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Isotype SRF231 Isotype SRF231 0.16 0.08 nm 0 0 200 400 600 800 Time(s) 1000 1200 1400 SRF231, a fully human, high affinity anti-CD47 antibody, exerts potent preclinical antitumor activity through engagement of the FcR, CD32a Marisa O. Peluso, Kshama A. Doshi, Caroline M. Armet, Li Zhang, Rachel W. O’Connor, Jamie Strand, Lilia Merbouche, Matthew Rausch, Jonathan A. Hill, Benjamin H. Lee, Pamela M. Holland, Vito J. Palombella, Alison M. Paterson Surface Oncology, Inc. Cambridge, MA, USA Presented at SITC2019 on November 9, 2019 Background SRF231 is a High Affinity CD47 Antibody with Slow Off Rate and No Agglutinating Properties SRF231 Exerts Antitumor Activity Through Phagocytosis and Cell Death Single-Dose Administration of SRF231 in the Raji Xenograft Model Leads to Sustained Antitumor Activity, Cytokine Induction, Macrophage Infiltration, and Tumor Necrosis SRF231-Induced Antitumor Activity is Dependent on Fc Engagement of FcγRIIa (CD32a) SRF231 Retains Antitumor Activity in Washout Conditions & Sub-Maximal Receptor Occupancy is Sufficient for Maximal Activity Conclusions • CD47 is a transmembrane protein that acts as a “Don’t Eat Me” signal to evade immune recognition • CD47 is overexpressed in multiple cancer subtypes and is associated with poor prognosis • Several anti-CD47 molecules designed to antagonize the CD47 axis are being tested in the clinic • Preclinical characteristics and antitumor mechanisms of the investigational agent SRF231, a fully human antibody targeting CD47, are described • SRF231 is a high affinity, CD47-targeting antibody that delivers an activating signal to myeloid cells via CD32a • SRF231 is a high affinity, CD47-targeting antibody with no agglutinating properties • SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a • SRF231 displays favorable preclinical characteristics regarding its RO/tumor exposure/efficacy relationship • SRF231 is currently being evaluated in a Phase I clinical trial [NCT03512340] in advanced solid tumors and lymphomas Figure 1. SRF231-hCD47 binding kinetics assessed by biolayer interferometry. (A) SRF231 immobilized using anti-hu Fc biosensor surface; CD47-his used as analyte. Binding traces (top) and calculated interaction parameters (bottom). (B) Tumor cell lines and primary cells stained with fluorescently labeled SRF231 or isotype control. MFI values obtained by flow cytometry and data reported as the MFI relative to isotype control. Figure 2. (A) RBCs from patients with hematologic cancers (n=3) incubated with CD47 mAbs overnight. Aggregation assessed by % doublet formation using flow cytometry. (B) Hu whole blood incubated with 100 μg/mL Ab for 1h. Smears imaged and analyzed qualitatively for evidence of RBC agglutination. (A and B) CD47 reference Abs included as positive (SRF4.2C11) and negative (2D3) controls. (C) Jurkat cells incubated with anti-CD47 mAbs for 72h and agglutination assessed by imaging. Anti-CD47 mAb (B6H12) was a positive control. Figure 3. (A-F) CFSE-Jurkat targets cultured with hu monocyte-derived macrophages (MoDM) at a 9:1 T:E ratio and incubated with mAb for 2 or 24h at 37°C. Cells stained with anti-CD14 and viability dye and analyzed by flow cytometry. (A) Phagocytosis denoted by % CFSE + tumor cells within CD14 + macrophages. (B) Tumor cell death denoted by % non-viable cells within non-phagocytosed target population. (C-F) Depletion of tumor cell population (arrow), relative to isotype at 2h (C and E) and 24h (D and F). Figure 4. Jurkat phagocytosis assays with hu MoDM (T:E 2:1) cocultured for 2h at 37°C. (A) Comparison of SRF231 (whole hIgG4) vs. SRF231 F(ab’) 2 . (B) Analysis of SRF231 Fc variants. (C) CD32a-specific blocking Ab (IV.3) abrogates SRF231-mediated phagocytosis. (D) IV.3 abrogates SRF231-mediated tumor cell death within the phagocytosis assay. Figure 5. (A and B) CFSE-Jurkat or Raji target cells co-cultured with macrophages in the presence of excess SRF231 or in "Washout" conditions where excess drug removed from target cells prior to coculture with hu MoDMs. Targets and macrophages co-cultured for 2h at 37°C. Phagocytosis reported as % CFSE + target cells within CD14 + macrophages via flow cytometry. (C) Correlation between % Max Phagocytosis vs. % Receptor Occupancy (RO) reveals that sub-maximal RO is sufficient for maximal phagocytosis induction. Figure 6. (A-C) SCID mice bearing 100-150 mm 3 Raji tumors treated once with SRF231 or isotype ip. (A) Mean tumor volume (n=8/grp). (B) Total hIgG4 plasma concentration (n=3/grp/timepoint). (C) SRF231 tumor exposure analyzed by total hIgG IHC staining (n=3/grp/timepoint). (D) SCID mice inoculated with luc -MM1.S cells iv. with BLI values 1.5x10 6 – 6.0x10 6 photon/second treated with SRF231 or isotype (n=4/grp). BLI imaging conducted 2-days following drug treatment. Figure 7. SCID mice bearing 100-150 mm 3 Raji tumors were treated once with SRF231 or isotype. (A) After 96h, tumors (n=3/grp) collected and tumor lysates analyzed for expression of mu IP-10 and MCP-1. (B-C) Raji tumor sections from isotype (168 h) and SRF231 (96 h) treated mice (n=3/grp) stained for F4/80 or H&E. (B) Representative F4/80 staining (left panel: 1X ,10X) and quantification (right panel) of % F4/80 + expression. (C) Necrotic tumor area (%) determined by quantitative image analysis of H&E stained tumor sections. P-values calculated using an unpaired t-test. CD47/SIRPα Blockade SIRPα SIRPα Signaling Phagocytic Function Cytokine Production Death Signals FcR Function SRF231 MCP-1 Fc Receptor (CD32α) Imbalance of “eat me/don’t eat me signals” Clusters of CD47 and IgGs enhance affinity for Fc receptor Recruitment of macrophages to tumor CD47 Clusters Active Cancer Cell Compromised Cancer Cells Tumor-Resident Macrophage (Scaffold) Tumor-Resident Macrophage Property SRF231 Isotype h I gG 4 (fully human) CD47 Binding A ffinity Kd (M) 6.2x10 -10 On-rate ka (1/Ms) 2.3x10 +05 Off- rate kdis (1/s) 1.7x10 -04 hIgG4 SRF231 2D3 SRF4.2C11 hIgG4 SRF231 mIgG1 B6H12 A A A B A B C D C A A B C B C D D E F B C B CFSE 10 3 10 3 0 0 10 4 10 4 10 5 10 3 0 10 4 10 5 10 5 10 3 0 10 4 10 5 CD14 CD14 CFSE CFSE 10 3 10 3 0 0 10 4 10 4 10 5 10 3 0 10 4 10 5 10 5 10 3 0 10 4 10 5 CD14 CD14 CFSE Efficacy (Raji) Efficacy A Cytokine Induction C Necrosis B Macrophage Infiltration Plasma PK (hIgG4 ELISA) Tumor Exposure (IgG IHC) IP-10 0 100 200 300 400 500 Tumor conc. (pg/mL) Isotype 300 μg SRF231 100 μg ** ** **: P<0.05 Isotype SRF231 1 x 1 x 10 x 10 x 0 5 10 15 F4/80 + (% ROI) Isotype 100 μg SRF231 100 μg ** **: P<0.05 0 5 10 15 20 25 Raji % necrotic tumor area Isotype 100 μg SRF231 100 μg *** ***: P<0.005 MCP-1 0.001 0.01 0.1 1 10 100 10000 20000 30000 40000 50000 SRF231 (µg/mL) Relative MFI PBMC CD34+ Bone Marrow Raji Jurkat U937 MM.1S Neutrophil HL-60 OPM-2 Tumor Normal 0.0001 0.01 1 100 10000 0 10 20 30 40 Antibody (μg/mL) % Doublets hIgG4 SRF231 2D3 SRF4.2C11 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 Phagocytosis (2h) Antibody (μg/mL) %CFSE + (of CD14 + ) SRF231 IgG4 0.001 0.01 0.1 1 10 100 0 10 20 30 40 Cell Death (2h) Antibody (μg/mL) %Cell Death (non-phagocytosed Jurkats) SRF231 IgG4 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 Jurkat Cell Depletion (2h) Antibody (μg/mL) %Non-Phagocytosed Targets SRF231 IgG4 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 Jurkat Cell Depletion (24h) Antibody (µg/mL) %Non-Phagocytosed Targets SRF231 IgG4 SRF231 F(ab’) 2 hIgG4 Antibody (µg/mL) %CFSE + (of CD14 + ) hIgG SRF231 (WT hIgG4) SRF231 (hIgG4; S288P, L235E) SRF231 (WT hIgG2) SRF231 (WT hIgG1) SRF231 (hIgG1; LLP-FES) SRF231 (hIgG1; N297A) 0 20 40 60 Antibody (µg/mL) %CFSE + (of CD14 + ) mIgG2b+SRF231 CD32a(IV.3) +SRF231 hIgG4 SRF231 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 0 5 10 15 20 Antibody (µg/mL) Dead (%non-phagocytosed) mIgG2b+SRF231 CD32a(IV.3) +SRF231 hIgG4 SRF231 Antibody (µg/mL) %CFSE + (of CD14 + ) 0.001 0.01 0.1 1 10 100 0 20 40 60 80 SRF231 hIgG4 hIgG4 Washout SRF231 Washout 0.001 0.01 0.1 1 10 100 0 20 40 60 Antibody (µg/mL) %CFSE + (of CD14 + ) hIgG4 SRF231 hIgG4 Washout SRF231 Washout 1 10 100 0 20 40 60 80 100 %Receptor Occupancy %Max Phagocytosis Raji Jurkat 0 10 20 30 0 500 1000 1500 2000 2500 Days after randomization Tumor volume (mm 3 ) Isotype control SRF231 30 μg SRF231 300 μg SRF231 100 μg SRF231 administration SRF231 10 μg 0.01 0.1 1 10 100 1000 Time (days) SRF231 plasma conc (µg/mL) 12 4 7 SRF231 300 μg SRF231 100 μg 21 SRF231 30 μg SRF231 10 μg 0 10 20 30 40 Time (days) hIgG4 positive (%) Isotype control SRF231 30 μg SRF231 100 μg SRF231 300 μg 12 4 7 21 SRF231 10 μg 0 1 2 0 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 (MM1.S-luc) Days BLI (photons/second) Isotype control SRF231 100 μg SRF231 30 μg SRF231 10 μg SRF231 3 μg Background BLI P272 Raji Jurkat 0.0001 0.001 0.01 0.1 1 10 100 0 20 40 60 SRF231 Antibody (µg/mL) %CFSE + (of CD14 + ) 0.001 0.01 0.1 1 10 100 0 20 40 60
