An Engineered Bi-Functional Fusion Protein (CD86-Fc-NKG2a) to Reverse NK Cell Tolerance of Malignant Cells

Casey Shuptrine, John Etchart, Zachary Opheim, Arpita Patel, Kyung Jin Yoo, Suresh de Silva, George Fromm, Josiah Hornblower, Taylor Schreiber Shattuck Labs, Inc. Austin, TX & Research Triangle Park, NC #P810 Contact: [email protected] Abstract In Vitro Characterization and Function Anti-Tumor Efficacy and Synergy with Checkpoint Blockade

Background: As clinical outcomes improve for immunotherapeutic A. HLA-E NKG2a/CD94 A. CT26 Growth in Balb/c Mice CT26 Survival in Balb/c Mice regimens based on PD-1/L1 blockade, the proportion of cancer patients 2000 100 who develop resistance to PD-1/L1 blockade will increase. Some resistance NK Cell 1500 mechanisms, including MHC-I and β2M downregulation, reflecting the Response Suppressed by NKG2a/CD94 Untreated *** nature of PD-1/L1 as a T-cell centric checkpoint. Recent clinical success Binding 1000 to HLA-E aQA-1 50 by groups targeting non-T cell immune cells has highlighted the potential mCD86-fc-NKG2a of enhancing macrophage or NK cell targeted immunotherapies for the CD28 500 Percent survival Tumor Volume (mm^3) Volume Tumor treatment of a wide variety of cancers. Future combinatorial approaches Tumor Cell NK Cell Tumor Cell NK Cell 0 0 will likely utilize agents targeting multiple different immune cells to 0 10 20 30 0 10 20 30 effectively arm both the adaptive and the innate immune systems. As a Days Following Engraftment Days Following Engraftment NKG2a ECD NKG2a/CD94 B. prime example of this, the anti-NKG2a monoclonal antibody EO771 Tumors in C57Bl/6 Mice at Day 34 B16F10 Tumors in C57Bl/6 Mice at Day 35 ✱✱✱ 1500 Monalizumab, has shown promising preclinical and clinical anti-cancer NKG2a Signal is 4000 B. Masked by Fusion ✱✱✱ effects, especially when combined with an ADCC competent antibody [1]. Construct, NK Cell Activated by ✱✱✱ CD86 ^3) 3000 Therefore, targeting non-classical innate immune checkpoints has the 1000 potential to synergize with currently approved cancer therapies. Here we CD28 2000 report the generation of a two-sided fusion protein, CD86-Fc-NKG2a, CD86 ECD 500 which was designed to provide competitive inhibition of the NK-centric Tumor Cell NK Cell 1000 Tumor Volume (mm^3) Volume Tumor HLA-E checkpoint, which providing co-stimulation to T cells via CD28. (mm Volume Tumor 0 0

Figure 3. MOA. (A) HLA-E can interact with NKG2a expressed on NK Vehicle aPD-1 Methods: Human and mouse variants of CD86-Fc-NKG2a were produced Vehicle aNKG2a/c and characterized using a variety of biochemical assays to determine the cells or T cells to suppress cytolytic cell activation. (B) CD86-Fc-NKG2a mCD86-fc-NKG2a correct molecular weight, subunit composition and binding affinity; can block the immune suppressive signals and simultaneously provide mCD86-fc-NKG2a NK/T cell co-stimulation. molecular assays to characterize in vitro cell binding, in vitro functional mCD86-fc-NKG2a + aPD-1 Figure 5. Anti-tumor efficacy (CT26, EO771, B16F10). 3 treatments of ARCs (300 activity, in vitro lysis capabilities; and anti-tumor efficacy in multiple Fc syngeneic model systems. A. CD86 NKG2a µg each) and/or antibodies (100 µg each) were given on days 7, 10, and 14 following tumor inoculations, when starting tumor volumes were equivalent. Monotherapies and Reducing: - + + - + + - + + combinations with (B) anti-PD1 (RMP1-14) are shown, along with survival. Results: The NKG2a domain bound recombinant and cell-expressed HLA- Deglycosylation: - - + - - + - - + E with high affinity. The CD86 domain bound recombinant CD28 and Boiled: + + + + + + + + + B. CTLA-4, with a higher affinity for CTLA-4. When tested head-to-head in a A. TIL Phenotyping Splenocyte Phenotyping therapeutic CT26 murine tumor model, mCD86-Fc-NKG2a demonstrated Perforin + Granzyme B + CD137+ Granzyme B+ IFN-γ + stronger anti-cancer activity compared to anti-NKG2a antibody controls. 4 0.3 20 5 5 The combination of the CD86-Fc-NKG2a with an anti-PD1 antibody was 4 4 3 15 effective in controlling tumor growth in immune competent mice. In vitro 0.2 cell lysis assays, with PBMCs, have further elucidated the effect of CD86- 3 3 10 2 2 2 Fc-NKG2a on NK cell mediated tumor cell death. αCD86 αFc αNKG2a 0.1 1 ** ** % of CD8+ Cells 5 % of CD8+ Cells % of CD8+ Cells % of CD8+ Cells 1 1 Fusion Protein Platform % of NK1.1 Cells ** 0.0 0 0 0 0 B. Capture: HLA-E-His Vehicle Vehicle Vehicle Detect: Anti-mCD86 Vehicle Vehicle 1.5 Fc Capture 2.5 CD28 Capture anti-NKG2a/c anti-NKG2a/c 2.0 anti-NKG2a/c anti-NKG2a/c anti-NKG2a/c EC50 = 0.442uM 2.0 EC50 = 10.18nM mCD86-Fc-NKG2a mCD86-Fc-NKG2a EC50 = 1.871 nM mCD86-Fc-NKG2a mCD86-Fc-NKG2a mCD86-Fc-NKG2a 1.0 1.5 1.5 1.0 0.5 1.0 Figure 5. Immune Phenotyping. (A) B16F10 tumors (day 7 post treatment) and Absorbance Absorbance Absorbance 0.5 0.5 Splenocytes (day 7 post treatment) collected from B16F10 bearing mice treated with 0.0 0.0 0.0 0 1 2 3 -2 0 2 mCD86-Fc-NKG2a and/antibodies as above, and phenotyped using flow cytometry. -2 0 2 Log [ARC] nM Log [ARC] nM Log [ARC] nM **p<0.01

C. Human CD86-Fc-NKG2a Binding to A431 Cells Mouse CD86-Fc-NKG2a Binding to WEHI3 Cells

2000 15000 Summary Detect: anti-hFc-PE Detect: anti-mCD86-APC 1500 10000 1000 - ARCs are a novel class of bi-functional biologics capable of targeting 5000 500 type-I and type-II membrane proteins; and can target all checkpoint Geometric Mean - PE Geometric Mean - APC molecules, the entire family of TNFR superfamily receptors, and NK 0 0 -2 0 2 -2 0 2 stimulatory molecules. D. Log [ARC] nM Log [ARC] nM Figure 1. ARC Platform. Agonist Redirected Checkpoints consist Caspase 3/7 Activity in A20 Cells Exposed to Splenocytes Caspase 3/7 Activity at 3.5 Hrs of a type I membrane protein extra-cellular domain (ECD), linked 100000 100000 ** - CD86-Fc-NKG2a can block HLA-E/NKG2a interactions on NK and T

to a type II ECD, via a central domain (i.e. Fc). Over 250 unique 80000 80000 cells, while simultaneously delivering a potent co-stimulatory signal mCD86-Fc-NKG2a constructs have been synthesized to date, and include mCD86-Fc-NKG2a 60000 Splenocytes Only 60000 through CD28, resulting in enhanced tumor cell killing and antitumor in combinations of all checkpoint molecules, the entire family of Splenocytes Only 40000 vivo activity. TNFSF ligands, and NK stimulatory molecules. 40000 No Splenocytes 20000 20000 Caspase 3/7 Activity

NKG2a CD86 Caspase 3/7 Activity 0 0 References 0:00:00 2:00:00 4:00:00 6:00:00 Time (Hrs) Fc Figure 4. ARC Characterization. (A) Western blot probing for all ARC 1. Andre´ P, Denis C, Soulas C, et al. Anti-NKG2A mAb is a Checkpoint Inhibitor that Promotes Anti-tumor domains. (B) Representative ELISA binding of HLA-E and CD28. (C) Immunity by Unleashing Both T and NK Cells. Cell. 2018 Dec 13; 175(7): 1731-1743.e.13. CD86-Fc-NKG2a binds to the cell surface of HLA-E (Qa-1 for mouse) positive tumor cells. (D) CD86-Fc-NKG2a enhances splenocyte Figure 2. Dimeric ARC Structure. Tertiary structure prediction of mediated killing of murine lymphoma cells in co-culture at 0.34 ug/ml. the CD86-Fc-NKG2a ARC. **p<0.01.