EZH2 Inhibition Compromises α4-1BB-Mediated Antitumor Efficacy by Reducing the Survival and Effector Programming of CD8+ T Cells

Enhancer of Zeste Homolog 2 (EZH2) inhibitors (EZH2i) are approved to treat certain cancer types. Previous studies have suggested the potential to combine EZH2i with immune checkpoint blockade targeting coinhibitory receptors like PD-(L)1 and CTLA-4, but whether it can also enhance the activity of agents targeting costimulatory receptors is not known. Here, we explore the combination between EZH2i and an agonist antibody targeting the T cell costimulatory receptor 4-1BB (α4-1BB). Our data show that EZH2i compromise the efficacy of α4-1BB in both CT26 colon carcinoma and in an in vivo protein immunization model. We link this to reduced effector survival and increased BIM expression in CD8+ T cells upon EZH2i treatment. These data support the requirement of EZH2 function in 4-1BB-mediated CD8+ T cell expansion and effector programming and emphasize the consideration that must be given when combining such antitumoral therapies.

780 ALTA-002, a SIRPα-directed TLR9 agonist antibody conjugate activates myeloid cells and promotes anti-tumor immunity

BackgroundNovel therapies engaging both innate and adaptive immune responses may engender durable anti-tumor immunity. Activation of toll-like receptor 9 (TLR9) by unmethylated CpG oligonucleotides promotes innate inflammatory responses and induces adaptive immunity. Immune cells expressing TLR9 encompass B cells and myeloid cells (including dendritic cells and plasmacytoid dendritic cells). Recently, several TLR9 agonists have demonstrated clinical benefit in patients with melanoma when administered intra-tumorally.1 Specifically designed for systemic administration, we developed a novel Toll-like Receptor Agonist Antibody Conjugate (TRAAC) molecule comprised of a differentiated TLR-9 agonist (T-CpG) conjugated to an antibody against SIRPα (ALTA-002). Signal regulatory protein α (SIRPα) is a myeloid inhibitory receptor that suppresses immune activation following binding of its ligand CD47. Blockade of CD47-SIRPα myeloid checkpoint pathway has been shown to promote myeloid-mediated anti-tumor functions leading to the induction of adaptive immunity.2 Additionally, SIRPα is highly expressed in various tumor types including renal cell carcinoma and melanoma.3 Here we present preclinical data demonstrating that ALTA-002 delivers T-CpG to SIRPα expressing myeloid cells, triggering TLR9 signaling, cell activation and immune modulation resulting in robust anti-tumor efficacy.MethodsIn vitro activity of ALTA-002 was evaluated using human PBMCs co-cultured in the presence of SIRPα positive and negative tumor cells. Anti-tumor efficacy of mouse ALTA-002 surrogate was evaluated in multiple syngeneic tumor models with varying immunogenicity profiles.ResultsIn vitro co-culture of human PBMC and SIRPα positive or negative tumor cells with ALTA-002 stimulates myeloid cells, leading to increased IRF7 induction, expression of co-stimulatory molecules, and cytokine secretion. In vitro treatment with ALTA-002 led to enhanced phagocytic engulfment by human monocyte-derived macrophages across multiple SIRPα positive and negative tumor cell lines. Following systemic delivery of a mouse ALTA-002 surrogate, durable anti-tumor activity was observed in both SIRPα positive and negative expressing tumors. ALTA-002 treated mice with eradicated tumors suppressed tumor growth upon tumor re-challenge, indicating tumor-specific immune memory.ConclusionsThese results demonstrate the unique properties of systemically administered ALTA-002, which integrates TLR9 activation and blockade of CD47-SIRPα interaction on myeloid cells to engender both innate and adaptive anti-tumor immune responses. These data support the development of ALTA-002 as an anti-cancer therapeutic for a variety of tumor malignancies.ReferencesHamid O, Ismail R, Puzanov I. Intratumoral Immunotherapy-Update 2019. Oncologist 2020;25(3):e423-e4382.Kuo T, Chen A, Harrabi O. Targeting the myeloid checkpoint receptor SIRPα potentiates innate and adaptive immune responses to promote anti-tumor activity. J Hematol Oncol 2020;13:160–1783.Yanagita T, Murata Y, Tanaka D. Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy. JCI Insight 2017;2(1):e89140.Ethics ApprovalIn vivo studies were approved by the Institutional Animal Care and Use Committee of Tallac Therapeutics.

Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.