasma, Plasmodium, Trypanosoma, Leishmania and from the phylum Microsporidia are, respectively, the causative agents of toxoplasmosis, malaria, Chagas disease, leishmaniasis and microsporidiosis, illnesses that kill millions of people around the globe. Crossing the host cell plasma membrane (PM) is an obstacle these parasites must overcome to establish themselves intracellularly and so cause diseases. The mechanisms of cell invasion are quite diverse and include (1) formation of moving junctions that drive parasites into host cells, as for the protozoans Toxoplasma gondii and Plasmodium spp., (2) subversion of endocytic pathways used by the host cell to repair PM, as for Trypanosoma cruzi and Leishmania, (3) induction of phagocytosis as for Leishmania or (4) endocytosis of parasites induced by specialized structures, such as the polar tubes present in microsporidian species. Understanding the early steps of cell entry is essential for the development of vaccines and drugs for the prevention or treatment of these diseases, and thus enormous research efforts have been made to unveil their underlying biological mechanisms. This Review will focus on these mechanisms and the factors involved, with an emphasis on the recent insights into the cell biology of invasion by these pathogens.

Targeting CD47 as a Novel Immunotherapy for Multiple Myeloma.

Multiple myeloma (MM) remains to be incurable despite recent therapeutic advances. CD47, an immune checkpoint known as the “don’t eat me” signal, is highly expressed on the surface of various cancers, allowing cancer cells to send inhibitory signals to macrophages and impede phagocytosis and immune response. In this study, we hypothesized that blocking the “don’t eat me” signaling using an anti-CD47 monoclonal antibody will induce killing of MM cells. We report that CD47 expression was directly correlated with stage of the disease, from normal to MGUS to MM. Moreover, MM cells had remarkably higher CD47 expression than other cell populations in the bone marrow. These findings indicate that CD47 is specifically expressed on MM and can be used as a potential therapeutic target. Further, blocking of CD47 using an anti-CD47 antibody induced immediate activation of macrophages, which resulted in induction of phagocytosis and killing of MM cells in the 3D-tissue engineered bone marrow model, as early as 4 hours. These results suggest that macrophage checkpoint immunotherapy by blocking the CD47 “don’t eat me” signal is a novel and promising strategy for the treatment of MM, providing a basis for additional studies to validate these effects in vivo and in patients.

Targeting CD47/TNFAIP8 by miR-155 overcomes drug resistance and inhibits tumor growth through induction of phagocytosis and apoptosis in multiple myeloma.

The mechanisms of drug resistance in multiple myeloma (MM) are poorly understood. Here we show that CD47, an integrin-associated receptor, is significantly up-regulated in drug resistant myeloma cells in comparison with parental cells, and that high expression of CD47 detected by immunohistochemistry is associated with shorter progression-free and overall survivals in MM patients. We show that miR-155 is expressed at low levels in drug resistant myeloma cells and is a direct regulator of CD47 through its 3´UTR. Furthermore, low miR-155 levels are associated with advanced stages of disease. MiR-155 overexpression suppressed CD47 expression on myeloma cell surface, leading to induction of phagocytosis of myeloma cells by macrophages and inhibition of tumor growth. MiR-155 overexpression also re-sensitized drug-resistant myeloma cells to bortezomib leading to cell death through targeting TNFAIP8, a negative mediator of apoptosis in vitro and in vivo. Thus, miR-155 mimics may serve as a promising new therapeutic modality by promoting phagocytosis and inducing apoptosis in patients with drug-refractory/relapsed MM.

Experimental evidences that P21 protein controls Trypanosoma cruzi replication and modulates the pathogenesis of infection

P21 is a protein secreted by Trypanosoma cruzi (T. cruzi). Previous studies have shown a spectrum of biological activities performed by P21 such as induction of phagocytosis, leukocyte chemotaxis and inhibition of angiogenesis. However, the activity of P21 in T. cruzi infection remains unknown. Here, we reported the role of P21 in mice harboring late T. cruzi infection. Treatment with recombinant P21 protein (rP21) reduced parasite load and angiogenesis, and induced fibrosis in the cardiac tissue of infected mice. In addition, rP21 reduced the growth of epimastigotes, inhibited intracellular replication of amastigotes and modulated the parasite cell cycle. Our data suggest that P21 controls parasite replication in the host, supporting the survival of both parasite and host.

Abstract 540: AO-176, a normal cell sparing humanized anti-CD47 antibody

Abstract AO-176 is a next generation humanized anti-CD47 IgG2 that binds human and cynomolgus monkey CD47 equivalently. AO-176, like other CD47 antibodies, blocks the interaction of CD47 with SIRPα, inducing phagocytosis of tumor cells by activated macrophages. Unique to AO-176 is its ability to directly kill tumor cells via programmed cell death type III and immunogenic cell death in a cell autonomous (non-ADCC) manner. AO-176 also exhibits preferential binding to tumor vs. normal cells, a notable characteristic, as CD47 is expressed on many normal cells including red blood cells (RBC), platelets, T cells and endothelial cells. While other CD47 antibodies induce hematological toxicities such as anemia and thrombocytopenia in both primate models and in patients, AO-176 negligibly binds RBCs and platelets and even at high doses, minimally impacts hematology in cynomolgus monkey toxicology studies. AO-176 is currently being evaluated in phase 1 clinical trials for the treatment of solid tumors. In recent head to head experiments conducted with other CD47 antibodies, AO-176 bound all normal cells tested to a significantly lower degree. For example, unlike anti-CD47 antibodies such as Hu-5F9-G4, we observed negligible and minimal ex vivo binding to healthy human RBCs and platelets respectively and significantly lower binding of AO-176 to other hematologic and non-hematologic cells such as T cells and endothelial cells. Although AO-176 binds cynomolgus monkey RBCs to a slightly greater degree than human RBCs ex vivo, AO-176 versus comparative published Hu5F9-G4 findings has demonstrated dramatically reduced receptor occupancy when evaluated in monkey toxicology studies. We have also developed a clinic-ready receptor occupancy assay to measure AO-176 binding to circulating PBMCs in patients treated with AO-176 and to demonstrate minimal binding to CD47 on normal human cells. We have begun to evaluate mechanisms that may underlie the normal cell sparing effects of AO-176. Published studies have demonstrated that in addition to CD47 protein expression level, clustering and mobility of CD47 at the cell surface may impact binding to ligands, downstream signaling and subsequent cellular responses such as apoptosis. For example, avidity for SIRPα and clearance by macrophages is modulated by CD47 clustering that can be modulated by protein glycosylation, association with lipid rafts, cytoskeleton, integrins or other cis-acting factors. Here, we compare the distribution of CD47 on tumor and RBCs to evaluate receptor association with other cellular membrane components that may impact distribution on the cell surface. In summary, AO-176 is a next generation anti-CD47 antibody that, aside from induction of phagocytosis, possesses additional attributes that include direct tumor cell killing and preferential binding to tumor versus normal cells. These attributes of AO-176 differentiate it from other CD47 axis targeting agents currently in clinical evaluation. Citation Format: Prabir Chakraborty, Myriam N. Bouchlaka, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Robyn J. Puro, Vicki Sung, Daniel S. Pereira. AO-176, a normal cell sparing humanized anti-CD47 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 540.

CD47/SIRPα blocking enhances CD19/CD3-bispecific T cell engager antibody-mediated lysis of B cell malignancies

T cell immunotherapies are promising options in leukemia, among which the CD19/CD3-bispecific T cell engager antibody blinatumomab (MT103) has shown high response rates at very low doses in patients with lymphoma. However, the high CD47 expression in human lymphoma cells has limited the curative effects of blinatumomab other antibodies. Here we report the combined use of blinatumomab with a CD47-blocking antibody. CD47 antibodies preferentially enabled phagocytosis of non-Hodgkin lymphoma cells by both human and murine macrophages. Treatment of human non-Hodgkin lymphoma cell-engrafted mice with CD47 antibody and blinatumomab separately inhibited lymphoma partially, while combination treatment led to persistent control of lymphoma. These antibodies enhanced the therapeutic efficacy through mechanisms combining both innate and adaptive immune responses by induction of phagocytosis and T cell cytotoxicity. The combination strategy in this study might be applicable to many other cancers.