Background CD47 checkpoint inhibition has yielded promising results in clinical trials, particularly when combined with other immunotherapeutics. Therefore, we here combined CD47 checkpoint inhibition with checkpoint inhibition of HLA-G using Dual Signaling Protein 216 (DSP216). DSP216 is a new immunotherapeutic composed of sequences selected from the SIRPα and LILRB2 extracellular domains fused to human IgG1. The SIRPα domain of DSP216 binds to and blocks CD47, an important don’t eat me signal, frequently overexpressed on cancer cells. The LILRB2 domain of DSP216 binds to and blocks HLA-G, a pivotal regulator of immune tolerance during pregnancy in placenta that is upregulated in many cancers. HLA-G binds to inhibitory receptors LILRB1 (ILT2) and LILRB2 (ILT4) expressed on macrophages, neutrophils, dendritic, T, NK and B cells and hereby, suppresses anti-cancer immunity in a multifold manner. Blocking the HLA-G checkpoint reverses this immune suppression and e.g. shift macrophages from a tumor-supportive (M2) towards a tumor-suppressive (M1) phenotype. To avoid off target activity, DSP216 is designed to binds cells in an ‘AND- gate’ fashion, which means that DSP216 should not or minimally bind to cells that express only CD47 or HLA-G, but should bind strongly to cells that express CD47 AND HLA-G due to enhanced avidity. Here, we tested if DSP216 follows this unique binding behavior and if DSP216 can prevent HLA-G mediated polarization of M0 macrophages to M2 macrophages.

Materials and Methods Binding of DSP216 to HLA-G⁺ CD47⁺ cancer cells was tested by flow cytometry. The binding was blocked with CD47 and HLA-G blocking antibodies to test if the binding was specific. Undesired binding of DSP216 to red blood cells (RBCs) and PBMCs was tested by flow cytometry. The effect of DSP216 on the polarization of M0 macrophages co-cultured with HLA-G⁺ CD47⁺ cancer cells was tested by monitoring the expression of CD163, CD206 and HLA-DR by flow cytometry.

Results DSP216 bound dose-dependently to HLA-G⁺ CD47⁺ cancer cells and blocking the binding with HLA-G or CD47 blocking antibodies showed that binding was specific to CD47 and HLA-G. When DSP216 was mixed with RBCs and PBMCs, it didn’t bind to single positive CD47⁺ RBCs and bound very weakly to CD47⁺ PBMCs. These experiments show that there is indeed strong binding through enhanced avidity when both targets, HLA-G and CD47, are present and weak binding if CD47 but no HLA-G is expressed.In co-culture of M0 macrophages with HLA-G⁺ CD47⁺ cancer cells, DSP216 prevented HLA-G induced upregulation of the M2 specific markers CD163 and CD206 and induced expression of the M1 specific marker HLA-DR. As tumor associated macrophages (TAMs) with an M2 like phenotype play an important role in the creation of an immunosuppressive tumor microenvironment, this is a first indication that DSP216 can inverse an immune suppressive tumor microenvironment.

Conclusions DSP216 binds exclusively to HLA-G+ CD47+ cells and not to single positive cells, like RBCs and PBMCs. DSP216 prevents HLA-G mediated polarization of M0 macrophages to M2 macrophages and instead promotes polarization to M1 macrophages. DSP216 is a novel bifunctional Fc-fusion therapeutic that has the potential to reverse tumor suppressive signaling by tumor associated macrophages and unleash the anti-tumor activity of various cells from innate and adaptive immune response.

Disclosure Information L.J. Jacob: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; European Unions Horizon 2020, I-direct-itn.eu. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; KAHR medical. L. Tamir: A. Employment (full or part-time); Significant; KAHR medical. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; KAHR medical. M. Abdeen: A. Employment (full or part-time); Significant; KAHR medical. G.A. Huls: None. A. Chajut: A. Employment (full or part-time); Significant; KAHR medical. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; KAHR medical. E. Bremer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; KAHR medical. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; KAHR medical. F. Consultant/Advisory Board; Significant; KAHR medical.