Polymorphisms in the human inhibitory signal-regulatory protein ? do not affect binding to its ligand CD47.
ABSTRACT: CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein ? (SIRP?), an inhibitory receptor on myeloid cells that gives a "don't-eat-me" signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRP? is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, but this is an important point for this interaction and other paired receptors being considered as targets for therapy. We show by x-ray crystallography that one human SIRP? allele differing in 13 amino acid residues has a very similar binding site and that several different alleles all bind CD47 with similar affinity as expected because the residues are mostly surface-exposed and distant from the binding site. A peptide from the binding site of CD47 has been reported to mimic the CD47 interaction with SIRP?, but we could find no binding. We discuss the possible pitfalls in determining the affinity of weak interactions and also speculate on how SIRP? polymorphisms may have been selected by pathogens and how this may also be true in other paired receptors such as the KIRs.
Project description:BACKGROUND:Accumulating preclinical data indicate that targeting the SIRP?/CD47 axis alone or in combination with existing targeted therapies or immune checkpoint inhibitors enhances tumor rejection. Although several CD47-targeting agents are currently in phase I clinical trials and demonstrate activity in combination therapy, high and frequent dosing was required and safety signals (acute anemia, thrombocytopenia) were recorded frequently as adverse events. Based on the restricted expression pattern of SIRP? we hypothesized that antibodies targeting SIRP? might avoid some of the concerns noted for CD47-targeting agents. METHODS:SIRP?-targeting antibodies were generated and characterized for binding to human SIRP? alleles and blockade of the interaction with CD47. Functional activity was established in vitro using human macrophages or neutrophils co-cultured with human Burkitt's lymphoma cell lines. The effect of SIRP? versus CD47 targeting on human T-cell activation was studied using an allogeneic mixed lymphocyte reaction and a Staphylococcus enterotoxin B-induced T-cell proliferation assay. Potential safety concerns of the selected SIRP?-targeting antibody were addressed in vitro using a hemagglutination assay and a whole blood cytokine release assay, and in vivo in a single-dose toxicity study in cynomolgus monkeys. RESULTS:The humanized monoclonal IgG2 antibody ADU-1805 binds to all known human SIRP? alleles, showing minimal binding to SIRP?1, while cross-reacting with SIRP?, and potently blocking the interaction of SIRP? with CD47. Reduced Fc?R binding proved critical to retaining its function towards phagocyte activation. In vitro characterization demonstrated that ADU-1805 promotes macrophage phagocytosis, with similar potency to anti-CD47 antibodies, and enhances neutrophil trogocytosis. Unlike CD47-targeting agents, ADU-1805 does not interfere with T-cell activation and is not expected to require frequent and extensive dosing due to the restricted expression of SIRP? to cells of the myeloid lineage. ADU-1805 is cross-reactive to cynomolgus monkey SIRP? and upon single-dose intravenous administration in these non-human primates (NHPs) did not show any signs of anemia, thrombocytopenia or other toxicities. CONCLUSIONS:Blocking the SIRP?-CD47 interaction via SIRP?, while similarly efficacious in vitro, differentiates ADU-1805 from CD47-targeting agents with respect to safety and absence of inhibition of T-cell activation. The data presented herein support further advancement of ADU-1805 towards clinical development.
Project description:CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRP?, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRP? engagement including humanized CD47 antibodies and a soluble SIRP? decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRP? or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRP? interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRP? interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRP? interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.
Project description:The function of the immune system in cancer initiation and progression has been widely examined. Notably, immunotherapy has become a promising approach for cancer treatment. CD47, a member of the immunoglobulin superfamily, plays an important role in the immune regulation of cancer by binding to SIRP?. Multiple studies have detected high CD47 expression on the surface of tumor cells, which indicates poor prognosis. Treatments that block the interaction of CD47 and SIRP? significantly suppress tumor growth and metastasis through diverse mechanisms, such as phagocytosis, antibody-dependent cellular cytotoxicity, and apoptosis. Recently, several studies have reported increased CD47 expression on different types of lymphoma cells, indicating that the CD47-SIRP? pathway can be used as a therapeutic target in lymphoma. This review focuses on the role of CD47-SIRP? in B-cell lymphoma and discusses promising therapeutic strategies targeting the CD47-SIRP? axis, which yield insights into the immunotherapy of B-cell lymphoma.
Project description:CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRP?. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRP? receptors, that block the SIRP?-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRP? and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRP? variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRP? binding. The SIRP?-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRP?-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.
Project description:Abstract CD47 belongs to immunoglobulin superfamily and is widely expressed on the surface of cell membrane, while another transmembrane protein SIRP? is restricted to the surface of macrophages, dendritic cells, and nerve cells. As a cell surface receptor and ligand, respectively, CD47 and SIRP? interact to regulate cell migration and phagocytic activity, and maintain immune homeostasis. In recent years, studies have found that immunoglobulin superfamily CD47 is overexpressed widely across tumor types, and CD47 plays an important role in suppressing phagocytes activity through binding to the transmembrane protein SIRP? in phagocytic cells. Therefore, targeting CD47 may be a novel strategy for cancer immunotherapy, and a variety of anti-CD47 antibodies have appeared, such as humanized 5F9 antibody, B6H12 antibody, ZF1 antibody, and so on. This review mainly describes the research history of CD47-SIRP? and focuses on macrophage-mediated CD47-SIRP? immunotherapy of tumors.
Project description:CD47 is overexpressed in various types of cancers and it can directly bind with SIRP?, which is mainly located on macrophages. The binding of CD47-SIRP? transmits a "don't eat me" signal, which can prevent cancer cells from immune clearance. Targeting the phagocytosis checkpoint of CD47-SIRP? axis has shown remarkable anticancer effect in preclinical and clinical research, which indicates the potential application of CD47-SIRP? blockade for cancer treatment. In this case, the comprehensive description of the regulation of CD47 in different types of cancer cells has significant implications for furthering our understanding of the role of CD47 in cancer. Based on the current reports, we summarized the regulatory factors, i.e., cytokines, oncogenes, microRNAs as well as enzymes, of CD47 expression in cancer cells. Accordingly, we also proposed several points needing further research, hoping to provide useful insights for the future investigation on the regulation of CD47 in cancers.
Project description:One common way to study human leucocytes and cancer cells in an experimental in vivo situation is to use mice that have been genetically engineered to lack an immune system and prevent human cell rejection. These mice lack CD132 and either RAG2 or the catalytic subunit of the DNA-dependent protein kinase, to make the mice deficient in lymphocytes and natural killer cells. The NOD mouse strain provides a better background for engraftment than other strains due to stronger engagement of the signal-regulatory protein-? (SIRP?) inhibitory receptor with human CD47 (hCD47) resulting in a 'don't-eat-me' signal. To determine the molecular parameters that determine this major functional effect in the NOD mouse we measured the affinity of hCD47 for SIRP? from various mouse strains. Human CD47 bound SIRP? from the NOD mouse with an affinity 65 times greater than SIRP? from other mouse strains. This is due mainly to the NOD SIRP? lacking two amino acids in domain 1 compared with other mouse strains. Remarkably the SIRP?(NOD) binds hCD47 with 10 times the affinity of the syngeneic hCD47/hSIRP? interaction. This affinity is outside the normal range for affinities for leucocyte surface protein interactions and raises questions as to what is the optimal affinity of this interaction for engraftment and what other xenogeneic interactions involved in homeostasis may also not be optimal.
Project description:CD47 is a widely expressed cell surface protein that functions as an immune checkpoint in cancer. When expressed by tumor cells, CD47 can bind SIRP? on myeloid cells, leading to suppression of tumor cell phagocytosis and other innate immune functions. CD47-SIRP? signaling has also been implicated in the suppression of adaptive antitumor responses, but the relevant cellular functions have yet to be elucidated. Therapeutic blockade of the CD47 pathway may stimulate antitumor immunity and improve cancer therapy. To this end, a novel CD47-blocking molecule, ALX148, was generated by fusing a modified SIRP? D1 domain to an inactive human IgG1 Fc. ALX148 binds CD47 from multiple species with high affinity, inhibits wild type SIRP? binding, and enhances phagocytosis of tumor cells by macrophages. ALX148 has no effect on normal human blood cells in vitro or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograft models, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies. Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with ALX148 induces multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation, macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune cells, causing increased T cell effector function, pro-inflammatory cytokine production, and a reduction in the number of suppressive cells within the tumor microenvironment. Taken together, these results show that ALX148 binds and blocks CD47 with high affinity, induces a broad antitumor immune response, and has a favorable safety profile.
Project description:Rapid clearance of adoptively transferred Cd47-null (Cd47(-/-)) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein ? (SIRP?) triggers inhibitory signaling through SIRP? cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47(-/-) mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirp?-KO (Sirp?(-/-)) mice, as well as Cd47(-/-) mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRP? axis dominantly controlling macrophage behavior. Sirp?(-/-) mice and Cd47(-/-) mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirp?(-/-) or Cd47(-/-) mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1?, and TNF?, but not IFN?, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirp? interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47(-/-) cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47(-/-) or Sirp?(-/-) mice.
Project description:CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRP?. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRP? blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1?mg/kg, whereas the effector function silent mAb IgG2? C47B222-(CHO) had minimal impact on RBC indices at 1 and 10?mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices.