Anti-SIRP? antibody immunotherapy enhances neutrophil and macrophage antitumor activity.
ABSTRACT: Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-? (SIRP?) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRP? with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRP? antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRP? blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.
Project description:Monoclonal antibodies are among the most promising therapeutic agents for treating cancer. Therapeutic cancer antibodies bind to tumor cells, turning them into targets for immune-mediated destruction. We show here that this antibody-mediated killing of tumor cells is limited by a mechanism involving the interaction between tumor cell-expressed CD47 and the inhibitory receptor signal regulatory protein-? (SIRP?) on myeloid cells. Mice that lack the SIRP? cytoplasmic tail, and hence its inhibitory signaling, display increased antibody-mediated elimination of melanoma cells in vivo. Moreover, interference with CD47-SIRP? interactions by CD47 knockdown or by antagonistic antibodies against CD47 or SIRP? significantly enhances the in vitro killing of trastuzumab-opsonized Her2/Neu-positive breast cancer cells by phagocytes. Finally, the response to trastuzumab therapy in breast cancer patients appears correlated to cancer cell CD47 expression. These findings demonstrate that CD47-SIRP? interactions participate in a homeostatic mechanism that restricts antibody-mediated killing of tumor cells. This provides a rational basis for targeting CD47-SIRP? interactions, using for instance the antagonistic antibodies against human SIRP? described herein, to potentiate the clinical effects of cancer therapeutic antibodies.
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, expressed on a variety of tumor cells, confers immune resistance by delivering an inhibitory "don't eat me" signal to phagocytic cells via its myeloid-specific receptor SIRP?. Recent studies have shown that blocking the CD47-SIRP? axis with CD47-directed antibodies or antibody-derivatives enhances phagocytosis and increases antitumor immune effects. However, CD47 expression on healthy cells creates an antigen sink and potential sites of toxicity, limiting the efficacy of CD47-directed therapies. In this study, we first characterized CD47 expression in Acute Myeloid Leukemia (AML) patients (n = 213) and found that CD47 is highly expressed on both AML bulk and stem cells irrespective of the disease state. Furthermore, to inhibit the CD47-SIRP? signaling pathway at the tumor site, we developed a so-called local inhibitory checkpoint monoclonal antibody (licMAB) by grafting the endogenous SIRP? domain to the N-terminus of the light chain of an antibody targeting CD33, a surface antigen expressed in AML. LicMABs selectively bind CD33-expressing cells even in the presence of a large CD33-negative CD47-positive antigen sink, stimulate phagocytosis of AML cells and eliminate AML cell lines and primary, patient-derived AML cells. Our findings qualify licMABs as a promising therapeutic approach to confine the benefit of disrupting the CD47-SIRP? axis to tumor antigen-expressing cells.
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:BACKGROUND:Signal regulatory protein ? (SIRP?) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47-SIRP? interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47-SIRP? interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRP?, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRP? polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRP?. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRP? antibody that binds human, cynomolgus monkey, and mouse SIRP? alleles with high affinity and blocks the interaction with CD47. METHODS:Human macrophages derived from donors with various SIRP? v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys. RESULTS:SIRP? blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg. CONCLUSIONS:The in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47-SIRP? axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.
Project description:Targeting the CD47-signal-regulatory protein ? (SIRP?) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRP? expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRP? may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRP? antagonists is restricted by polymorphisms within the CD47-binding domain of SIRP?, necessitating pan-allele reactive anti-SIRP? antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRP? regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRP? antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRP? reactivity from which approximately 70 antibodies with diverse SIRP? binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRP? antibodies bound to both human SIRP? v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRP? interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRP? antigen-binding fragments, each with unique epitopes, in complex with SIRP? (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRP? antibodies cross-react with cynomolgus SIRP? and various mouse SIRP? alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRP? antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; Fc?R: Fc? receptor; Ig: immunoglobulin; IND: investigational new drug; MDM?: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.
Project description:CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRP?, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRP? variants with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRP?. As high-affinity SIRP? monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.
Project description:The inhibitory immunoreceptor SIRP? is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRP? interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRP? on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRP? signaling (SIRP??CYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRP? signaling in atherosclerosis development. Bone marrow (SIRP??CYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRP? as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRP? as a potential therapeutic target in atherosclerosis.
Project description:The macrophage checkpoint is an anti-phagocytic interaction between signal regulatory protein alpha (SIRP?) on a macrophage and CD47 on all types of cells - ranging from blood cells to cancer cells. This interaction has emerged over the last decade as a potential co-target in cancer when combined with other anti-cancer agents, with antibodies against CD47 and SIRP? currently in preclinical and clinical development for a variety of hematological and solid malignancies. Monotherapy with CD47 blockade is ineffective in human clinical trials against many tumor types tested to date, except for rare cutaneous and peripheral lymphomas. In contrast, pre-clinical results show efficacy in multiple syngeneic mouse models of cancer, suggesting that many of these tumor models are more immunogenic and likely artificial compared to human tumors. However, combination therapies in humans of anti-CD47 with agents such as the anti-tumor antibody rituximab do show efficacy against liquid tumors (lymphoma) and are promising. Here, we review such trials as well as key interaction and structural features of CD47-SIRP?.
Project description:Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2-/- ?c-/- mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein ? (SIRP?) is a key modulator of the recipient's innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRP? to recipient CD47 and was modulated by the strength of the SIRP?-CD47 interaction. Therefore, sensing SIRP? polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.