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 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-SIRPα signaling pathway at the tumor site, we developed a so-called local inhibitory checkpoint monoclonal antibody (licMAB) by grafting the endogenous SIRPα 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-SIRPα axis to tumor antigen-expressing cells.
Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.
BackgroundDespite advances in the development of novel strategies against acute myeloid leukemia (AML), treatment options are limited and most patients relapse. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate the outgrowth of the disease, highlighting the need of targeting LSCs to improve patient survival. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen. CD47 is a ubiquitously expressed immune checkpoint upregulated on LSCs where it acts as an immune escape mechanism. CD47 transmits a ‘don’t eat me’ signal upon its interaction with the signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123+ AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity.Materials and MethodsThe bifunctional SIRPα-CD123 antibody was generated by fusing an extracellular domain of the SIRPα receptor, which functions as the CD47 blocking domain, to the CD123 antibody. The biological activity of the SIRPα-CD123 antibody was examined using AML-derived MOLM-13 cells, primary AML patient material and patient-derived xenografted (PDX) AML cells with NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice.ResultsThe SIRPα fusion improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody. The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity via NK cells. Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells. Furthermore, an improved cytotoxicity towards leukemia initiating AML PDX cells was observed with the SIRPα-CD123 antibody using luciferase bioluminescence in vivo imaging. With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in both allogenic and autologous setting. We were also able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity.ConclusionsThe bifunctional SIRPα-CD123 fusion antibodies target the CD123+CD47+ cells and stimulate their phagocytosis by blocking the inhibitory CD47 signal. The dual mode of action of the SIRPα-CD123 has the potential to deplete the AML LSCs through NK cell cytotoxicity and macrophage-mediated phagocytosis while restricting the CD47 related on-target off-leukemia toxicity.SupportH2020-EU grant agreement no 641549Disclosure InformationS. Tahk: None. S.M. Schmitt: None. B. Vick: None. C. Augsberger: None. L. Pascual Ponce: None. I. Jeremias: None. G. Wittmann: None. M. Subklewe: None. N.C. Fenn: None. K. Hopfner: None.
Background: 4-1BB (CD137; TNFRSF9) binds 4-1BBL, which triggers subsequent proliferation and activation of immune cells, T and NK cells in particular. We designed and generated PVR-4-1BBL, a hexameric protein of 4-1BBL fused to PVR (CD155), the major ligand for TIGIT that marks exhausted T cells. This fusion protein efficiently bind to its targets TIGIT and 4-1BB on cells as well as in soluble form.
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