A differentiated CD47 therapeutic antibody recognizing a novel and sparing erythrocytes and platelets Taylor B. Guo, Zhengyi Wang, Lei Fang and Jingwu Zang I-MAB Biopharma, Inc., Shanghai, China

ABSTRACT Anti-tumor activity of TJ-C4 in Raji B cell lymphoma xenograft Tumor cells overexpress CD47 which engages signal-regulatory (SIRPa) on and liver cancer patient derived xenograft model (mf) to deliver a “do-not-eat” signal to avoid being phagocytosed. A 10 10 IgG ( 3 m g/ k g) Day 11 Day 14 Day 17 Blocking CD47 using SIRPa-Fc or anti-CD47 antibodies (Ab) has emerged as a Ref ( 3 m g/ k g) i.v injection of 1e6 CD47 mAb 10 9 TJ- C4 ( 3 m g/ k g) IgG promising strategy to neutralize CD47 and promote tumor eradication. However, Luci-Raji cells treatment i.p q2d 10 8 CD47 is also expressed on red blood cells (RBC) and platelets (PLT) which can act 10 7 Ref

10 6 Luminescenceintensity as a large Ab sink. Targeting CD47 also led to anemia and thrombocytopenia in 10 5 5 6 7 8 9 10 11 12 13 14 15 16 17 TJ-C4 animal studies and phase I trials, which is of serious concern. Here we report the Day 0 Day 5 Da y s p o s t t u m o r e n g r a f t m e n t discovery of a new CD47 Ab (TJ-C4) with a novel epitope that endows it with B IgG TJ-C4

enhanced phagocytic and RBC-sparing properties, thus differentiating itself from 2500 IgG ( 10 m g/ k g) 2500 2500

)

)

)

3 3 s.c injection of liver CD47 mAb Ref ( 10 m g/ k g) 3 2000 TJ- C4 ( 10 m g/ k g) 2000 2000 current CD47-targeting therapies. PDX samples treatment i.v q2d 1500 ~40% 1500 1500

1000 1000 1000 **

500 500 500

Tumorvolume(mm Tumor(mm volume MATERIALS AND METHODS Tumor(mm volume 0 0 0 Day 0 The day of tumor 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Antibody generation. A naïve human PBMC scFv phage library was subjected to several Days post t reat m ent Days post t reat m ent Days post t reat m ent growing to 100-300 mm3 rounds of solution panning for binding to biotinylated recombinant human CD47 TJ-C4 anti-CD47 mAb treatment completely eliminated the tumor cells in Raji B cell lymphoma xenograft model extracellular domain (ECD). All binders with unique VH and VL sequences were converted (A). TJ-C4 anti-CD47 mAb demonstrated significant efficacy in liver PDX model (B). to full IgG1 antibody and screened through a series of functional assays. A reference antibody 5F9 was prepared based on sequence information from Forty-seven patent and Differentiated RBC sparing properties of TJ-C4 was included in all experiments described below. A g/ml 15 3 0.03 0.003 IgG Affinity determination by BIAcore. mAbs were captured on CM5 sensor chip via anti- 100 30 10 1 0.3 0.1 0.01 Ref TJ-C4 IgG-Fc. The CD47 ECD monomeric were serially titrated from 50 nM down to 1.6 IgG 10 nM and injected for 3 min. The dissociation was monitored for 10 min. Data were fitted to

1:1 Langmuir binding model. Ref 5 Agglutination index Agglutination In vitro assay. Raji cells or primary AML patient cells were CFSE-labeled TJ-C4 0 1 3 0.1 0.3 10 30 0.01 0.03 100 and incubated with PBMC-derived mfs in the presence of anti-CD47 or control antibodies 0.003 g/ml for 3 h at 37 °C. Phagocytosis was measured by flow cytometry (FACS) by gating on B Unstained IgG TJ-C4 Ref CD14+ mfs and then assessing the percentages of CFSE+ cells. 100 In vitro human RBC binding. Purified RBCs were incubated with anti-CD47 or control 80 antibodies for 1 h at 4 °C followed by addition of fluorochrome-conjugated secondary Donor1 60 antibodies. Binding of CD47 antibodies to RBCs was analyzed by FACS. 40

20 In vivo antibody treatment of Raji xenograft (CDX) or liver cancer patient derived binding RBC of % xenograft (PDX) in mice. NSG mice were engrafted with 106 luciferase-labelled Raji cells 0 IgG Ref

by i.v. injection. Treatment of CD47 antibodies started from 5 days post engraftment at a TJ-C4 Donor2 dose of 10 mg/kg every other day i.p. (n=6/group). All mice were imaged in vivo for tumor Unstained growth by IVIS Lumina III imaging system. For PDX model, NOD-SCID mice were C

RBC Hemoglobin Reticulocytes Platelets /L)

3 6 16 9 800 800 PBS

/L) 9 transplanted s.c. with liver tumor samples. When engrafted tumors grew to 100-300 mm , /L) Ref (15 mg/kg)

12 14 5 600 600 TJ-C4 (15 mg/kg) mice were randomized to receive treatment of anti-CD47 or control antibodies at a dose of 12 4 10 400 400 10 mg/kg every other day i.v. (n=5/group). Tumor growth was monitored by tumor 8 3 200 200

6 Hemoglobin (g/dL) Hemoglobin

dimension measurements. (10 counts RBC 2 4 0 (10 counts Platelet 0

-8 -4 0 4 8 12 16 20 24 -8 -4 0 4 8 12 16 20 24 (10 counts Reticulocyte -8 -4 0 4 8 12 16 20 24 -8 -4 0 4 8 12 16 20 24 Hematology in non-human primates. Naive cynomolgus monkeys were given a single 1- Day Day Day Day hour i.v. infusion of 15 mg/kg of CD47 mAb or PBS control (3 animals/group) on Day 1. (A) TJ-C4 did not induce hemagglutination in vitro. (B) TJ-C4 minimally binds to human RBCs. (C) A single i.v. Peripheral blood was withdrawn at pre-dose Days 7 and 2, and at Days 3, 6, 10, 14 and 21 dose of TJ-C4 at 15 mg/kg did not alter hematological parameters throughout the study in contrast with a ref. Ab. post administration. A complete CBC panel was examined. Epitope binning of anti-CD47 antibodies. CD47 protein and first anti-CD47 mAb were An unique binding epitope of TJ-C4 in complex with CD47 pre-incubated followed by the addition of a second biotinylated anti-CD47 mAb. Antibodies A Competing with ref 1 B were deemed to occupy the same or overlapping epitope bin if the biotinylation signal was 1.5 TJ-C4 Ref 1 Description reduced. TJ-C4 Ref The structure of TJ-C4/CD47 1.0 complex adopts straighter Crystallization and structure determination. CD47-ECD/TJ-C4 complex was co- head to head orientation, OD 450 OD Structure of 0.5 unlike the structure of crystallized by combining 1 l protein solution with 1 l precipitation solution. mAb/CD47 ref/CD47 complex (PDB 5iwl) Crystallographic data were collected at beamline BL19U1, Shanghai Synchrotron 0.0 Ctrl. TJ-C4 Ref 1 presenting tilted head to Radiation Facility at a resolution of 2.7 Å. CD47 CD47 head orientation. Competing with ref 2 1.5 The epitope of TJ-C4 on RESULTS CD47 is conformationally 1.0 Binding discontinuous and includes a epitope on TNMEAQ loop (residues 26-

General properties of TJ-C4 450 OD 0.5 CD47 31), T34, E35, L74, and an LTR hinge (residues 101-103) 3 100 0.0 JCI 2016.126(7):2610 of CD47. A B Ctrl. TJ-C4 Ref 2 80 2 (A) Epitope binning of TJ-C4 with ref Ab 1 or 2. (B) Crystal structure and epitope regions of TJ-C4/CD47 complex. 60 Ref: 0.038 nM 40 Ref: 0.44 nM Ka = 5.33E+05 (1/Ms) OD 450 OD 1 TJ-C4: 0.045 nM TJ-C4: 0.71 nM Kd = 2.47E-04 (1/s) 20 KD = 4.6E-10 (M) CONCLUSIONS

0 cells Raji CD47+ of % 0 -4 -2 0 2 -2 -1 0 1 2 Concentration Concentration  TJ-C4 is an affinity-matured, sub-nanomolar, fully human, CD47 blocking mAb Log (nM) Log (nM) C D E IgG Ref TJ-C4 that strongly competes with SIRPa binding and promotes mf phagocytosis.  TJ-C4 demonstrated significant efficacy in the complete eradication of tumor 2.5 6 IgG Ref cells in the Raji CDX and reduction of tumor growth in the liver PDX model. 2.0 TJ-C4 4 Ref: 0.15 nM Binding 1.5 TJ-C4: 0.19 nM  TJ-C4 is endowed with a differentiated RBC-sparing property with minimal RBC- 2

1.0 binding and no agglutination in vitro, and no RBC-depletion in vivo.

Ratio 665/620 nm 665/620 Ratio Phagocytosis index Phagocytosis 0.5 0 AML  TJ-C4 binds a novel epitope on CD47 with a unique orientation distinct from all -3 -2 -1 0 1 2 + 2 2 10 0.4 10 0.4 10 Concentration 0.08 0.08 current CD47 antibodies. A series of patents have been filed around TJ-C4, its Log (nM) g/ml CFSE unique properties and the novel epitope. (A) Binding of CD47 mAb to soluble and cell surface CD47. (B) Affinity of TJ-C4 to CD47 by Biacore. (C)  TJ-C4 has good drug-like properties and the molecule is at CMC/preclinical Competition of CD47 binding with SIRPa by CD47 mAb. (D) Phagocytosis of Raji cells by human MΦ in response to CD47 mAb. (E) Binding and phagocytosis of primary AML cells by CD47 mAb at 10 g/ml. stage of development.