Natasja Nielsen Viller

TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Anti-Tumor Activity and Minimal Erythrocyte Binding

Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47–SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication. Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails. Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies. Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068–79. ©2016 AACR.

TTI-621 (SIRPαFc), a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by multiple polarized macrophage subsets

Tumor-associated macrophages (TAMs) are heterogeneous and can adopt a spectrum of activation states between pro-inflammatory and pro-tumorigenic in response to the microenvironment. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein that blocks the CD47 “do-not-eat” signal, promotes tumor cell phagocytosis by IFN-γ-primed macrophages. To assess the impact of CD47 blockade on diverse types of macrophages that are found within the tumor microenvironment, six different polarized human macrophage subsets (M(-), M(IFN-γ), M(IFN-γ+LPS), M(IL-4), M(HAGG+IL-1β), M(IL-10 + TGFβ)) with distinct cell surface markers and cytokine profiles were generated. Blockade of CD47 using TTI-621 significantly increased phagocytosis of lymphoma cells by all macrophage subsets, with M(IFN-γ), M(IFN-γ+LPS) and M(IL-10 + TGFβ) macrophages having the highest phagocytic response. TTI-621-mediated phagocytosis involves macrophage expression of both the low- and high-affinity Fcγ receptors II (CD32) and I (CD64), respectively. Moreover, macrophages with lower phagocytic capabilities (M(-), M(IL-4), M(HAGG+IL-1β)) could readily be re-polarized into highly phagocytic macrophages using various cytokines or TLR agonists. In line with the in vitro study, we further demonstrate that TTI-621 can trigger phagocytosis of tumor cells by diverse subsets of isolated mouse TAMs ex vivo. These data suggest that TTI-621 may be efficacious in triggering the destruction of cancer cells by a diverse population of TAMs found in vivo and support possible combination approaches to augment the activity of CD47 blockade.

Abstract 2653: The anti-myeloma activity of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, is enhanced when combined with a proteasome inhibitor

CD47 is transmembrane glycoprotein that delivers an anti-phagocytic (“do not eat”) signal by binding to its receptor, signal-regulatory protein α (SIRPα), on the surface of macrophages. Many tumors, including multiple myeloma (MM), express high levels of CD47 as a means to exploit the CD47-SIRPα pathway and escape macrophage-mediated immune surveillance. TTI-621 (SIRPαFc) is a soluble recombinant fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG1. It is designed to promote anti-tumor responses by blocking the CD47 “do not eat” signal and engaging activating Fcγ receptors on macrophages. We have previously shown that TTI-621 enhances phagocytosis of malignant cells in vitro and exhibits anti-tumor activity in acute myeloid leukemia and B lymphoma xenograft models. In this study we investigated the anti-myeloma activity of TTI-621 as both a single agent and in combination with bortezomib or carfilzomib, two proteasome inhibitors that are approved for use in MM patients. The ability of TTI-621 to trigger macrophage phagocytosis of MM cells was assessed using a flow cytometry-based phagocytosis assay. Blockade of CD47 using TTI-621 effectively triggered macrophage-mediated phagocytosis of MM cells, and this anti-tumor effect was significantly enhanced by pre-treatment of MM cells with bortezomib or carfilzomib. In order to understand the molecular mechanism behind this enhanced phagocytic effect, we performed immunophenotyping of the tumor cells and observed an upregulation of pro-phagocytic “eat” signals on the tumor cell surface that potentially augment the phagocytic response. To investigate whether CD47 blockade in the context of proteasome inhibition translates to enhanced anti-tumor activity in vivo, we employed a MM xenograft model. NOD.SCID mice were subcutaneously engrafted with human MM cells followed by treatment with TTI-621, in the absence or presence of concurrent administration of a proteasome inhibitor. As monotherapies, both TTI-621 and the proteasome inhibitor reduced tumor burden relative to vehicle controls. Moreover, the combination of CD47 blockade and proteasome inhibition resulted in a greater reduction in tumor growth as compared to the two drugs alone. In conclusion, these data demonstrate that TTI-621 exhibits anti-myeloma activity that is further enhanced by combination with bortezomib or carfilzomib. TTI-621 monotherapy is currently being evaluated in a Phase 1b study in patients with MM and other hematological malignancies (NCT02663518). These data provide a rationale to evaluate a combination study of TTI-621 and a proteasome inhibitor in MM patients. Citation Format: Emma Linderoth, Simone Helke, Vivian Lee, Tapfuma Mutukura, Mark Wong, Gloria H. Lin, Lisa D. Johnson, Xinli Pang, Jeff Winston, Penka S. Petrova, Robert A. Uger, Natasja N. Viller. The anti-myeloma activity of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, is enhanced when combined with a proteasome inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2017-2653

Abstract B028: Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro

High expression of the CD47 “do-not-eat” signal is a mechanism commonly used by tumor cells to escape phagocytosis by macrophages. CD47 transmits an inhibitory signal upon binding to its receptor signal-regulatory protein α (SIRPα) on the surface of macrophages. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein, neutralizes the suppressive effects of CD47 and triggers macrophage-mediated phagocytosis of tumor cells in vitro, and effectively controls tumor growth in vivo. Using a human culture system, we investigated whether this increase in macrophage-mediated phagocytosis results in augmented T cell responses. To evaluate the T cell response to a model tumor antigen, Jurkat, a human leukemia cell line, was stably transfected with a construct containing the human cytomegalovirus phosphoprotein pp65 (CMV-Jurkat). Consistent with our previous studies, blockade of CD47 on CMV-Jurkat cells using TTI-621 led to a dramatic increase in tumor cell phagocytosis by primary macrophages derived from peripheral blood monocytes of healthy, HLA-A2+ donors. Using a high-affinity soluble TCR multimer that specifically recognizes an immunodominant epitope from pp65 complexed with HLA-A2, we found that TTI-621-mediated phagocytosis of CMV-Jurkat resulted in increased pp65 antigen presentation on the surface of macrophages. In cultures treated with a control Fc fragment, where macrophages only exhibited a low level of phagocytosis, no presentation of pp65 peptide could be detected. Moreover, mock-transfected tumor cells were efficiently phagocytosed in the presence of TTI-621, yet did not result in presentation of CMV pp65 peptide. To assess whether this increase in antigen presentation results in augmented T cell responses, macrophages from the phagocytosis assay were co-cultured with autologous CD8+ T cells for five days. We observed robust proliferation of CMV pp65-specific CD8+ T cells following co-culture with macrophages that had phagocytosed CMV-Jurkat in the presence of TTI-621 compared to control Fc treatment. In contrast, no proliferation of CMV pp65-specific CD8+ T cells occurred when macrophages had phagocytosed mock-transfected tumor cells, suggesting that the proliferation of CMV-specific CD8+ T cells is a tumor antigen-specific response. In addition, preliminary data indicate that primed CMV-specific CD8+ T cells are fully functional and are capable of exhibiting cytotoxicity against CMV-Jurkat. Collectively, our study demonstrates for the first time that in a human culture system, blockade of the CD47 “do not eat” signal results in increased phagocytosis, augmented tumor antigen presentation and enhanced anti-tumor CD8+ T cell responses. These results provide further support for the concept that CD47 lies at the intersection of the innate and adaptive immune systems. TTI-621, which is currently in a Phase I trial in patients with advanced hematological malignancies (NCT02663518), may thus be able to harness the anti-tumor activity of both macrophages and T cells. Citation Format: Natasja Nielsen Viller, Tran Truong, Emma Linderoth, Lisa D. Johnson, Stephane Viau, Gloria H. Y. Lin, Mark Wong, Xinli Pang, Penka S. Petrova, Robert A. Uger. Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B028.

Abstract 2345: SIRPαFc, a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by both classically (M1) and alternatively (M2) activated macrophages

Macrophages are characterized by their heterogeneity and plasticity in response to the microenvironment. Although macrophages have the capacity to phagocytose cancer cells that express pro-phagocytic signals, tumor cells often evade macrophage-mediated destruction by increased cell surface expression of CD47, which delivers an anti-phagocytic (“do not eat”) signal by binding the inhibitory signal-regulatory protein α (SIRPα) receptor on the surface of macrophages. We have previously shown that soluble SIRPα-Fc fusion protein (SIRPαFc) neutralizes the suppressive effects of CD47 and promotes macrophage-mediated phagocytosis of tumor cells both in vitro and in vivo. In an attempt to recapitulate the functional and phenotypic heterogeneity of tumor infiltrating macrophages, we have examined the ability of SIRPαFc to trigger phagocytosis of lymphoma cells by six distinctly polarized macrophage populations. We generated human monocyte-derived macrophages (MDMs) and polarized them into M0, M1, M2a, M2b and M2c subsets. These MDMs varied greatly in their expression of myeloid surface markers including CD14, CD11b, CD80, CD86, HLA-DR, CD206, CD200R and CD163; as well as in expression of the Fc gamma receptors (FcγRs) CD16, CD32 and CD64. Next, the ability of SIRPαFc to trigger MDM phagocytosis of lymphoma cells was examined using a flow cytometry-based assay. Blockade of CD47 on the tumor cells using SIRPαFc dramatically increased phagocytosis of tumor cells by all subsets, with M1 and M2c MDMs being superior at phagocytosis. Moreover, we found that M0, M2a and M2b MDMs, which exhibited slightly lower phagocytic capabilities, were remarkably plastic in nature and could readily be re-polarized into highly phagocytic MDMs using a variety of agents. This suggests that SIRPαFc will be efficacious in triggering the destruction of cancer cells by the diverse population of MDMs found in vivo. To further understand what drives the phagocytic capacity of polarized MDMs, we analyzed FcγR expression and observed a positive correlation between MDM expression of the high-affinity FcγRI (CD64) and phagocytic activity following SIRPαFc treatment. Moreover, re-polarization of M0, M2a and M2b MDMs resulted in upregulation of FcγRs and enhanced tumor cell phagocytosis. Finally, by individually blocking CD16, CD32 and CD64 on MDMs prior to the phagocytosis assay, we found that the low-affinity FcγRs CD16 and CD32 also contribute to SIRPαFc-mediated phagocytosis of lymphoma cells. In conclusion, SIRPαFc triggered phagocytosis of lymphoma cells by a diverse panel of polarized MDMs, which required MDM expression of FcγRs. These data support the evaluation of SIRPαFc in cancer patients and a clinical study of SIRPαFc in patients with lymphoma and other hematological malignancies is currently in progress. Citation Format: Gloria H. Y. Lin, Vien Chai, Vivian Lee, Karen Dodge, Tran Truong, Mark Wong, Lisa D. S. Johnson, Xinli Pang, Penka S. Petrova, Robert A. Uger, Natasja N. Viller. SIRPαFc, a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by both classically (M1) and alternatively (M2) activated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2345.