HUMAN ANTI-CD47 ANTIBODIES AND USES THEREOF

FIELD OF THE INVENTION

[OOOll The present invention pertains to anti-CD47 antibodies.

BACKGROUND OF THE INVENTION

[0002] CD47, a cell membrane protein, belongs to the immunoglobulin superfamily containing an extracellular N-terminal Ig variable (IgV) domain with five transmembrane domains and a short C-terminal intracellular tail. Four alternatively spliced isoforms of CD47 differ in the length of their cytoplasmic tails have been identified so far [Brown, E. (2001 ) J Clin Invest

107(12): 1499-500; Reinhold, M.I., et al. (1995) J Cell Sci 108 (Part ll):34l9-25]. Recent studies demonstrated CD47 is a ligand of SIRP-D expressed on the phagocytic cells of immune system including macrophages, dendritic cells, and neutrophils [Tsai et al. (2008) J Cell Biol 180(5):989- 1003] The CD47-SIRP-D interactive axis has been shown to be involved in several cellular processes including apoptosis [Wang et al. (2016) J Dent Res 95(6):697-703], proliferation [Sick et al. (2011) Glia 59(2):308-l9; Kaur et al. (2013) Sci Rep 3: 1673], adhesion, and migration [Rebres et al. (2005) J Cell Physiol 205(2): 182-93; Sick et al. (2012) Br J Pharmacol 167(7): 1415-30] .

Additionally, studies also suggested CD47-SIRP-D interaction plays important role angiogenic processes as well [Zhang et al. (2015) Brain Res 1623:74-80; Chao et al. (2012) Curr Opin Immunol 24(2): 225-32]

[0003] CD47 is ubiquitously expressed by all human cells and has been shown to be over expressed in various tumor cells. Indeed, most human cancers studied to date, including acute myeloid leukemia (AML), chronic myeloid leukemia [Jaiswal et al. (2009) Cell l38(2):27l-85], acute lymphoblastic leukemia (ALL) [Chao et al. (2011) Cancer Res 71(4): 1374-84], non-Hodgkin’s lymphoma (NHL) [Chao et al. (2010) Cell 142(5):699-713], multiple myeloma (MM) [Rendtlew et al. (2007) Br J Haematol l38(6):756-60], bladder cancer, and other solid tumors (Chan et al. (2009) Proc Natl Acad Sci U SA 106(33): 14016-21), overexpress surface CD47, making CD47 an universal target to treat human cancers. High levels of CD47 were shown to allow cancer cells to avoid phagocytosis due to engage of the SIRP-a on phagocytes with CD47. The interaction between CD47 and SIRP-a, provides a“don't-eat-me” signal to phagocytes and prevents the phagocytic elimination of cancer cells and inhibits the T-cell immune response, subsequently

[Oldenborg et al. (2000) Science 288(5473):205l-4, Blazar et al. (2001 ) J Exp Med 194(4): 541-9.]. Since tumor cells overexpress CD47 to escape surveillance of host immune system, CD-47 targeted therapy aiming to restore the clearance of tumor cells were actively tested in clinical currently.

[0004] A number of therapeutics targeting the CD47-SIPR-D axis is currently under- developing including anti-CD47 antibodies, engineered decoy receptor, anti-SIRPa antibodies, and bispecific agents. Upon administration, anti-CD47 antibody or derivatives selectively inhibits the interaction between CD47 and SIRP-D. The blockage of CD47-SIRP-D interaction abrogates SIRP-D mediated inhibitory signals to phagocytes and results in, for example, macrophage activation and phagocytosis of tumor cells [Chao et al. (2011) Cancer Res 71(4): 1374-84; Chao et al. (2010) Cell 142(5):699-713; Majeti et al. (2009) Cell 138(2): 286-99, Chao et al. (2010) Sci TramlMed 2(63):63ra94] Furthermore, blocking CD47 transmitted signaling with antibodies also activates both an anti-tumor T-lymphocyte immune response and T cell-mediated killing of CD47-expressing tumor cells [Matozaki et al (2009) Trends Cell Biol l9(2):72-80; Latour et al. (2001) J Immunol l67(5):2547-54]

BRIEF SUMMARY OF THE INVENTION

[0005] Accordingly, the present invention provides new human CD47 blocking antibodies identified from a cancer patient antibody Fab library, hereinafter called as the anti-CD47 antibodies.

[0006] In one aspect, the present invention provides an isolated human anti-CD47 antibody, or an antigen-binding fragment thereof, comprising (a) a heavy chain variable (Vh) region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13; (b) an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14; or (c) a reformatted H chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, and 21.

[0007] In one example of the invention, the antibody or antigen-binding fragment thereof blocks the interaction of CD47 with signal-regulatory protein alpha (SIRP-D).

[0008] In another aspect, the invention provides IgG4-reformatted CD47 antibodies, which are prepared and obtained from the anti-CD47 antibodies.

[0009] In one example of the invention, the IgG4-reformatted CD47 blocking antibody were confirmed to be effective in treatment of a cancer since it is found in the invention that the IgG4- reformatted CD47 blocking antibody treated cancer cells, such as Jurkat-l or HL60 cells, and induced robust phagocytosis activity explicated by polarized THP-l macrophages as well as peripheral blood mononuclear cell (PBMC), but not antibody-dependent cell-mediated cytotoxicity (ADCC) and apoptosis of tested cancer cells, in vitro.

[OOIO] In one yet aspect, the present invention provides a method for treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the anti- CD47 antibodies.

[0011] In one embodiment, the broad spectrum of mammalian cancers to be treated is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, multiple myeloma, bladder cancer, breast cancer, head-and-neck squamous cell carcinoma, ovarian cancer, and colon cancer.

[0012] In one further aspect, the present invention provides a pharmaceutical composition comprising a therapeutically acceptable amount of the anti-CD47 antibodies and one or more pharmaceutically acceptable carriers. In particular, the pharmaceutical composition is effective in the treatment of a cancer.

[0013] In the examples of the present invention, the anti-CD47 antibody comprises a heavy chain variable (Vh) region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13.

[0014] In the examples of the present invention, the anti-CD47 antibody comprises a light chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2,

4, 6, 8, 10, 12, and 14.

[0015] In the examples of the present invention, the anti-CD47 antibody comprises a heavy chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, and 21.

[0016] In one yet aspect, the present invention provides a fragment binding to CD47, including a Fab, Fab’, F(ab) ₂ , F(ab’)2 or scFv of the antibody according the prevent invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0017] Fig. 1 shows the gel analysis of purified IgG4-reformatted CD47 antibodies; wherein the transiently HEK293F-expressed CD47 antibodies were purified from culture supernatant using Protein-G chromatography and purified antibody (~2 pg/lane) was PAGE-gel analyzed under reducing and non-reducing condition and visualized using Coomassie blue staining (M: Protein molecular weight marker; 1: CD47 antibody under non-reducing condition; 2: CD47 antibodies under reducing condition).

[0018] Fig. 2 provides the results of the binding of the purified antibodies to CD47-Fc protein (R&D Systems, USA) using ELISA assay. Fig. 2A shows the results of the direct ELISA binding assay of purified antibodies against CD47-Fc protein; wherein the purified antibodies and Hu5F9-G4 were shown to bind, specifically, to CD47-Fc protein, but not to SIRP-D. Fig. 2B shows the titration ELISA analysis of purified antibodies against CD47-Fc protein; wherein the purified antibodies and Hu5F9-G4 were shown to bind recombinant CD47 in a dose-dependent manner with variable binding activities among purified CD47 antibodies.

[0019] Fig. 3 shows the flow cytometry analysis of cell-surface CD47 binding using the anti- CD47 antibodies; wherein the anti-CD47 antibodies from library bound to cell-surface CD47 in a dose-dependent manner as analyzed using flow cytometry; and Jurkat-l cells (Fig. 3A) or HL-60 cells (Fig. 3B) were stained with the anti-CD47 antibodies, Hu5F9-G4 antibody, or purified human IgG4 isotype control and analyzed for surface binding by flow cytometry, and FITC-conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) was used for detection.

[0020] Fig. 4 shows the competition analysis of antibody binding between cell-surface CD47 and recombinant CD47-Fc protein using flow cytometer; wherein Jurkat-l cells were stained with the anti-CD47 antibodies (1 pg/ml) in the absence (blue color) or presence (red color) of 20 pg/ml CD47-Fc fusion proteins and analyzed for surface binding using flow cytometry; and FITC- conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) was used for detection. Hu5F9-G4 was included for comparison and purified human IgG4 was used as negative control (black). A representative flow analysis was shown using selected CD47 antibodies, Hu5F9-G4, or a human IgG4 isotype control.

[002l] Fig. 5 shows the blockage of CD47-SIRPa interaction by the anti-CD47 antibodies. Human CD47-Fc binding to SIRP-D Dpre-coated wells was detected by ELISA in the absence or presence of increasing concentrations of the anti-CD47 antibodies or Hu5F9-G4 as control. The anti- CD47 antibodies and Hu5F9-G4, with different degree of activities, could block the interaction of CD47 with SIRP-a.

[0022] Fig. 6 shows the anti-CD47 antibodies enhanced antibody-dependent phagocytosis activities of HL-60 or Jurkat-l cells using polarized THP-l macrophages or PBMC. The anti-CD47 antibodies, CwPlAl, CwP2E8, BrPlF3, BrPlFll, and Hu5F9-G4 were able to enhance, significantly, polarized THP-l- or PBMC-mediated phagocytosis against antibody -treated cells.

[0023] Fig. 7 shows the anti-CD47 antibodies did not induce ADCC. The ADCC Reporter

Bioassay Complete Kit (Promega, USA) with Raj i cells as target was used. Six to one ratio of effector-to-target cells were incubated with Hu5F9-G4, CwPlAl~lgG4, or BrPlFl l-IgG4 at different concentrations as indicated (x-axis) for 6 hours. CwPlAl-IgGl with a human IgGl Fc fragment and HuIgG4 were also included as controls.

[0024] Fig. 8 shows apoptosis assay of Jurkat-l or HL-60 cells in the presence of the anti- CD47 antibodies. Jurkat-l cells were incubated with 10 ug/ml of the anti-CD47 antibodies, Hu5F9- G4, or IgG4 isotype control. Apoptotic cells were identified by staining with Annexin-V and analyzed using flow cytometry. The anti-CD47 antibodies or Hu5F9-G4 treatment did not induce apoptosis of Jurkat-l or HL-60 cells.

[0025] Fig. 9 shows RBC surface antigen-binding assay (A) and RBC aggregation assay (B). Human anti-CD47 antibodies (CwPlAl, CwP2Fl2 and BrPlFl 1) bound to surface CD47 of RBCs in a dose-dependent manner. Further, CwPlAl and BrPlFl 1 did not induce hemagglutination of human RBCs.

[0026] Fig. 10 shows anti-tumor activities with human anti-CD47 antibodies, CwPlAl and BrPlFl 1, in HL-60 mouse xenograft model. Male SCID mice were injected s.c. in the right flank with 100 pl Matrigel plus 1.0 x 10 ⁷ HL-60 cells, and then isotype control antibody (400 pg/mouse), Hu5F9-G4, or human anti-CD47 antibodies, BrPlFl 1-G4 and CwPlAl in IgGl or IgG4 isotype (400 pg/mouse) were injected i.p. three times per week for three weeks. CwPlAl-Gl, CwPlAl -G4 and BrPlFl 1-G4 antibodies exhibited significant antitumor activity against the HL60 xenograft.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0028] As used herein, the term "antibody" refers to immunoglobulin molecules comprised of four polypeptide chains, including two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Antibodies may include intact immunoglobulins and the variants and portions of antibodies well known in the art. In the present invention, a fragment binding to CD47 is provided, including a Fab, Fab’, F(ab) ₂ , F(ab ) ₂ or scFv of the antibody according the prevent invention. [0029] As used herein, the term“Fc” is the tail region of an antibody that interacts with cell surface receptors. This property allows antibodies to activate the immune system. In contrast to Fab, the Fc regions of all antibodies in a class are the same for each species; they are constant rather than variable.

[0030] As used herein, the term“Fragment antigen-binding” or“Fab” refers to an antigen binding fragment on an antibody which binds to antigens. The Fab fragment is an antibody structure that still binds to antigens but is monovalent with no Fc portion. Fab is composed of one constant and one variable domain of each of the heavy and the light chain, wherein the variable domain contains the antigen-binding site, comprising a set of complementarity determining regions, at the amino terminal end of the monomer. An antibody digested by the enzyme papain yields two Fab fragments of about 50 kDa each and an Fc fragment.

[0031] The term“F(ab')2” as used herein refers to a fragment antibody that is generated by pepsin digestion of a whole IgG antibody to remove most of the Fc region while leaving intact some of the hinge region. The F(ab‘)2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 1 10 kDa.

[0032] As used herein, the term“single-chain variable fragment” or“scFv” refers to a fusion protein of the variable regions of the havevy and light chains of an antobody or an immunoblobulin, connected with a short linker peptide, such as a linker having 10 to 25 amino acids. This single chain variable fragment retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.

[0033] As used herein, the term“antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to an immune mechanism through which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells expressing tumor- or pathogen-derived antigens on their surface.

[0034] The present invention provides the anti-CD47 antibodies that specifically bind to human CD47 and block the interaction of CD47 with SIRPa. According to analyses as shown in Example 8 and Example 9, the anti-CD47 antibodies of the invention are capable to enhance macrophage-mediated phagocytosis and do not induce apoptosis.

[0035] According to the invention, the anti-CD47 antibody binds specifically to human and mouse CD47.

[0036] According to the invention, the anti-CD47 antibody comprises a heavy chain variable (Vh) region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13.

[0037] According to the invention, the anti-CD47 antibody comprises an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

[0038] According to the invention, the anti-CD47 antibody is one containing the amino acids selected from the group consisting of :

a Vh region having an amino acid sequence of SEQ ID NO: 1, and an L chain region having an amino acid sequence of SEQ ID NO: 2;

a Vh region having an amino acid sequence of SEQ ID NO: 3, and an L chain region having an amino acid sequence of SEQ ID NO: 4;

a Vh region having an amino acid sequence of SEQ ID NO: 5, and an L chain region having an amino acid sequence of SEQ ID NO: 6;

a Vh region having an amino acid sequence of SEQ ID NO: 7, and an L chain region having an amino acid sequence of SEQ ID NO: 8;

a Vh region having an amino acid sequence of SEQ ID NO: 9, and an L chain region having an amino acid sequence of SEQ ID NO: 10;

a Vh region having an amino acid sequence of SEQ ID NO: 11, and an L chain region having an amino acid sequence of SEQ ID NO: 12; and

a Vh region having an amino acid sequence of SEQ ID NO: 13, and an L chain region having an amino acid sequence of SEQ ID NO: 14.

[0039] In addition, the anti-CD47 antibody of the present invention comprises a reformatted H chain having the amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, and 21.

[0040] The amino acid sequences are listed in the following tables:

[0041] Table 1. Amino Acid Sequences of the anti-CD47 Fabs binders identified from library screening.

Table 2. Amino Acid Sequences of the heavy chains of the IgG4-reformatted CD47 Antibodies.

[0042] According to the invention, the anti-CD47 antibody is one containing the amino acids selected from the group consisting of :

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

15, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 2;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

16, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 4;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

17, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 6;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

18, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 8;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

19, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 10;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

20, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 12; and

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO:

21, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO: 14.

[0043] Throughout this document, reference is made to the following representative anti- CD47 antibodies of the invention. CwPlAl represents an antibody having an H chain region corresponding to SEQ ID NO: 15, and an L chain region corresponding to SEQ ID NO: 2. CwPlC4 represents an antibody having an H chain region corresponding to SEQ ID NO: 16, and an L chain region corresponding to SEQ ID NO: 4. CwP2E8 represents an antibody having an H chain region corresponding to SEQ ID NO: 17, and an L chain region corresponding to SEQ ID NO: 6. CwP2F8 represents an antibody having an H chain region corresponding to SEQ ID NO: 18, and an L chain region corresponding to SEQ ID NO: 8. CwP2Fl2 represents an antibody having an H chain region corresponding to SEQ ID NO: 19, and an L chain region corresponding to SEQ ID NO: 10. BrPlF3 represents an antibody having an H chain region corresponding to SEQ ID NO: 20, and an L chain region corresponding to SEQ ID NO: 12. BrPlFll represents an antibody having an H chain region corresponding to SEQ ID NO: 21, and an L chain region corresponding to SEQ ID NO: 14. [0044] In the invention, the anti-CD47 antibodies promote macrophage-mediated phagocytosis of a CD47-expressing cell.

[0045] In the invention, the anti-CD47 antibodies do not induce either an antibody-dependent cell-mediated cytotoxicity or an apoptosis activity.

[0046] In the invention, the antibody may comprise an IgG isotype selected from the group consisting of IgG 1 isotype and IgG2 isotype.

[0047] In the invention, the antibody may be prepared as an IgG4-reformatted CD47 antibody.

[0048] In one embodiment, the present invention provides a method for treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the anti- CD47 antibodies.

[0049] In one embodiment, the broad spectrum of mammalian cancers to be treated is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, multiple myeloma, bladder cancer, breast cancer, head-and-neck squamous cell carcinoma, ovarian cancer, and colon cancer.

[0050] In further embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically acceptable amount of the anti-CD47 antibodies and one or more pharmaceutically acceptable carriers. In particular, the pharmaceutical composition is effective in the treatment of a cancer.

[0051] In one embodiment of the present invention, TRB800-01, a research antibody Fab library, was screened with commercially available recombinant CD47 protein (R&D Systems, USA) to isolate fully human antibodies against CD47. TRB800-01 was constructed using blood samples collected from clinically diagnosed patients containing 16c oral, 22x esophageal, and 15c prostate cancers (IRB No. VGHKS17-CT11-13) with a capacity of ~l x 10 ¹⁰ .

[0052] In another embodiment of the present invention, the titer of the eluted phages increased significantly through panning, indicating enrichment in CD47-specific binders.

Randomly selected 384 clones from the 3 ^rd -round panning was further confirmed using direct ELISA analysis against CD47 protein, and positive binders with OD450 greater than 0.5 were selected for sequencing analysis. Seven unique clusters were identified and were chosen to be reformatted into full-length human IgG4 for expression using HEK293 cells in the FreeStyle™ 293-F system

(Invitrogen, USA), and for further characterizations.

[0053] The present invention is further illustrated by the following examples, which should be construed as illustrative only and not in any way limit the remainder of the present invention. Without further illustration, it is believed that those skilled in the art will be able to make the best use of the present invention based on the description herein.

[0054] Example 1 Human antibody Fab library screening against recombinant CD47 protein

[0055] Human antibody Fab library was constructed from selected cancer patient bloods with diversity ~l0 ¹⁰ . Briefly, the identification of CD47 antibodies from the antibody library was done as described below. ELISA well was coated with 500 ng/well (in PBS) of human CD47-Fc protein (R&D Systems, USA) for l6hr at 4°C. After blocking, -10 ¹⁰ phages were added and incubated at room temperature for lhr on shaker. Unbound phages were removed and the wells were washed 10 times with PBS containing 0.05% Tween-20 (PBS-T). After washing, bound phages were eluted by adding 100 pl of 0.1M TEA solution (Sigma, USA) and neutralized with 50 mΐ of 1M Tris-HCl, pH7.4. Eluted phages were then used to infect log phase TG-l cells, plated on 1.5% agar plates containing antibiotic and glucose, and incubated at 30°C for overnight. On the next morning, scrapped bacteria (30 Oϋboo) were used for phage rescuing using Ml 3 Hyperphages (PROGEN, Germany). After centrifugation, rescued phages were PEG-pelleted and reconstituted in PBS, titrated, and used for the next round of panning. The panning processes were repeated for two additional times. After final round of selection, individual clone was selected, grown, and induced with 1 mM IPTG for Fab productions. The supernatant containing expressed Fabs from each individual clone were analyzed by ELISA against CD47-Fc protein for binder identification.

[00561 Example 2 CD47 binder sequencing analysis and reformatting

[0057] ELISA positive binders with OD450 >0.5 were selected for sequencing analysis.

Heavy chains of identified CD47 binders were engineered onto a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions such as ADCC and CDC. The amino acid sequences of the light chain and heavy chain variable (Vh) region, and the reformatted full length heavy chain sequences (in IgG4 isotype) of identified CD47 binders were listed in Table 1 and Table 2. Individual reformatted CD47 antibody heavy chain and corresponding light chain were then sub cloned onto separate pCI-neo vector (Promega, USA) for pairwise HEK293 transfection, expression, purification, and characterization.

[0058] Example 3 Antibody expression and purification

[0059] HEK 293 cells and FreeStyle™ 293 Expression Medium (Invitrogen, USA) were used for recombinant antibody production. Transient transfection was done according to the

manufacturer’s instruction (Invitrogen, USA). To purify antibodies, the culture supernatant was applied to Pierce protein G agarose resin (ThermoFisher, USA) and were dialyzed against PBS. Purified antibody was analyzed by SDS-PAGE on 4-12% Bolt Bis-Tris plus gel (Invitrogen, USA) under reducing or non-reducing condition, and visualized by Coomassie brilliant blue staining (Invitrogen, USA).

[0060] Results from Fig. 1 showed all IgG4-reformatted anti-CD47 antibodies were properly expressed and formed. And also, greater than 90% purity of each individual antibody was obtained using Protein-G chromatography.

[006l] Example 4 CD47 binding study

[0062] To examine and confirm the binding specificity, purified anti-CD47 antibodies were firstly assayed using direct ELISA assay. Briefly, 96-well ELISA plate (Nunc, Denmark) was coated with 1 mg/ml of recombinant CD47-Fc or SIRP-a (R& D Systems, USA) for 16 hours at 4°C.

CD47 or SIRP-a pre-coated wells were blocked with 5% non-fat milk in PBS at room temperature for lhr, and then lOOul of 0.5 pg/ml human anti-CD47 antibodies were added and incubated for 1 hour at room temperature with shaking. After incubation and washes, 100 pl of 1 : 2500 diluted HRP -conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) were added to each well and incubated for 1 hour at room temperature. After final washing, bound antibodies were detected using TMB solution (Invitrogen, USA). The reaction was stopped by adding 50 mΐ of 1M HC1 and read the absorbance of each well at OD450 nm.

[0063] Results in Fig. 2A indicated all candidate antibodies and Hu5F9-G4, though binding activity varies among clones, were shown to be specific against recombinant CD47 with no cross reactivity against SIRP-D protein (R&D Systems, USA). Hu5F9-G4, a humanized antibody targeting CD47 with extraordinary bioactivity in vitro and in vivo, was used as a positive control for comparison.

[0064] Titration ELISA assay was used to examine the binding activity of these candidate antibodies. For titration ELISA assay, wells were coated with 1 pg/ml of recombinant CD47-Fc for l6hr at 4°C. CD47 precoated wells were blocked with 5% non-fat milk in PBS at room temperature for lhr, and then lOOul 1 :3 serial diluted anti-CD47 antibody starting from 30 nM were added and incubated for 1 hour at room temperature with shaking. After incubation and washes, lOOul of 1 :2500 diluted HRP -conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) were added to each well and incubated for 1 hour at room temperature. After final washing, bound antibodies were detected using TMB solution (Invitrogen, USA). The reaction was stopped with 1M HC1 and read the absorbance of each well at OD450 nm. EC50, the antibody concentration required for half of max absorbance, was then calculated by GraphPad Software for each tested CD47 antibody.

[0065] As shown in Fig. 2B, all tested Anti-CD47 antibodies bound to recombinant CD47 in a dose-dependent manner, and the measured EC50 for Hu5F9-G4, CwPlAl, CwPlC4, CwP2E8, CwP2F8, CwP2Fl2, BrPlF3, and BrPlFll were 0.1442 nM, 0.1835 nM, 0.2271 nM, 0.4694 nM, 0.3264 nM, 0.4413 nM, 10.1 nM, and 1.21 nM, respectively.

[0066] Example 5 Cell surface antigen-binding assay

[0067] In one embodiment of the invention, Cell surface CD47 binding by flow cytometer was used to examine if discrepancies between recombinant and natural CD47 protein. The binding activity with increasing concentrations of the Anti-CD47 antibodies against cell-surface CD47 on Jurkat-l or HL-60 cells (BCRC, Taiwan). For cell surface antigen-binding assay, 1 x 10 ⁵ of Jurkat-l cells (BCRC, Taiwan) were incubated with 0.001, 0.01, 0.1, 1, 10, or 100 pl/ml of anti-CD47 antibody in 100 pL at 4°C for l5min. After incubation, the cells were washed with ice-cold staining (IX PBS+2% FBS+0.05% NaNh) buffer for three times, followed by incubation with FITC- conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA). Lastly, cells were washed and analyzed using BD Accuri™ C6 Plus flow cytometer (BD Biosciences, USA). Mean fluorescence intensity (MFI) values were ploted.

[0068] The results collected from Fig. 3 suggested a dose-dependent manner on cell surface binding of the anti-CD47 antibodies and Hu5F9-G4, but not the human IgG4 isotype control (BioLegend, USA), on both tested cell lines.

[0069] Example 6 Competition assay of CD47 binding using flow cytometry

[0070] In another embodiment of the invention, the binding specificity of the anti-CD47 antibodies against natural CD47 was performed by antibody binding competition assay using flow cytometry with or without excess amount of recombinant CD47-Fc protein was performed. 1 xlO ⁵ of Jurkat-l cells were incubated with 1 pg/ml of human anti-CD47 antibody in the absence or presence of lOpg/ml recombinant CD47-Fc protein at 4°C for 15 minutes. After that, the cells were washed and stained with FITC conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) before analyzed using BD Accuri™ C6 Plus (BD Biosciences, USA) flow cytometer. [0071] Fig. 4 shows that surface CD47 binding activity of the anti-CD47 antibodies and Hu5F9-G4 were shown to be blocked by the presence of 10 pg/ml of CD47-Fc fusion proteins, indicating the specific binding of the all tested antibodies against CD47

[0072] Example 7 The CD47-SIRPa interaction blocking assay

[0073] The CD47-SIRPa interaction blocking activities of recombinant anti-CD47 antibodies were tested using ELISA assay. ELISA wells (Nunc, Denmark) were coated with 1 pg/ml of recombinant human His-tagged SIRP-a protein (R&D Systems, USA) for l6hr at 4°C. After blocking, 1 pg/ml of recombinant CD47-Fc protein and 3-fold serially diluted of anti-CD47 antibody starting from 30 nM were added and incubated at room temperature for lhr. After incubation, wells were washed and incubated with HRP-conjugated goat anti-human Fc antibody (1:2500 dilution, Jackson ImmunoResearch, USA) for lhr. After final washing, the bound CD47-Fc proteins were detected with TMB substrate. The reaction was stopped by adding 1M HC1 and OD450 readings were obtained. IC50, the antibody concentration required to inhibit half of max absorbance, was calculated by GraphPad Software for each tested CD47 antibody.

[0074] The anti-CD47 antibodies as shown in Fig. 5 were able to block the interaction between recombinant human CD47 and recombinant human SIRP-a. The calculated IC50S for Hu5F9-G4, CwPlAl, CwPlC4, CwP2E8, CwP2F8 and CwP2Fl2 is 0.063 nM, 0.053 nM, 0.155 nM, 0.556 nM, 0.068 nM and 0.063 nM, respectively. Results from this study indicated CwPlAl, CwP2F8 and CwP2Fl2 have similar or even beter blocking activities than that of Hu5F9-G4.

[0075] Example 8 In vitro antibody-mediated phagocytosis assay

[0076] Whether the CD47 and SIRP-a interaction blocking antibodies could induce macrophage-mediated phagocytosis of CD47 ⁺ cancer cells were then examined. In vitro phagocytosis assays were done as described below briefly. The differentiation of THP-l (BCRC, Taiwan) monocytes to macrophages was induced by PMA, LPS (Sigma, USA) and IFN-D□ (R&D Systems, USA). The polarized THP-l cells were then stained with CellTracker Red (CTR, Life Technology, USA) before use. CFSE-labeled human cells, Jurkat-l or HL-60 cells (BCRC, Taiwan), in the presence of 5 pg/ml isotype control antibody, Hu5F9-G4, or human anti-CD47 antibodies (IgG4) were incubated with polarized THP-l macrophages for 3hr at 37°C. Cells were washed before analyzed by flow cytometry to determine the phagocytic index (number of cells ingested per 100 macrophages). PBMC-derived macrophages were induced by 50 ng/ml rhM-CSF (Peprotech,

USA) treatment for 7 days and confirmed by anti-CDl4 antibody (Abeam, USA) staining. CFSE- labeled HL-60 cells in the presence of 10 pg/ml isotype control antibody, Hu5F9-G4, or human anti- CD47 antibodies were incubated with macrophages for 2hr at 37°C. Then, the cells were analyzed by flow cytometry to determine the phagocytic index.

[0077] The HL-60 AML and Jurkat-l ALL cells were used as target cells. Polarized THP-l cells as well as human peripheral blood-derived macrophages were used to examine the phagocytotic activity. Phagocytic activity was measured by flow cytometry. As shown in Fig. 6A-C, human anti- CD47 antibodies and Hu5F9-G4 significantly promoted phagocytosis of tumor cells by polarized THP-l and PBMC-derived macrophages.

[0078] Example 9 ADCC assay

[0079] To evaluate if human CD47 antibodies also enable ADCC in addition to phagocytosis, CwPlAl-IgG4, BrPlFll-IgG4, and Hu5F9-G4 with IgG4 scaffold were examined for their ADCC activities using an ADCC reporter bioassay. In this study, CwPlAl-IgGl with a human IgGl Fc fragment and HuIgG4 were also included as controls, respectively. The ADCC Reporter Bioassay Complete Kit (Promega, USA) with Raji cells as target was used according to the manufacturer’s instructions. Six to one ratio of effector-to-target cells were incubated with HuigG4, Hu5F9-G4, CwPlAi-lgG! , CwPJ AI-IgG4, or BrPIFl 1-1 gG4 at different concentrations as indicated (x-axis) for 6 hours. After incubation, Bio-Glo luciferase assay reagent was added and luminescence was read. Samples were run in duplicate and averages of the duplicate values were graphed with error bars displaying standard error of the mean (SEM) [0080] Results shown in Fig. 7 indicated CwPlAl-IgGl is able to induce an ADCC activity in a dose-dependent manner. In contrast, CwPlAl-IgG4, BrPlFl l-IgG4, Hu5F9-G4, and HuIgG4 did not induce ADCC. Collectively, the mechanism of action of TRB human CD47 antibodies, CwPlAl-IgG4 and BrPlFl l-IgG4, do not induce either ADCC or apoptosis, but rather activation of antibody-dependent cellular phagocytosis.

[0081] Example 10 Apoptosis assay

[0082] Apoptosis assay was carried out to examine if the binding of the anti-CD47 antibodies to CD47 directly induce apoptosis of cancer cells. Human cells, Jurkat-l, HL-60, or Raji cells, were resuspended at lxlO ⁶ cells/ml in RPMI-1640 medium containing 10% FBS. 10 pg/ml isotype control, 2D3, Hu5F9-G4, or human anti-CD47 antibodies (IgG4) were added and the cells were incubated at 37°C for 3hr. In the case of HL-60 cell line, staurosporine was used as a positive control. Apoptotic cells were identified by staining with Annexin-V and PI according to the manufacturer’s instructions (BD Biosciences, USA) and analyzed using flow cytometry.

[0083] As shown in Fig. 8A, it is found that as similar to the isotype control, the tested anti- CD47 antibodies and Hu5F9-G4 did not induce apoptosis of Jurkat-l cells. Fig. 8B showed that human anti-CD47 antibodies, unlike treatment with staurosporine, did not induce apoptosis of HL-60 cells. Similar results also obtained using Raji cells for assay (data not shown). Collectively, our results indicated anti-human CD47 antibodies induce phagocytosis rather than apoptosis activity.

[0084] The anti-CD47 antibodies with therapeutic potentials in this invention are currently tested in xenograft animal models for their efficacies against acute myeloid leukemia and solid tumors.

[0085] Example 11 RBC surface antigen-binding assay and RBC aggregation assay

[0086] For RBC surface antigen-binding assay, cell surface CD47 binding activity was measured using flow cytometer. Human red blood cells were isolated by Ficoll-Paque Plus (Sigma, Sweden) using density gradient centrifugation. Purified human RBCs were incubated with 1, 10 and l00pg/ml of anti-CD47 antibodies (IgG4) in IOOmI at 4°C for l5min. After incubation, the cells were washed with ice-cold staining buffer (IX PBS+2% FBS+0.05% NaN ₃ ) for three times, followed by incubation with FITC-conjugated goat anti-human Fc antibody (Jackson

ImmunoResearch, USA). Cells were washed and analyzed using BD Accuri™ C6 Plus flow cytometer (BD Biosciences, USA). Mean fluorescence intensity (MFI) values were plotted. For RBC aggregation assay, human RBCs were first diluted in PBS and then added to a 96-well round bottom plate (Thermo, Denmark) with various concentrations of antibodies. The plate was incubated at 37°C for 4 h. Non-hemagglutinated RBCs were defined as punctuate dot, and the HA index was calculated by Image J software.

[0087] As shown in Fig. 9A, human anti-CD47 antibodies (CwPlAl, CwP2Fl2 and BrPlFl 1) bound to surface CD47 of RBCs in a dose-dependent manner. Figure 9B showed CwPlAl and BrPlFl 1 did not induce hemagglutination of human RBCs. On the contrary, Hu5F9- G4, an anti-CD47 antibody under clinical investigations, induced serious RBC hemagglutination. Results indicated, anti-CD47 antibodies, CwPlAl and BrPlFl 1, could have better safety profile than that of Hu5F9-G4 regards to the hemagglutination of RBCs.

[0088] Example 12 In vivo anti-CD47 antibody efficacy evaluation using mouse xenograft model

[0089] Male SCID mice (BioLASCO, Taiwan) were injected s.c. in the right flank with 100 mΐ Matrigel (Coming, USA) plus 1.0 x 10 ⁷ HL-60 cells (0.1 mL cell suspension). Mice were intraperitoneally injected with isotype control antibody (400 ug/mouse), Hu5F9-G4, or human anti- CD47 antibodies, BrPlFl 1-G4 and CwPlAl in IgGl or IgG4 isotype (400 pg/mouse) three times per week for three weeks. Tumor volumes were measured twice per week. Tumor volumes were calculated using formula V=LW ² /2. After sacrificed, tumor tissues were resected and fixed with formalin.

[0090] To evaluate the anti-tumor activities, anti-CD47 antibodies, BrPlFl 1-G4, CwPlAl- Gl and CwPlAl-G4 were tested using HL-60 xenograft model and were compared with Hu5F9-G4 antibody. As shown in Fig. 10A, CwPlAl-Gl, CwPlAl-G4 and BrPlFl l-G4 antibodies administered intraperitoneally at 400 pg/mouse demonstrated significant antitumor activity against the human acute promyelocytic leukemia HL60 xenograft and which is comparable to that of Hu5F9- G4. Representative tumors from each treatment were shown in Fig. 10B.

[0091] Example 12 Pharmacokinetics assessment

[0092] The PK of the anti-CD47 antibody will be evaluated in rats (n=5). By tail vein injection, 10 mg/kg the anti-CD47 antibodies will be administered into rat. Blood samples will be collected from each rat via the caudal vena cava at 0 min, 1 h, 4 h, 8 h, 12 h, lday, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days after the injection. The concentration of antibody in serum will be determined by enzyme-linked immunosorbent assay (ELISA). Serum concentrations of the anti-CD47 antibody were interpolated from a 4-parameter logistic regression of the standard curve on the same plate.

[0093] Given the above, the anti-CD47 antibodies were engineered on a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions (ADCC and CDC), and did not involve apoptosis, and therefore, it is suggested in the invention that the anti-CD47 antibodies (and so does Hu5F9-G4) is able to initiate an activation of macrophage-mediated phagocytosis through blocking the interaction of CD47 with SIRP-□ .