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SIRP?-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells.

ABSTRACT: 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.

SUBMITTER: Ponce LP

PROVIDER: S-EPMC5355265 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells.

Ponce Laia Pascual LP Fenn Nadja C NC Moritz Nadine N Krupka Christina C Kozik Jan-Hendrik JH Lauber Kirsten K Subklewe Marion M Hopfner Karl-Peter KP

Oncotarget 20170201 7

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 SIRPα. Recent studies have shown that blocking the CD47-SIRPα 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 ther ...[more]

PMID: 28061465

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Project description:CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

Project description:BackgroundSignal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47-SIRPα 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-SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47.MethodsHuman macrophages derived from donors with various SIRPα 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.ResultsSIRPα 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.ConclusionsThe 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-SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.

Dataset Information

SIRP?-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells.

Publications

SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells.

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