WO2023056969A1

WO2023056969A1 - Bispecific antibodies specifically binding to cd47 and cd20, and uses thereof

Info

Publication number: WO2023056969A1
Application number: PCT/CN2022/124082
Authority: WO
Inventors: Yizhen Yang; Yu Cai; Xianwen YANG; Tianshu XU; Xiong LI; Wei-Guo Su
Original assignee: Hutchmed Limited
Priority date: 2021-10-09
Filing date: 2022-10-09
Publication date: 2023-04-13
Also published as: TW202325735A; AR127271A1

Abstract

Provided are novel bispecific antibodies, comprising at least one binding domain capable of specifically binding to CD47 and at least one binding domain capable of specifically binding to CD20. Also provided are methods of producing these bispecific antibodies and to methods of using the same.

Description

BISPECIFIC ANTIBODIES SPECIFICALLY BINDING TO CD47 AND CD20, AND USES THEREOF

Field of the invention

The invention relates to novel bispecific antibodies, comprising at least one binding domain capable of specifically binding to CD47 and at least one binding domain capable of specifically binding to CD20. The invention further relates to methods of producing these bispecific antibodies and to methods of using the same.

Background

CD47 (Cluster of Differentiation 47) was firstly identified as the tumor antigen of human ovarian cancer in 1980s. CD47, also known as an integrin-associated protein (IAP) , ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multiple membrane receptor belonging to an immunoglobulin superfamily that has a single immunoglobulin-like domain and five membrane spanning regions. As a ligand of signal regulatory protein α (SIRPα) , CD47 binds to the V-like domain at the NH2 terminus of SIRPα. SIRPα is expressed primarily on bone marrow cells, including macrophages, granulocytes, dendritic cells (DCs) , mast cells and their precursors, e.g. hematopoietic stem cells. CD47 on normal cells binds to SIRPα on macrophages, which releases the “don’ t eat me” signal, and thereby inhibits the phagocytic function of macrophages. It is an important mechanism how macrophages distinguish self from non-self in the innate immune system. CD47 is widely expressed on human tumor cells and tissues, including acute myelogenous leukemia (AML) , chronic granulocytic leukemia, acute lymphocytic leukemia (ALL) , non-Hodgkin’s lymphoma (NHL) , multiple myeloma (MM) , bladder cancer and other solid tumors. The tumor cells escape from the phagocytosis of macrophages though the binding of highly expressed CD47 to SIRPα on the surface of macrophages, which favors tumor growth. The immune checkpoint CD47 is considered to be a target which is potentially effective and can be widely used for tumor immunotherapy. At present, a variety of specific blockers have been developed to target the CD47/SIRPα interaction. There are a number of preclinical and clinical trials being carried out, which relate to the drugs including anti-CD47 antibodies and SIRPα fusion proteins for treatment in diffuse large B cell lymphoma, acute myelogenous leukemia, and advanced solid tumors.

CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa located on pre-B and mature B lymphocytes (Valentine et al, J. Biol. Chem. 264 (1989) 11282-11287; and Einfeld et al, EMBO J. 7 (1988) 711-717) . The antigen is also expressed on greater than 90 %of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al, Blood 63 (1984) 1424-1433) , but is not found on hematopoietic stem cells, pro-B-cells, normal plasma cells or other normal tissues (Tedder et al, J. Immunol. 135 (1985) 973-979) . CD20 is thought to regulate an early step (s) in the activation process for cell cycle initiation and differentiation (Tedder et al, supra) and possibly functions as a calcium ion channel (Tedder et al, J. Cell. Biochem. 14D (1990) 195) .

Given the expression of CD20 in B-cell lymphomas, this antigen has been a useful therapeutic target to treat such lymphomas. Given the expression of CD20 in B-cell lymphomas, this antigen can serve as a candidate for "targeting" of such lymphomas. Anti-CD20 antibodies specifically binding to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B-cells. Rituximab was approved by the US Food and Drug Administration (FDA) on November 26, 1997, for the indication of NHL. During the past decades, its indications have expanded, including chronic lymphocytic leukemia (CLL) , Waldenstrom’s macroglobulinemia and some autoimmune diseases, with the administration of either alone or in combination with other agents.

The present invention provides a novel anti-CD20/CD47 bispecific antibody specifically binding to both CD47 and CD20, e.g., CD47 and CD20 co-expressed on tumor cells. The bispecific antibody with dual functions of targeting CD20-expressing cancer cells and blocking the CD47 “don’ t eat me signal” on the same cancer cells is expected to activate the macrophages of innate immunity to specifically kill cancer cells that express both CD20 and CD47. The phagocytosis of cancer cells is expected to increase cancer neoantigen presentation and indirectly stimulate adaptive immunity to kill cancer cells. The bispecific antibody has a high anti-tumor activity and does not cause a significant agglutination of red blood cells only expressing CD47. So the present invention can satisfy more clinical demands.

Summary of the invention

The present invention provides a bispecific antibody comprising at least one antigen-binding domain capable of specifically binding to CD47 and at least one antigen-binding domain capable of specifically binding to CD20.

In one aspect, the bispecific antibody provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47, and

(b) at least one antigen-binding domain capable of specifically binding to CD20, wherein the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the bispecific antibody is provided herein, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the bispecific antibody is provided herein, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the bispecific antibody is provided herein, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD47 are selected from

(1) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 2;

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 3, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4;

(3) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 5, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10;

(4) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 6, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 9 or 10; or

(5) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.

In some embodiments, the bispecific antibody is provided herein, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the bispecific antibody is provided herein, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(1) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 25; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 28; or

(2) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 29, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 30, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 31; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 33, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 34.

In some embodiments, the bispecific antibody is provided herein, wherein the VH of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37, and the VL of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.

In some embodiments, the bispecific antibody is provided herein, wherein the bispecific antibody comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL of the antigen-binding domain capable of specifically binding to CD47comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16; and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7; and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10, and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37; and the VL of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.

In some embodiments, the bispecific antibody is provided herein, wherein

(a) the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7; and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8, and

(b) the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(1) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 35 and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 36, or

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 37 and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 38.

In some embodiments, the bispecific antibody is provided herein, wherein any one of the antigen-binding domain is chimeric, fully human or humanized.

In some embodiments, the bispecific antibody is provided herein, wherein any one of the antigen-binding domain comprises a Fab fragement or a scFv fragement.

In some embodiments, the bispecific antibody is provided herein, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement and the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement or a Fab fragement.

In some embodiments, the scFv fragement comprises a peptide linker connecting the C terminus of a VH to the N-terminus of a VL.

In some embodiments, the bispecific antibody provided herein comprises an Fc domain.

In some embodiments, the Fc domain is an IgG, particularly a human IgG1 Fc domain or a human IgG4 Fc domain.

In some embodiemnts, the first and the second subnit of the two subunits of the Fc domain are identical or non-identical.

In some embodiments, the Fc domain comprises one or more amino acid modification (s) altering the binding affinity of the antibody to an Fc receptor and/or effector function.

In some embodiments, the Fc domain comprises one or more amino acid modification (s) increasing the binding affinity of the antibody to an FcRn.

In some embodiments, the Fc domain comprises amino acid substitutions at positions 428 and 434 (numbering according to Kabat EU index) .

In some embodiments, the Fc domain comprises amino acid substitutions at positions 428 and 434, wherein said amino acid substitutions are M428L and N434S (numbering according to Kabat EU index) .

In some embodiments, one or two of the subunit (s) of the Fc domain (preferably the two of the subunits are identical) comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 66, 67, 68, 69 or 77, preferably, the subunit comprises M428L and N434S (numbering according to Kabat EU index) .

In some embodiments, each of the two subunits of the Fc domain comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 77.

In some embodiments, the Fc domain comprises one or more amino acid modification (s) promoting the association of the first and second subunit of the Fc domain.

In some embodiments, the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knob-into-hole method.

In some embodiments, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index) .

In some embodiments, the first subunit of the Fc domain comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 70, and/or the second subunit of the Fc domain comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 71.

In some embodiments, the bispecific antibody provided herein is bivalent, trivalent or tetravalent.

In some embodiments, the bispecific antibody provided herein comprises

(a) a first Fab fragement capable of specifically binding to CD47,

(b) a second Fab fragement capable of specifically binding to CD20, and

(c) an Fc domain composed of a first and a second subunit capable of stable association.

In some embodiments, one or two of the Fab fragement (s) is CrossFab, e.g., CrossFab (CL–CH1) .

In some embodiments, the second Fab fragement is CrossFab (CL-CH1) .

In some embodiments, the bispecific antibody provided herein comprises

(i) a first polypeptide chain in the format of: VH (anti-CD47) –CL–Fc domain subunit,

(ii) a second polypeptide chain in the format of: VL (anti-CD47) –CH1,

(iii) a third polypeptide chain in the format of: VH (anti-CD20) –CH1–Fc domain subunit, and

(iv) a forth polypeptide chain in the format of: VL (anti-CD20) –CL.

In some embodiments, the bispecific antibody provided herein comprises

(i) a first polypeptide chain in the format of: VH (anti-CD47) –CH1–Fc domain subunit,

(ii) a second polypeptide chain in the format of: VL (anti-CD47) –CL,

(iii) a third polypeptide chain in the format of: VH (anti-CD20) –CL–Fc domain subunit, and

(iv) a forth polypeptide chain in the format of: VL (anti-CD20) –CH1.

In some embodiments, the bispecific antibody provided herein comprises

(i) a first polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 60,

(ii) a second polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61,

(iii) a third polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 62, and

(iv) a forth polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 63.

In some embodiments, the bispecific antibody provided herein comprises

(a) two scFv fragments capable of specifically binding to CD47, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain, wherein each of the two scFv fragments is connected to the whole antibody.

In some embodiments, each of the two scFv fragements is connected via a peptide linker to the whole antibody (e.g., the C-terminus of the whole antibody) .

In some embodiments, each of the two scFv fragements is connected via a peptide linker to the C-terminus of the two heavy chains of the whole antibody.

In some embodiments, the peptide linker comprises a GS linker.

In some embodiments, the peptide linker comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 39, 40, 41, 42, 43, or 44.

In some embodiments, the scFv fragement comprises a peptide linker connecting the C terminus of a VH to the N-terminus of a VL, or a peptide linker connecting the N terminus of a VH to the C-terminus of a VL.

In some embodiments, the scFv fragement comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 45 or 46.

In some embodiments, the bispecific antibody provided herein comprises two heavy chains and two light chains, wherein

(1) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, 48, 49 or 78; and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50, or

(2) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51 or 52; and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.

In some embodiments, the bispecific antibody provided herein comprises

(a) two scFv fragments capable of specifically binding to CD47, each of the two scFv fragments comprises a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain, each of the two Fab fragments comprises a VH and a VL, wherein the VH and the VL are selected from

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 37, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 38,

wherein each of the two scFv fragments is connected via a peptide linker to the C-terminus of the two heavy chains of the whole antibody.

In some embodiments, the bispecific antibody provided herein comprises

(a) two scFv fragments capable of specifically binding to CD47, each of the two scFv fragements comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 45 or 46, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain, wherein

(1) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50,

(2) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 48, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in as set forth in SEQ ID NO: 50,

(3) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 49, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50,

(4) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 78, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50,

(5) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53, or

(6) each of the heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 52, and each of the light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53,

In some embodiments, the bispecific antibody provided herein comprises

(a) two scFv fragments capable of specifically binding to CD47, each of the two scFv fragements comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 45, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain, wherein each of the heavy chains comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 49 or SEQ ID NO: 78, and each of the light chains comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 50,

In one aspect, the present invention also provides an isolated nucleic acid, encoding the bispecific antibody of the present invention.

In one aspect, the present invention also provides a recombinant vector or an expression vector, comprising one or more nucleic acids of the present invention, wherein the vector is suitable for the recombinant production of the bispecific antibody of the present invention.

In one aspect, the present invention also provides a host cell, comprising one or more recombinant vectors or expression vectors of the present invention.

In one aspect, the present invention also provides an immunoconjugate, comprising the bispecific antibody of the present invention.

In one aspect, the present invention also provides a pharmaceutical composition, comprising the bispecific antibody of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, or the immunoconjugate of the present invention, and optionally comprising a pharmaceutically acceptable excipient.

In one aspect, the present invention also provides a method for treating or preventing disease or condition in a subject, comprising administering to an individual an effective amount of the bispecific antibody of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, or the immunoconjugate of claim the present invention, or the pharmaceutical composition of the present invention.

In some embodiments, the disease or condition is CD47-related and/or CD20-related.

In some embodiments, the disease or condition is cancer.

In some embodiments, the cancer comprises hematological cancer, for example acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , Non- Hodgkin lymphoma (e.g., Burkitt’s lymphoma) , B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL) , chronic myelocytic leukemia (CML) , follicular lymphoma, small lymphotic lymphoma (SLL) , central nervous system (CNS) lymphoma, Richter’s Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and anaplastic large cell lymphoma. chronic lymphoid lymphoma (CLL) and non-Hodgkin’s lymphoma (NHL) .

In one aspect, the present invention also provides a use of the bispecific antibody of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, or the immunoconjugate of claim the present invention, or the pharmaceutical composition of the present inventionin the manufacture of a medicament for treating cancer in a subject.

Brief Description of the Drawings

Figure 1 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Raji cells.

Figure 2 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Toledo cells.

Figure 3 shows the binding affinity of antibody HMA02h14-48 to CD47 on the surface of REC-1 cells.

Figure 4 shows the activity of antibody HMA02h14-48 in blocking the interaction between human CD47 and SIRPα.

Figure 5 shows the effect of antibody HMA02h14-48 on phagocytosis of Raji cells by human MΦ.

Figure 6 shows the effect of antibody HMA02h14-48 on phagocytosis of Toledo cells by human MΦ.

Figure 7 shows the effect of antibody HMA02h14-48 on phagocytosis of REC-1 cells by human MΦ.

Figure 8 shows the effect of antibody HMA02h14-48 on phagocytosis of HL-60 cells by human MΦ.

Figure 9 shows the effect of antibody HMA02h14-48 on agglutination of the red blood cell in vitro.

Figure 10 shows the ability of antibody HMA02h14-48 to bind to CD47 on the surface of human red blood cells.

Figure 11 shows the inhibition of Toledo tumor growth by Hu5F9 and HMA02h14-48.

Figure 12 shows the inhibition of REC-1 tumor growth by Hu5F9 and HMA02h14-48.

Figure 13 shows the binding activities of anti-CD20/CD47 bispecific antibodies to human CD47 protein by ELISA.

Figure 14 shows the binding activities of anti-CD20/CD47 bispecific antibodies to human CD20 protein by ELISA.

Figure 15 shows the binding activities of anti-CD20/CD47 bispecific antibodies to both human CD47 and human CD20 protein by ELISA.

Figure 16 shows the binding activities of anti-CD20/CD47 bispecific antibodies to SU-DHL-6 cells.

Figure 17 shows the binding activities of anti-CD20/CD47 bispecific antibodies to human CD47 on CHO-K1/hCD47 OE cells.

Figure 18 shows the blocking activity of anti-CD20/CD47 bispecific antibodies in the CD47 (SU-DHL-6) /SIRPα interaction by Jurkat SIRPα Signaling Assay.

Figure 19 shows the blocking activity of anti-CD20/CD47 bispecific antibodies in the CD47 (CHO-K1/CD47 OE) /SIRPα interaction by Jurkat SIRPα Signaling Assay

Figure 20 shows the effect of anti-CD20/CD47 bispecific antibodies on phagocytosis of CD20 ⁺CD47 ⁺ lymphoma tumor cells by human MΦ.

Figure 21 shows the effect of anti-CD20/CD47 bispecific antibodies on hemagglutination of the red blood cell in vitro.

Figure 22 shows the ability of anti-CD20/CD47 bispecific antibodies to bind to CD47 on the surface of human red blood cells.

Figure 23 shows the pro-phagocytic activities of anti-CD20/CD47 bispecific antibodies on human red blood cells.

Figure 24 shows the pro-phagocytic activities of anti-CD20/CD47 bispecific antibodies on human red blood cells.

Figure 25 shows effect of BsAb1 on tumor growth of SU-DHL-6 xenograft subcutaneously implanted in NOD-SCID mice.

Figure 26 shows effect of BsAb8 on tumor growth of SU-DHL-6 xenograft subcutaneously implanted in nude mice.

Detailed description of the invention

Definitions

Unless otherwise stated, the present invention will be implemented using conventional techniques in molecular biology (including recombinant techniques) , microbiology, cell biology, biochemistry and immunology, which are within the technical scope in the art.

In order that the invention may be more readily understood, some of the scientific and technical terms are defined as follows. Unless otherwise explicitly defined elsewhere herein, all scientific and technical terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. With respect to definitions and terms in the art, reference can be made to Current Protocolsin Molecular Biology (Ausubel) by professionals. The abbreviation of an amino acid residue is a standard 3-letter and/or 1-letter code used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used in the present application (including the claims) includes the corresponding plural form thereof, unless otherwise explicitly specified.

When used to connect two or more optional items, the term “and/or” should be understood to mean any one of the optional items or any two or more of the optional items.

As used herein, the term “comprise” or “include” means to include the mentioned elements, integers, or steps, but does not exclude any other elements, integers, or steps. As used herein, when the term “comprise” or “include” is used, unless otherwise indicated, it also encompasses instances composed of the mentioned elements, integers or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to encompass an antibody variable region composed of the specific sequence.

The terms “CD47” or “CD47 protein” refers to any natural CD47 from any vertebrate source, including mammals (such as primates (e.g., humans) and rodents (e.g., mice and rats) ) , unless otherwise stated. The term covers a “full length” unprocessed CD47 and any form of CD47 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD47, such as splice variants or allelic variants. In some embodiments, CD47 refers to a full length CD47 or fragment thereof (such as a mature fragment thereof lacking a signal peptide) from a human. In some embodiments, a human CD47 (hCD47) refers to CD47 or a fragment thereof identical to the amino acid sequence as set forth in NCBI accession number NP_001768.1 or a fragment thereof. In some embodiments, the term also covers a fusion protein comprising CD47 or a fragment thereof.

The terms “CD20” or “CD20 protein” refers to any natural CD20 from any vertebrate source, including mammals (such as primates (e.g., humans) and rodents (e.g., mice and rats) ) , unless otherwise stated. The term covers a “full length” unprocessed CD20 and any form of CD20 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD20, such as splice variants or allelic variants. In some embodiments, CD20 refers to a full length CD20 or fragment thereof (such as a mature fragment thereof lacking a signal peptide) from a human. In some embodiments, a human CD20 (hCD20) refers to CD20 or a fragment thereof identical to the amino acid sequence as set forth in NCBI accession number NP_068769.2 or a fragment thereof. In some embodiments, the term also covers a fusion protein comprising CD20 or a fragment thereof.

The term "anti-CD20/CD47 bispecific antibody" as used herein refers to an antibody comprising multiple antigen-binding domain capable of specifically binding to both CD47 and CD20.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) , and antibody fragments so long as they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the various antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations generally include a large number of antibodies being directed against different epitopes (or specific for different epitopes) . The modifier “monoclonal” indicates the feature of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring any particular method to produce the antibody.

The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term "bispecific" means that the antibody is able to specifically bind to at least two distinct antigenic determinants. A bispecific antibody comprises at least two antigen-binding domains, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants in different antigens, e.g., expressed on two distinct cells. For example, the antibodies of the present invention are bispecific, comprising two antigen-binding domains capable of specifically binding to CD20, and two antigen-binding domains capable of specifically binding to CD47.

The terms "whole antibody" and "intact antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. Each of the heavy chains consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3) . Each of light chains consists of a light chain variable region (abbreviated herein as VL) and a light-chain constant region. The light chain constant region consists of a domain CL. The VH region and the VL region can be further divided into hypervariable regions (complementarity determining regions, or CDRs) , with more conservative regions (framework regions, or FRs) inserted therebetween. Each VH or VL consists of three CDRs and four FRs, arranged from the N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of an antibody to an antigen, but shows multiple effector functions. The heavy chain of an antibody may be assigned to one of five types, called a (IgA) , δ (IgD) , ε (IgE) , γ (IgG) , or μ (IgM) , some of which may be further divided into subtypes, e.g.

(IgGl) , γ2 (IgG2) , γ3 (IgG3) , γ4 (IgG4) , al (IgAl) and a2 (IgA2) . The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ) , based on the amino acid sequence of its constant domain.

A “complementary determining region” or “CDR region” or “CDR” is a region in an antibody variable domain, which is hypervariable in sequence and forms a structurally established loop ( “hypervariable loop” ) and/or contains an antigen contact residue ( “antigen contact point” ) . CDR is mainly responsible for binding to epitopes. CDRs of heavy chain and light chain are generally called CDR1, CDR2 and CDR3, which are numbered sequentially from the N-terminus. The CDRs located in an antibody heavy chain variable domain are called HCDR1, HCDR2 and HCDR3 respectively, while the CDRs located in the antibody light chain variable domain are called LCDR1, LCDR2 and LCDR3 respectively. Each VH or VL consists of three CDRs and 4 FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In a given VH or VL amino acid sequence, the accurate amino acid sequence boundary of each CDR can be determined by using any one of the various well known schemes or a combination thereof, including, for example: Chothia scheme (Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins” , Journal of Molecular Biology, 196, 901-917 (1987) ) ; Kabat scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) and Contact (University College London) ; North scheme (North et al., A New Clustering of Antibody CDR Loop Conformations” , Journal of Molecular Biology, 406, 228-256 (2011) ) . The boundary of the CDR of the antibody in the present invention can be determined according to any schemes or a combination thereof in the art and personal evaluation.

An "antibody fragment" refers to a molecule other than a whole antibody that comprises a portion of a whole antibody that binds the antigen to which the whole antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F (ab') 2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv) ; and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-134 (2003) . For a review of scFv fragments, see e.g. Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) ; see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F (ab') 2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404, 097; WO 1993/01161; Hudson et al, Nat Med 9, 129-134(2003) ; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-6448 (1993) . Triabodies and tetrabodies are also described in Hudson et al, Nat Med 9, 129-134 (2003) . Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage) , as described herein.

Papain digestion of whole antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy and light chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL and a constant domain of a light chain (CL) , and a VH and a first constant domain (CH1) of a heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteins from the antibody hinge region. Fab'-SH are Fab'fragments wherein the cysteine residue (s) of the constant domains bear a free thiol group. Pepsin treatment yields an F (ab') ₂ fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc domain. According to the present invention, the term "Fab fragment" also includes "cross-Fab fragments" or "crossover Fab fragments" .

A "single chain variable fragment" or "scFv fragment" is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short peptide linker of 10 to about 25 amino acids. The peptide linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96) . In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH, namely being able to assemble together with a VL, or of a VL, namely being able to assemble together with a VH to a functional antigen binding domain and thereby providing the antigen binding property of full length antibodies.

The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) , and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) . This crossover Fab molecule is also referred to as CrossFab (VL-VH) . On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) , and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) . This crossover Fab molecule is also referred to as CrossFab (CL-CH1) .

The term "valent" as used within the current application denotes the presence of a specified number of binding domains in an antibody. As such, the terms "bivalent" , "tetravalent" , and "hexavalent" denote the presence of two binding domains, four binding domains, and six binding domains, respectively, in an antibody. Valency of an antibody may also be expressed in relation to the number of binding domains for a given antigenic determinant. For example, in some embodiments the antibodies of the present invention are bivalent with respect to CD47, and bivalent with respect to CD20 (i.e. 2+2) .

By "fused" , "connected" or "linked" is meant that the components (e.g. a heavy chain of an antibody and a scFv fragment) are linked by peptide bonds, either directly or via one or more peptide linkers.

The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 60 amino acids. Peptide linkers are known in the art or are described herein. Suitablely, non-immunogenic linker comprises, for example, glycine polymers (G) n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers. A peptide linker comprising glycine-serine polymers, including, for example, (GS) _n, (GSGGS) _n, (GGGS) _n, (GGGGS) _n (SEQ ID NO: 39) or (GGGSGGGGS) _n, (GGGGS) _n G, wherein "n" is an integer ranging from 1 to 10, perferably ranging from 1 to 4, more preferably 3 or 4, are collectively defined as GS linker. In some embodiments, a peptide linker comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 39, 40, 41, 42 or 44. Peptide linker can futher comprises a naturally or a non-naturally occurring sequence, for example, a naturally sequence derived from the CH1 domain (such as an amino acid sequence consisiting of ATG) or hinge region of an IgG. In some embodiments, a peptide linker comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 43.

The term "antigen-binding domain" or "antigen-binding site" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen-binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions) . Preferably, an antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) . Examples of antigen-binding domain include, without limitation, a diabody, a Fab, a Fab', a F (ab') ₂, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , and an scFv dimer (bivalent diabody) .

By "specifically binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000) ) , and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002) ) . In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10%of the binding of the antibody to the antigen as measured, e.g. by SPR. In certain embodiments, an antibodies that binds to the antigen has a dissociation constant (KD) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, <0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10 ^～7M or less, e.g. from 10 ^～7M to 10 ^～13M, e.g. from 10 ^～8M to 10 ^～13M) .

"Affinity" or "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen) . Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g. antibody and antigen) . The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD) , which is the ratio of dissociation and association rate constants (Kd and Ka, respectively) . Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR) .

The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc domains and variant Fc domains. An IgG Fc domain comprises an IgG CH2 and an IgG CH3 domain. The "CH2 domain" of a human IgG Fc domain usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG) . In one embodiment, a human IgG heavy chain Fc domain extends from Cys226, or from Pro230, to the C-terminus of the heavy chain. In one embodiment, the C-terminal lysine (Lys447) of the Fc domain may or may not be present. In one embodiment, the C-terminal lysine (Lys447) and glycine (Gln446) of the Fc domain may or may not be present. In one embodiment, the C-terminal lysine (Lys447) of the Fc domain is substituted with alanine. Unless otherwise specified herein, numbering of amino acid residues in the Fc domain or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term “chimeric” as used herein, means an antibody or antigen-binding domain, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster. In some embodiments, an antigen-binding domain capable of specifically binding to CD20 comprises a variable region derived from a mouse and a constant region derived from human.

The term “humanized” as used herein means that the antibody or antigen-binding domain comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.

The term “fully human” as used herein, with reference to antibody or antigen- binding domain, means that the antibody or the antigen-binding domain has or consists of amino acid sequence (s) corresponding to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires or other human antibody-encoding sequences. In certain embodiments, a fully human antibody does not comprise amino acid residues (in particular antigen-binding residues) derived from a non-human antibody.

The terms “carcinoma” or “cancer” refer to or describe physiological disorders in mammals, generally characterized by unregulated cell growth. This definition includes benign and malignant cancers and resting tumors or micrometastasis. The “cancer” includes, but is not limited to, solid tumors and blood or hematological cancers. Examples of various cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia.

The term “subject” , “patient” or “individual” herein includes any human or non-human animals. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, bovine, chicken, amphibians, reptiles, etc.

The terms therapeutically “effective amount” , therapeutically “effective dose” and “effective amount” herein refer to the amount of the anti-CD20/CD47 bispecific antibody of the present invention that effectively prevents or ameliorate the symptoms of one or more diseases or conditions or the development of the diseases or conditions when given to cells, tissues or subjects alone or in combination with other therapeutic drugs. Therapeutically effective dose also refers to the amount of the antibody or antigen-binding fragment thereof that is sufficient to result in improvement of the symptoms, such as the amount to treat, cure, prevent or improve related medical conditions or to increase the speed of treatment, cure, prevention or improvement of such conditions. When the active ingredient alone is administered to an individual, the therapeutically effective dose refers only to the ingredient. When administered in combination, the therapeutically effective dose refers to the comprehensive amount of active ingredients contributing to therapeutic effects, regardless of administration in combination, in sequence or at the same time. The effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameter by at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

As used herein, “treatment” includes 1) therapeutic measures (therapeutic treatment) , which cure, alleviate and relieve the symptoms of the diagnosed pathological condition or disease and/or stop the progress of the diagnosed pathological condition or disease, and 2) preventive or prophylactic measures (preventive or prohylatic treatment) , which prevent and/or alleviate the development of the pathological condition or disease. Therefore, the subject receiving the treatment include an individual who has suffered from the disease, an individual who is prone to suffer from the disease, and an individual who wants to prevent the disease. In some embodiments, the present invention relates to the treatment of a disease or condition. In some other embodiments, the present invention relates to the prevention of a disease or condition.

In some embodiments according to the present invention, the “treatment” of a disease or condition refers to the amelioration of the disease or condition (i.e., alleviating or preventing or reducing the progression of the disease or at least one of its clinical symptoms) . In some other embodiments, “treatment” refers to relieving or improving at least one body parameter, including those physical parameters that may not be discernible by the patient. In some other embodiments, “treatment” refers to the regulation of a disease or condition physically (e.g., stabilization of discernible symptoms) , physiologically (e.g., stabilization of body parameters) , or both. Methods for evaluating the treatment and/or prevention of a disease are generally known in the art unless explicitly described herein.

In yet other embodiments according to the present invention, “prevention” of a disease or condition includes inhibition of the occurrence or development of the disease or condition or the symptom of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term “prevention” refers to administration of drugs to a subject prior to the onset of conditions or symptoms of cancer, in particular, in a subject at risk of cancer.

In some embodiments, after “treating” the cancer by the method of the present invention, an individual patient is considered to have been successfully treated if the individual shows one or more of the following: the number of cancer cells was decreased or cancer cells disappeared completely; tumor size was decreased; infiltration of cancer cells into peripheral organs was inhibited or absent, including, for example, the spread of cancer cells to soft tissues and bones; tumor metastasis was inhibited or absent; tumor growth was inhibited or absent; one or more symptoms associated with the specific cancer were relieved; incidence and mortality were reduced; the quality of life was improved; the tumor incidence, frequency or tumorigenicity was reduced; the number or frequency of cancer stem cells in tumor was reduced; tumor cells were differentiated into a non-tumorigenic state; or a combination of some of the effects.

“Inhibition of tumor growth” refers to any mechanism by which tumor cell growth can be inhibited. In some embodiments, tumor cell growth is inhibited by delaying tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by stopping tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis. In some embodiments, tumor cell growth is inhibited by inducing tumor cell differentiation. In some embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

As used herein, “sequence identity” refers to the degree of identity of sequences based on one by one nucleotide or amino acid comparing in the comparison window. The “ (percentage) sequence identity” can be calculated as follows: comparing the two optimally aligned sequences in the comparison window, determining the number of positions with the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size) , and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment for purposes of determining the percentage of sequence identity can be achieved in various ways known in the art, for example, using publicly available computer softwares such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art is able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences or the target sequence area being compared. In the present invention, for antibody sequences, the percentage of identity of two amino acid sequences is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and then performing an optimal alignment in accordance with a numbering rule in a preferred embodiment.

The term “agglutination” as used herein refers to cell agglomeration, and the term “hemagglutination” refers to agglomeration of a particular class of cells (i.e., red blood cells) . Therefore, hemagglutination is a type of agglutination.

The term “conservative substitution” or conservative amino acid substitution"as used herein refers to the substitution of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as "conservatively modified variants. " Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. The conservative substitutions for one another are exemplified in Table A.

“Hu5F9” herein is an anti-CD47 antibody in the form of IgG4P, formed by recombinant expression by GenScript according to the variable region sequence of 5F9 disclosed in patent US 2015/0183874 A1. “SRF231” is an anti-CD47 antibody in the form of IgG4P, formed by recombinant expression by GenScript according to the variable region sequence of 2.3D11 disclosed in patent US 20180201677 A1.

Bispecfic antibody of the invention

The present invention provides a bispecific antibody comprising at least one antigen-binding domain capable of specifically binding to CD47 and at least one antigen-binding domain capable of specifically binding to CD20. In some embodiments, the bispecific antibodies provided herein further comprise Fc domain.

Antigen-binding domain capable of specifically binding to CD47

The term "antigen-binding domain capable of specifically binding to CD47" refers to polypeptides that specifically bind to CD47. In one aspect, the term "antigen-binding domain capable of specifically binding to CD47" refers to the portion of a molecule (e.g., a bispecific antibody) that contains regions specifically bind to patial or whole length of CD47. For example, one or more antibody variable regions may provide antigen-binding domains that can specifically bind to CD47. In particular, the antigen-binding domains that specifically bind to CD47 comprise the light chain variable region (VL) and the heavy chain variable region (VH) . In some embodiments, the "antigen-binding domain capable of specifically binding to CD47" is a scFv fragment or a Fab fragment, especially a scFv fragment.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 derives from any anti-CD47 antibodies known in the prior arts. "anti-CD47 antibody" refers to an antibody that can modulate, e.g., inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and/or signaling. These antibodies can modulate, e.g., inhibit, block, antagonize, neutralize or otherwise interfere with the interaction between CD47 and SIRPα (signal-regulatory-protein α) (for example, human CD47 and human SIRPα) .

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 binds to the same epitope as an anti-CD47 antibody known in the prior arts and described herein. In some embodiments, the antigen-binding domain capable of specifically binding to CD47 competes for binding to CD47 with an antibody known in the prior arts and described herein. In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises three HCDRs and/or three LCDRs derived from any of the anti-CD47antibodies in the prior arts (such as IBI-188, TJC4, SHR-1603, SRF231, IMC-002, CC-90002, A0-176 or Hu5F9, or the variants thereof) and described herein.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises the HCDR1, HCDR2 and HCDR3 of a VH and the LCDR1, LCDR2 and LCDR3 of a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises the HCDR1, HCDR2 and HCDR3 of a VH and comprises the LCDR1, LCDR2 and LCDR3 of a VL, wherein the VH and VL are selected from:

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 7, the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH and VL are selected from

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 1, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 2.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 3, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 4.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the antigen-binding domain capable of specifically binding to comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 10.

Antigen-binding domain capable of specifically binding to CD20

The term "antigen-binding domain capable of specifically binding to CD20" refers to polypeptides that specifically bind to CD20. In one respect, the term "antigen-binding domain capable of specifically binding to CD20" refers to the portion of a molecule (e.g., a bispecific antibody) that contains regions that specifically bind to patial or whole length of CD20. For example, one or more antibody variable regions may provide antigen-binding domain that can specifically bind to CD20. In particular, the antigen-binding domain specifically binding to CD20 contains the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH) . In some embodiments, the "antigen-binding domain capable of specifically binding to CD20" can be scFv fragment or Fab fragment, especially Fab fragments.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 is derived from any anti-CD20 antibodies reported in the prior arts. The terms "anti-CD20 antibody" refer to an antibody that is capable of binding CD20 with sufficient affinity. In some embodiments, anti-CD20 antibodies are type I anti-CD20 antibodies or type II anti-CD20 antibodies. Examples of type I anti-CD20 antibodies include e.g. rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO 131921, ublituximab, HI47 IgG3 (ECACC, hybridoma) , 2C6 IgGl (as disclosed in WO 2005/103081) , 2F2 IgGl (as disclosed in WO 2004/035607 and WO 2005/103081) and 2H7 IgGl (as disclosed in WO 2004/056312) . In some embodiments, anti-CD20 antibodies are type II anti-CD20 antibodies. Examples of type II anti-CD20 antibodies include e.g. obinutuzumab, tositumumab, humanized B-Lyl antibody IgGl (achimeric humanized IgGl antibody as disclosed in WO 2005/044859) , 11B8 IgGl (as disclosed in WO 2004/035607) and AT80 IgGl.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises the HCDR1, HCDR2 and HCDR3 of a VH and the LCDR1, LCDR2 and LCDR3 of a VL, wherein the VH and VL are selected from:

(1) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 35, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 36; or

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 37, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 38.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 25, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 28.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 29, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 30, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 31, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 33, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 34.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20, comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 37, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 38.

In one aspect, the bispecific antibody provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47, comprising the HCDR1、HCDR2 and HCDR3 of a VH and the LCDR1、LCDR2 and LCDR3 of a VL, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 2, 4, 8, 9 or 10, and

(b) at least one antigen-binding domain capable of specifically binding to CD20, comprising the HCDR1、HCDR2 and HCDR3 of a VH and the LCDR1、LCDR2 and LCDR3 of a VL, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO: 35 or 37, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 36 or 38.

In one aspect, the bispecific antibody provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47, comprising a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16, and

(b) at least one antigen-binding domain capable of specifically binding to CD20, comprising a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 25, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 28.

In one aspect, the bispecific antibodies provided herein comprises

(b) at least one antigen-binding domain capable of specifically binding to CD20, comprising a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 29, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 30, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 31, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 33, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 34.

In one aspect, the bispecific antibodies provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10, and

(b) at least one antigen-binding domain capable of specifically binding to CD20, comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 37, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.

In one aspect, the bispecific antibodies provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47, comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8, and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH and VL are selected from

(1) a VH comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35, and a VL comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36; or

(2) a VH comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 37, and a VL comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 38.

In one aspect, the bispecific antibodies provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH and the VL are selected from

(5) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10; and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH and the VL are selected from

In one aspect, the bispecific antibody provided herein comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8; and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 36.

In one aspect, the bispecific antibody provided herein comprises

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 37, and a VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 38.

Format of bispecific antibodies

In one aspect, the bispecific antibody is provided herein, wherein the antigen-binding domains capable of specifically binding to CD47 and/or the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement or a scFv fragement.

In some embodiments, the Fab fragement is a Cross-Fab fragment. In some embodiments, the Fab fragment is a CrossFab (CL-CH1) .

In some embodiments, the scFv fragement comprises a peptide linker connecting the C-terminus of a VH to the N-terminus of a VL (as from N-terminus to C-terminus, in the form of "VH-peptide linker-VL" ) . In some embodiments, a scFv fragement comprises a peptide linker connecting the C-terminus of a VL to the N-terminus of a VH (as from N terminus to C terminus, in the form of "VL-peptide linker-VH" ) . In some embodiments, the peptide linker connecting a VH and a VL comprises a GS linker. In some embodiments, the GS linker comprises one selected from (G ₄S) n, (SG ₄) nor G ₄ (SG ₄) _n peptide linkers, wherein "n" is an integer ranging from 1 to 10, perferably ranging from 2 to 4, more perferably 3 or 4. In some embodiments, the GS linker comprises (G ₄S) ₂ (SEQ ID NO: 40) . In some embodiments, the GS linker comprises (G ₄S) ₃ (SEQ ID NO: 41) . In some embodiments, the peptide linker comprises (G ₄S) ₄ (SEQ ID NO: 42) . In some embodiments, the GS linker comprises AST (G ₄S) ₃ (SEQ ID NO: 43) . In some embodiments, the GS linker comprises (G ₄S) ₄G (SEQ ID NO: 44) .

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising a VH linked via peptide linker to a VL. In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising a peptide linker connecting the C terminus of a VH to the N-terminus of a VL.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising a peptide linker connecting the C terminus of a VH to the N-terminus of a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising a peptide linker connecting the C terminus of a VH to the N-terminus of a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 45.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 46.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 and/or the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 36.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 37, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 38.

In some embodiments, the antigen-binding domain capable of specifically binding to CD20 is CrossFab (CL-CH1) comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 36.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a Fab fragement comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a Fab fragement comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises a CrossFab (CL-CH1) comprising a VH and a VL, wherein the VH comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

In one aspect, the bispecific antibody provided herein is multivalent, such as bivalent, trivalent or tetravalent.

Bispecific antibody, (1+1) format

In one aspect, the bispecific antibody provided herein is bivalent.

In one aspect, the bispecific antibody provided herein comprises

(a) one antigen-binding domain capable of specifically binding to CD47, and

(b) one antigen-binding domain capable of specifically binding to CD20.

In some embodiments, the bispecific antibody provided herein futher comprises a Fc domain composed of a first and a second subunit capable of stable association, wherein one antigen-binding domain is connected directly or via a peptide linker to the N-terminus of one of the Fc domain subunit, and the other antigen-binding domain is connected to the N-terminus of the other Fc domain subunit. In some embodiments, the Fc domain comprises knob-into-hole modification. In a particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (knob) , and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (hole) (numbering according to Kabat EU index) .

In one aspect, the bispecific antibody provided herein comprises

(a) a first Fab fragement capable of specifically binding to CD47,

(b) a second Fab fragement capable of specifically binding to CD20, and

In some embodiments, the first Fab fragement and the second Fab fragement are each directly connected to the N-terminus of the Fc domain subunits. In some embodiments, one of the Fab fragement is CrossFab fragement. In some particular embodiments, one of the Fab fragement is CrossFab (CL-CH1) .

In some embodiments, the bispecific antibody provided herein comprises

(a) a first Fab fragement capable of specifically binding to CD47 ,

(b) a second Fab fragement capable of specifically binding to CD20 , and

(c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second Fab fragement is CrossFab (CL-CH1) and directly connected to the N-terminus of one of the Fc domain subunit, and the second Fab fragement directly connected to the N-terminus of the other Fc domain subunit.

In some embodiments, the bispecific antibody provided herein comprises

(i) a first polypeptide chain in the format of: VH (anti-CD47) –CH1-Fc domain subunit ,

(ii) a second polypeptide chain in the format of: VL (anti-CD47) –CL,

(iv) a forth polypeptide chain in the format of: VL (anti-CD20) –CH1,

wherein one Fc domain subunit comprises amino acid substitutions S354C and T366W (knob) , and the other Fc domain subunit comprises amino acid substitutions Y349C, T366S, L368A and Y407V (hole) (numbering according to Kabat EU index) .

As used herein, VH (anti-CD47) and VL (anti-CD47) refer respectively to the heavy and light chain variable domain of the antigen-binding domain capable of specifically binding to CD47; VH (anti-CD20) and VL (anti-CD20) refer respectively to the heavy and light chain variable domain of the antigen-binding domain capable of specifically binding to CD20. In some embodiments, the CL in the third polypeptide chain comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 74, and the CH1 in the forth polypeptide chain comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 75.

In some eombodiment, the Fc domain subunit in the first polypeptide chain comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 70, and/or the Fc domain subunit in the second polypeptide chain comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 71.

In some embodiments, the bispecific antibody provided here comprises

(i) a first polypeptide chain comprising or consisiting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 60,

(ii) a second polypeptide chain comprising or consisiting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61,

(iii) a third polypeptide chain comprising or consisiting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 62, and

(iv) a forth polypeptide chain comprising or consisiting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 63.

Bispecific antibody, (2+2) format

In one aspect, the bispecific antibody provided herein is tetravalent.

In one aspect, the bispecific antibody provided herein comprises

(a) two antigen-binding domains capable of specifically binding to CD47,

(b) two antigen-binding domains capable of specifically binding to CD20, and

(c) an Fc domain.

In one aspect, the bispecific antibody provided herein comprises

(a) two scFv fragments capable of specifically binding to CD47, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain.

In some embodiments, each of the two heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, 48, or 49 or 78, and each of the two light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50.

In some embodiments, each of the two heavy chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51 or 52, and each of the two light chains comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.

In some embodiments, each of the two scFv fragments is connected to the terminus respectively, such as N-terminus of the two heavy chains, C-terminus of the two heavy chains, N-terminus of the two light chains, or C-terminus of the two light chains of the whole antibody. In some embodiments, each of the two scFv fragments is connected to the C-terminus of the two heavy chains of the whole antibody. In some embodiments, each of the two scFv fragments is connected via a peptide linker to the C-terminus of the two heavy chains of whole antibody.

In some embodiments, the peptide linker linking a scFv fragment to a chain of whole antibody comprises one selected from (G ₄S) n, (SG ₄) nor G ₄ (SG ₄) n peptide linkers, wherein "n" is an integer ranging from 1 to 10, perferably ranging from 2 to 4, more perferably 3 or 4. In some embodiments, the peptide linker comprises (G ₄S) ₂ (SEQ ID NO: 40) . In some embodiments, the peptide linker comprises (G ₄S) ₃ (SEQ ID NO: 41) . In some embodiments, the peptide linker comprises (G ₄S) ₄ (SEQ ID NO: 42) . In some embodiments, the peptide linker comprises (G ₄S) ₄G (SEQ ID NO: 44) .

Variants

In certain instances, amino acid sequence variants of the bispecific antibodies of the invention are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any of deletion, insertion, and substitution or combination thereof can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, for example, antigen-binding.

In certain instances, antigen-binding moiety variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs, FRs, Fc, or constant regions. Conservative substitutions are shown in Table A under the heading of “preferred substitutions. ” More substantial changes are provided in Table A under the heading of “exemplary substitutions, ” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC) .

Table A.

Amino acids may be grouped according to common side-chain properties:

(1 ) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; or

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

In some embodiments, the antigen-binding domains of the bispecific antibody provided herein also cover an antigen-binding domain, in which the three CDRs of the heavy chain variable region contain at least one, and no more than 5, 4, 3, 2 or 1amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) , compared with the CDRs disclosed herein; and/or in which the three CDRs of the light chain variable region comprise, at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions) , compared with the CDRs disclosed herein.

In some embodiments, the antigen-binding domains of the bispecific antibody provided herein also cover an antigen-binding domain, in which the heavy chain variable region and/or light chain variable region contain one or more (preferably no more than 10, more preferably no more than 6, 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) , compared with the heavy chain variable region and/or light chain variable region of the antibody specifically disclosed herein, wherein the amino acid change does not occur in the CDR region.

Fc domain variant

In embodiments in accordance with various aspects of the present invention, the bispecific antibody further comprises an Fc domain.

In some embodiemnts, the first and the second of the two subunits of the Fc domain are identical or non-identical.

In certain aspects, one or more amino acid modifications may be introduced into the Fc domain of an antibody provided herein, thereby generating an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

The present invention provides antibody variants possessing some but not all effector functions that make the antibody variants desirable candidates for applications wherein, e.g., the in vivo half-life of the antibody is important, and certain effector functions (such as complements and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, an Fc receptor (FcR) binding assay can be conducted to ensure that an antibody lacks FcγR binding (and therefore may lack ADCC activity or antibody cross-linking activity) , but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, only express Fc γ RIII, while monocytes express Fcγ RI, Fcγ RII and Fcγ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Tmmunol ·9: 457-492 (1991) . The binding sites with FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been depicted, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276: 6591-6604, 2001) .

Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149) . Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent Nos. 7,332,581 and 8,219,149) .

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2) : 6591 -6604 (2001) . ) In certain instances, an antibody variant comprises an Fc domain with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc domain (EU numbering of residues) . In some instances, alterations are made in the Fc domain that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) , e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .

Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) , which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) . Those antibodies comprise an Fc domain with one or more substitutions therein which improve binding of the Fc domain to FcRn. Such Fc variants include those with substitutions at one or more of Fc domain residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 or 434, e.g., substitution of Fc domain residue 434 (US Patent No. 7,371,826) . See also Duncan &Winter, Nature 322: 738-40 (1988) ; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821, and WO 94/29351 concerning other examples of Fc domain variants.

In one aspect, the antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of the glycosylation sites of an antibody can be conveniently achieved by changing the amino acid sequence so as to produce or remove one or more glycosylation sites. Glycosylation can be changed, for example, to increase affinity of the antibody for the “antigen” . Such carbohydrate modification can be accomplished, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which results in the elimination of one or more variable region framework glycosylation sites, thereby eliminates glycosylation at this site. This aglycosylation can increase affinity of the antibody for the antigen. Such a method is described in, for example, U.S. Patent No. 5,426,300. When the antibody comprises an Fc domain, the saccharides attached to same can be changed. In some applications, modifications to remove undesired glycosylation sites are useful, such as removal of fucose modules to improve antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In other applications, galactosylation modification can be made to modify complement-dependent cytotoxicity (CDC) .

Fc domain modifications promoting heterodimerization

Bispecific antibodies may comprise different components (e.g. antigen binding domains) fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.

In one aspectθthe Fc domain of the bispecific antibodies provided herein comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain. The invention can use any of the known approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, e.g. which are well described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291.

In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain. The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) .

In a particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index) .

Modifications in the Fab domains

Multispecific antibodies with a domain replacement/exchange in one binding arm (CrossMabVH-VL or CrossMabCL-CH1) are described in detail in WO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 1 1187-1191. They clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange) .

In one aspect, the invention relates to a bispecific antibody comprising

(a) a first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to CD20, and

(c) an Fc domain composed of a first and a second subunit capable of stable association, wherein in one of the Fab fragments, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged. The bispecific antibodies are prepared according to the Crossmab technology. In one aspect, in one of the Fab fragments, the constant domains CL and CH1 are replaced by each other so that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain. More particularly, in the first Fab fragment or in the second Fab fragment, the constant domains CL and CH1 are replaced by each other so that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain.

In another aspect, and to further improve correct pairing, the bispecific antibody comprising a Fab fragment can contain different charged amino acid substitutions (so-called "charged residues" ) . These modifications are introduced in the crossed or non-crossed CHI and CL domains. In a particular aspect, the invention relates to a bispecific antibody, wherein in one of CL domains the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been substituted by lysine (K) and wherein in one of the CHI domains the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been substituted by glutamic acid (E) .

Cysteine engineered antibody variants

In one aspect, it may be desirable to produce cysteine engineered antibody, e.g., “thioMAb” , in which one or more residues of the antibody are substituted with cysteine residues.

Antibody derivatives

In one aspect, the antibody provided herein may be further modified to comprise additional non-protein moieties that are known in the art and readily available. A moiety suitable for antibody derivatization includes, but is not limited to, a water soluble polymer. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , ethylene glycol/propylene glycol co-polymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dialkane, poly-1, 3, 6-trialkane, ethylene/maleic anhydride co-polymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol) , polyvinyl alcohol, and mixtures thereof.

Immunoconjugates

The invention also provides immunoconjugates comprising bispecific antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) , radioactive isotopes , or biologically active polypeptides.

In one aspect, an immunoconjugate is an antibody-drug conjugate (ADC) in which a bispecific antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1) ; an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298) ; a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701 , 5,770,710, 5,773,001 , and 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993) ; and Lode et al., Cancer Res. 58: 2925-2928 (1998) ) ; an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006) ; Jeffrey et al., Bioorganic &Med. Chem. Letters 16: 358-362 (2006) ; Torgov et al., Bioconj. Chem. 16: 717-721 (2005) ; Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000) ; Dubowchik et al., Bioorg. &Med. Chem. Letters 12: 1529-1532 (2002) ; King et al., J. Med. Chem. 45: 4336-4343 (2002) ; and U.S. Patent No. 6,630,579) ; methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC-1065.

In one aspect, an immunoconjugate comprises a bispecific antibody as described herein conjugated to one or more enzymatically active toxins or fragments thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In one aspect, an immunoconjugate comprises a bispecific antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸ Re, ¹⁵³Sm, ²¹²Bi, and ³²P and radioactive isotopes of Lu.

In one aspect, an immunoconjugate comprises a bispecific antibody as described herein conjugated to one or more polypeptides. In some embodiments, polypeptide is a ligand, such as a lymphokine and/or a cellular factor which interacts with a specific cellular receptor. Lymphokines are low molecular weight proteins which are secreted by T cells when antigens or lectins stimulate T cell growth. Examples of lymphokines include, but are not limited to, interferon-α, interferon-γ, interleukin-1 (IL-1) , interleukin-2 (IL-2) , interleukin-3 (IL-3) , tumor necrosis factor (TNF) , a colony stimulating factor (e.g. CSF-1, G-CSF or GM-CSF) , a chemotaxin, macrophage migration inhibitory factor (MIF) , macrophage-activating factor (MAF) , NK cell activating factor, T cell replacing factor, leukocyte-inhibitory factor (LIF) , a lymphotoxin, osteoclast-activating factor (OAF) , soluble immune response suppressor (SIRS) , growth-stimulating factor, monocyte growth factor.

Techniques for conjugating a therapeutic moiety to bispecific antibody are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy" , cited in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (authoring) , pp. 243-256 (Alan R. Liss, Inc. 1985) .

Production and purification of antibody

The invention further provides isolated polynucleotides encoding an antibody of the invention as described herein, or a fragment thereof.

The isolated polynucleotides encoding antibodies of the invention may be expressed as a single polynucleotide that encodes the entire antibody or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional antibody.

In certain embodimentsθthe polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA) . RNA of the present invention may be single stranded or double stranded.

According to another aspect of the invention, there is provided an isolated polynucleotide encoding an antibody as defined herein before or a fusion polypeptide as described herein before. The invention further provides a vector, particularly an expression vector, comprising the isolated polynucleotide of the invention and a host cell comprising the isolated polynucleotide or the vector of the invention. In one aspect the host cell is a eukaryotic cell, particularly a mammalian cell.

Antibodies of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the antibody or polypeptide fragments thereof, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one aspect of the invention, a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antibody (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the bispecific antibody or polypeptide fragments thereof (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.

In a further aspect of the invention, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one aspect, a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) an antibody of the invention. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the fusion proteins of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of antibodies are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the antibody for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO) , human embryonic kidney (HEK) cells, insect cells, or the like.

In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, expi-293 cell , a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NSO, Sp20 cell) . Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain, may be engineered so as to also express the other of the immunoglobulin chains such that the expressed product is an antigen binding domain that has both a heavy and a light chain.

In another aspect, provided is a method for producing the antibody of the invention, comprising the steps of (i) culturing the host cell of the invention under conditions suitable for expression of said antibody, and (ii) isolating said antibody form the host cell or host cell culture medium.

Antibodies of the invention prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the antibody binds. For example, for affinity chromatography purification of antibodiesof the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an antibody essentially as described in the Examples. The purity of the antibody or fragments thereof can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the antibodies expressed as described in the examples were shown to be intact and properly assembled as demonstrated by reducing and non-reducing SDS-PAGE.

Pharmaceutical composition

The term “pharmaceutical composition” refers to such a preparation/formulation that allows the active ingredient contained therein existing in a form to be biological activity effective and does not contain additional ingredients that have unacceptable toxicity to the subject to whom the preparation/formulation is administrated.

The term “pharmaceutical excipient” refers to a pharmaceutical carrier, a diluent, an adjuvant (e.g., Freund’s adjuvant (complete and incomplete) ) , or an excipient, which is administered with a therapeutic agent.

The pharmaceutical composition of the present invention may include the antibody of the present invention and a pharmaceutical excipient. These pharmaceutical compositions can be included in kits, such as diagnostic kits.

As used herein, a “pharmaceutical carrier” includes any and all solvents, dispersion medium, isotonic agents, absorption delaying agents, etc., that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. It is also possible to use saline solutions, aqueous dextrose and glycerol solutions as liquid carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, diol, water, ethanol, etc. For the application of excipients and uses thereof, see also “Handbook of Pharmaceutical Excipients” , fifth edition, R. C. Rowe, P. J. Seskey and S. C. Owen, Pharmaceutical Press, London, Chicago. The composition may also contain a small amount of a wetting agent or an emulsifier, or a pH buffering agent. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release agents, etc. Oral formulation can comprise standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, etc.

The present invention provides a pharmaceutical composition comprising one or more bispecific antibodies, and nucleic acids, vectors or host cells, or immunoconjugates or fusions of the present invention. It should be understood that the bispecific antibodies, a nucleic acid, a vector or a host cell thereof, or an immunoconjugate, or a fusion, or a pharmaceutical composition thereof provided by the present invention can be integrated with suitable pharmaceutical carriers, excipients and other reagents in the preparation for co-administration, so as to provide improved transfer, delivery, tolerance, etc.

The pharmaceutical preparation/formulation comprising the bispecific antibodies described herein can be prepared by mixing the bispecific antibodies of the present invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of aqueous solutions or lyophilized preparations. Exemplary lyophilized antibody preparations/formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody preparations/formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter preparation/formulation including a histidine-acetate buffering agent.

The pharmaceutical compositions or preparations/formulations of the present invention may also comprise one or more other active ingredients that are required for the treatment of specific diseases, preferably those active ingredients with complementary activities that do not adversely affect each other. For example, it is desirable that other therapeutic agents are also included. In one aspect, the other therapeutic agents are chemotherapeutic agents, radio therapeutic agents, cytokines, vaccines, other antibodies, immunomodulators or other biomacromolecular drugs.

In one aspect, the pharmaceutical composition of the present invention may also comprise nucleic acids encoding the bispecific antibodies.

Method of use

In one aspect, the present invention provides a method for preventing, diagnosing or treating CD47-related and/or CD20-related disease or condition in a subject. The method comprises administering to a patient in need thereof an effective amount of the bispecific antibody described herein, or an immunoconjugate or an immune fusion comprising the antibody or a pharmaceutical composition comprising the bispecific antibody, or immunoconjugate or immune fusion, or a nucleic acid, a vector or a host cell described herein.

In one aspect, the present invention provides the use of the bispecific antibodies in the production or preparation of drugs for the prevention, diagnosis or treatment of CD47-related and/or CD20-related disease or condition in subjects.

In one aspect, the bispecific antibody described herein, or the immunoconjugate or immune fusion comprising the antibody or a pharmaceutical composition comprising the bispecific antibody, or immunoconjugate or immune fusion provided by the present invention can be used as therapeutic agents to prevent or treat CD47-related and/or CD20-related disease or condition in subjects. For CD47-related and/or CD20-related disease or condition in subjects identified by using standard methods, the bispecific antibodies, and pharmaceutical compositions or immunoconjugates or immune fusion disclosed in the present invention, or the nucleic acids, vectors or host cells described herein can be administered.

In one aspect, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent or therapeutic mode. In one aspect, the therapeutic agents are, for example, chemotherapeutic agents, radio therapeutic agents, cytokines, vaccines, other antibodies, immunomodulators or other biomacromolecular drugs. In one aspect, the therapeutic mode includes surgery; and radiation therapy, local irradiation or focus irradiation, etc.

The above-mentioned combination therapy includes combined administration (in which two or more of therapeutic agents are contained in the same or separate preparations/formulations) and separate administration, wherein the administration of the bispecific antibody of the present invention may occur prior to, simultaneously with, or after administration of additional therapeutic agent and/or adjuvant and/or treatment.

In one aspect, the CD47-related disease or condition of the present invention refer to disease or condition related to abnormal CD47 expression, activity and/or signal transmission in a subject, including but not limited to cancer. In one aspect, in CD47-related disease or condition, the (level or content) of nucleic acid encoding CD47 is increased, or CD47 expression is increased, or CD47 protein level is increased, or activity is increased, or activity signal transmission is increased, e.g., compared to those in the healthy subject, or to the adjacent tissue, cell or organ that has no such diseases or conditions (e.g., non-cancer tissue, cell or organ) ..

In one aspect, the CD20-related disease or condition of the present invention refer to disease or condition related to abnormal CD20 expression, activity and/or signal transmission in a subject, including but not limited to cancer. In one aspect, in CD20-related disease or condition, the (level or content) of nucleic acid encoding CD20 is increased, or CD20 expression is increased, or CD20 protein level is increased, or activity is increased, or activity signal transmission is increased, e.g., compared to those in the healthy subject, or to the adjacent tissue, cell or organ that has no such diseases or conditions (e.g., non-cancer tissue, cell or organ) .

In one aspect, the treatment of the disease will benefit from the inhibition of CD47 in nucleic acid or protein levels, or benefit from blocking of the binding of CD47 to its ligand or CD47-mediated signal transmission. In one aspect, the treatment of the disease will benefit from the inhibition of CD20 in nucleic acid or protein levels, or benefit from blocking of the binding of CD20 to its ligand or CD20-mediated signal transmission. In one aspect, the treatment of the disease will benefit from the inhibition of CD47 and CD20 in nucleic acid or protein levels, or benefit from blocking of the binding of CD47 and CD20 to its ligands, or, CD47-and CD20-mediated signal transmission.

In one aspect, the subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or having a risk of suffering from a disease described herein) . In one embodiment, the subject suffers from or has a risk of suffering from a disease described herein (e.g., cancer) . In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy.

In one aspect, the cancer includes various hematological cancer and solid tumors, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be at an early stage, a middle stage or a late stage, or a metastatic cancer.

In a specific embodiment, the hematological cancer is including but not limited to acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , Non-Hodgkin lymphoma (e.g., Burkitt’s lymphoma) , B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL) , chronic myelocytic leukemia (CML) , follicular lymphoma, small lymphotic lymphoma (SLL) , central nervous system (CNS) lymphoma, Richter’s Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and anaplastic large cell lymphoma.

In a specific embodiment, the solid tumors is including but not limited to breast cancer, gastric cancer, an osteosarcoma, desmoplastic small round cell cancer, squamous cell carcinoma of head and neck cancer, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, melanoma, Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma.

The antibody of the present invention may be administered in any suitable manner, including oral, parenteral, intrapulmonary and intranasal administration, and, if topical treatment is needed, it can be administered intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various administration regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

The antibody of the present invention will be formulated and administered in a manner consistent with good medical practice. Not necessarily, but optionally, the antibody is formulated with one or more agents currently used to prevent or treat the disease. The effective amount of these other agents depends on the amount of antibody present in the preparation/formulation, the type of condition, or disease to be treated, and other factors discussed above. In order to prevent or treat disease or condition, the antibody or antigen-binding fragment of the present invention will be administered in a suitable dose depending on the type of disease or condition to be treated, the type of antibodies, the severity and course of the disease, whether the antibody is for the purpose of prevention or treatment, the previous treatment, the patient’s clinical history and response to antibodies and the judgment of the attending physician. The antibody is appropriately administered to the patient at one time or over a series of treatments.

In certain embodiments, any bispecific antibody provided herein can be used to detect the presence of CD47 and/or CD20 in a sample. In one aspect, the detection method includes:

(a) contacting the sample with the antibody, or an antigen-binding fragment or conjugate or fusion thereof of the present invention; and

(b) detecting the formation of the complex of the antibody or antigen-binding fragment thereof or the conjugate or the fusion and the CD47 and/or CD20 protein.

The term “detection” when used herein includes quantitative or qualitative detection. In certain embodiments, the sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the sample comprises cells or tissues. In one aspect, the sample is from hyperproliferative or cancerous focus related focus.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention can be used to diagnose CD47-related and/or CD20-related disease or condition, such as cancer, for example to evaluate (e.g., monitor) the treatment response or progression of the disease or condition described herein, and diagnosis and/or staging thereof in an individual. In certain embodiments, a labeled bispecific antibody is provided. Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) , and parts that are indirectly detected, such as enzymes or ligands, for example, by enzymatic reactions or intermolecular interactions. In one aspect, provided herein is a kit for diagnosing CD47-related and/or CD20-related disease or condition, which kit comprises the antibody or antigen-binding fragment thereof of the present invention.

In one aspect provided herein, the sample is obtained prior to treatment with the bispecific antibody. In one aspect, the sample is obtained prior to treatment with other therapies. In one aspect, the sample is obtained during or after treatment with other therapies.

Kits and Articles of Manufacture

In another aspect of the invention, a kit or article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The kit or article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle) . At least one active agent in the composition is a bispecific antibody of the invention.

The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the kit or article of manufacture may comprise a first container with a composition contained therein, wherein the composition comprises a bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.

Alternatively, or additionally, the kit or article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The present invention includes any combinations of specific embodiments described herein. It should be understood that although the specific content and examples are described to illustrate the preferred embodiments of the present invention, these are merely illustrative and used as examples. The present invention further covers embodiments modified on the basis of the preferred embodiments of the present invention that are obvious to a person skilled in the art. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety.

EMEMPLARY EMBODIMENTS

Embodiment 1. A bispecific antibody comprises

Embodiment 2. The bispecific antibody of Embodiment 1, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

Embodiment 3. The bispecific antibody of

Embodiments

1 or 2, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

Embodiment 4. The bispecific antibody of any one of Embodiments 1 to 3, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD47 are selected from

Embodiment 5. The bispecific antibody of any one of Embodiments 1 to 4, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.

Embodiment 6. The bispecific antibody of any one of Embodiments 1 to 5, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

Embodiment 7. The bispecific antibody of Embodiment 6, wherein the VH of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37, and the VL of the antigen-binding domain capable of specifically binding to CD20 comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.

Embodiment 8. The bispecific antibody of any one of Embodiments 1 to 7, wherein the bispecific antibody comprises

Embodiment 9. The bispecific antibody of any one of Embodiments 1 to 8, wherein the bispecific antibody comprises

Embodiment 10. The bispecific antibody of Embodiment 9, wherein

Embodiment 11. The bispecific antibody of any one of Embodiments 1 to 10, wherein any one of the antigen-binding domain is chimeric, fully human or humanized.

Embodiment 12. The bispecific antibody of any one of Embodiments 1 to 11, wherein any one of the antigen-binding domain comprises a Fab fragement or a scFv fragement.

Embodiment 13. The bispecific antibody of any one of Embodiments 1 to 12, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement and the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement or a Fab fragement.

Embodiment 14. The bispecific antibody of any one of Embodiments 12 to 13, wherein the scFv fragement comprises a peptide linker connecting the C terminus of a VH to the N-terminus of a VL.

Embodiment 15. The bispecific antibody of Embodiment 1 to 14, wherein the bispecific antibody is bivalent, trivalent or tetravalent.

Embodiment 16. The bispecific antibody of any one of Embodiments 1 to 15, wherein the bispecific antibody comprises

(a) a first Fab fragement capable of specifically binding to CD47,

(b) a second Fab fragement capable of specifically binding to CD20, and

Embodiment 17. The bispecific antibody of Embodiment 16, wherein the second Fab fragement is CrossFab (CL-CH1) .

Embodiment 18. The bispecific antibody of any one of Embodiments 1 or 17, wherein the bispecific antibody comprises

(i) a first polypeptide chain in the format of: VH (anti-CD47) –CH1-Fc domain subunit,

(ii) a second polypeptide chain in the format of: VL (anti-CD47) –CL,

(iv) a forth polypeptide chain in the format of: VL (anti-CD20) –CH1.

Embodiment 19. The bispecific antibody of any one of Embodiments 1 to 18, wherein the bispecific antibody comprises

(ii) a second polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61,

Embodiment 20. The bispecific antibody of any one of Embodiments 1 to 15, wherein the bispecific antibody comprises

(a) two scFv fragments capable of specifically binding to CD47, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain,

wherein each of the two scFv fragments is connected to the whole antibody.

Embodiment 21. The bispecific antibody of Embodiment 20, wherein each of the two scFv fragements is connected via a peptide linker to the whole antibody.

Embodiment 22. The bispecific antibody of any one of Embodiments 20 to 21, wherein each of the two scFv fragements is connected via a peptide linker to the C-terminus of the two heavy chains of the whole antibody.

Embodiment 23. The bispecific antibody of any one of Embodiments 14, 21 to 22, wherein the peptide linker comprises a GS linker.

Embodiment 24. The bispecific antibody of any one of Embodiments 14, 21 to 23, wherein the peptide linker comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 39, 40, 41, 42, 43, or 44.

Embodiment 25. The bispecific antibody of any one of Embodiments 20 to 24, wherein the scFv fragement comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 45 or 46.

Embodiment 26. The bispecific antibody of any of Embodiments 20-25, wherein the two heavy chains and two light chains of a whole antibody are selected from:

(1) each of the heavy chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, 48, 49 or 78 and each of the light chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50; or

(2) each of the heavy chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51 or 52 and each of the light chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.

Embodiment 27. The bispecific antibody of any one of Embodiments 1 to 15, wherein the bispecific antibody comprises an Fc domain.

Embodiment 28. The bispecific antibody of any one of Embodiments 16 to 27, wherein the Fc domain is human IgG1Fc domain.

Embodiment 29. The bispecific antibody of any one of Embodiments 16 to 28, wherein the Fc domain comprises one or more amino acid modification (s) promoting the association of the first and the second subunits of the Fc domain and/or one or more amino acid modification (s) increasing the binding affinity of the antibody to an FcRn.

Embodiment 30. The bispecific antibody of any one of Embodiments 16-29, wherein the Fc domain comprises one or more amino acid substitution (s) at positions 428 and 434, preferably said amino acid substitutions are M428L and N434S (numbering according to Kabat EU index) .

Embodiment 31. The bispecific antibody of any one of Embodiments 16 to 30, wherein each of the two subunits of the Fc domain comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 66, 67, 68, 69 or 77 .

Embodiment 32. An isolated nucleic acid, encoding the bispecific antibody of any one of Embodiments 1-31.

Embodiment 33. A recombinant vector or an expression vector, comprising one or more nucleic acids of Embodiment 32, wherein the vector is suitable for the recombinant production of the bispecific antibody of any one of Embodiments 1-31.

Embodiment 34. A host cell, comprising one or more recombinant vectors or expression vectors of Embodiment 33.

Embodiment 35. An immunoconjugate, comprising the bispecific antibody of any one of Embodiments 1-31.

Embodiment 36. A pharmaceutical composition, comprising the bispecific antibody of any one of Embodiments 1-31, the nucleic acid of Embodiment 32, the vector of Embodiment 33, the host cell of Embodiment 34, or the immunoconjugate of Embodiment 35, and optionally comprising a pharmaceutically acceptable excipient.

Embodiment 37. A method for treating or preventing disease or condition in a subject, comprising administering to an individual an effective amount of the bispecific antibody of any one of Embodiments 1-31, the nucleic acid of Embodiment 32, the vector of Embodiment 33, the host cell of Embodiment 34, or the immunoconjugate of Embodiment 35, or the pharmaceutical composition of Embodiment 36.

Embodiment 38. The method of Embodiment 37, wherein the disease or condition is CD47-related and/or CD20-related.

Embodiment 39. The method of any one of Embodiments 37 -38, wherein the disease or condition is cancer.

Embodiment 40. The method of Embodiment 39, wherein the cancer is hematological cancer, for example acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , Non-Hodgkin lymphoma (e.g., Burkitt’s lymphoma) , B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL) , chronic myelocytic leukemia (CML) , follicular lymphoma, small lymphotic lymphoma (SLL) , central nervous system (CNS) lymphoma, Richter’s Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and anaplastic large cell lymphoma. chronic lymphoid lymphoma (CLL) and non-Hodgkin’s lymphoma (NHL) .

Embodiment 41. Use of the bispecific antibody of any one of Embodiments 1-31 , the nucleic acid of Embodiment 32, the vector of Embodiment 33, the host cell of Embodiment 34, or the immunoconjugate of Embodiment 35, or the pharmaceutical composition of Embodiment 36 in the manufacture of a medicament for treating a disease or condition in a subject.

Example 1 Preparation and screening of hybridoma-derived antibody

The anti-CD47 antibodies were obtained by hybridoma technique. The recombinant protein CD47-Fc (ACROBiosystems, Cat: CD7-H5256) containing the extracellular domain of human CD47 with a Fc tag was used as an antigen to immunize mice. After mixing and emulsifying the recombinant protein CD47-Fc with complete or incomplete Freund’s adjuvant (Sigma-Aldrich) , SJL mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) and BALB/c mice (Yangzhou University Medical Center) were immunized. The mice were subjected to one round of immunization (complete Freund’s adjuvant) and two rounds of booster immunization (incomplete Freund’s adjuvant) and taken blood after each booster immunization. The binding activity of the serum of the mice after immunization to the recombinant human CD47-Fc (ACROBiosystems, Cat: CD7-H5256) protein is detected by ELISA assay, and at the same time, the binding potency of mice serum to CHO cells (constructed by GenScript) overexpressing human CD47 was detected by flow cytometry (FACS) . Spleen cells of the mice with a higher serum titer were selected to fuse with myeloma cell line SP2/0 (ATCC) . Four days before fusion, the recombinant protein CD47-Fc of human CD47 extracellular domain was intraperitoneally injected into mice for booster immunization. On the day of fusion, mice were euthanized, and then mouse spleen cells were homogenized to obtain a single cell suspension. The mouse spleen cells were fused with murine myeloma cell line SP2/0 (3: 1) by means of an electrofusion apparatus. The fused cells were resuspended in a medium containing HAT (hypoxanthine, aminopterin and thymidine deoxynucleotide, GIBCO, Cat: 21060016) to screen the successfully fused hybridoma cells. The supernatant of hybridoma cells was collected and the hybridoma cells that secreted antibodies specifically binding to human CD47 were screened by two rounds of ELISA. Then, the activity of secretion supernatant of the hybridoma was determined by CD47-related functional screening tests (such as binding specificity with human CD47 or cynomolgus monkey CD47; no activity in induction of red blood cell agglutination; activity in promoting phagocytosis of tumor cells by macrophages) , and then the positive hybridoma clones were selected and subcloned for single or multiple rounds to obtain monoclone. After screening, 125G4A4 was finally chosen as a hybridoma clone.

The candidate hybridoma cell 125G4A4 was subjected to an expanded culture, and after 7-10 days of culturing, the supernatant was collected, centrifuged and filtered to remove cells and debris. The supernatants were passed through a Protein A purification column (GenScript) , then cleaned and equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0) , and then eluted with 0.1 M sodium citrate (pH 3.5) ; and the eluent was immediately neutralized with one ninth volume of 1 M Tris-HCl (pH 9) , and then dialyzed with PBS buffer. Finally, the hybridoma-derived antibody 125G4A4 was obtained for further characterization.

1.1 Detection of binding activities of antibodies to CHO-K1 cells overexpressing human CD47 proteins by FACS

A human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in hamster ovary cell line CHO-K1 to establish CHO-K1 cell line overexpressing the human CD47 protein. The cells were co-incubated with serially diluted antibody 125G4A4 and reference antibody C0774CK230-C (i.e., Hu5F9) (the highest concentration being 300 nM, three fold dilution, 12 concentration points in total) at 4℃ for 50 minutes. After washing twice with iced PBS, the cells were incubated with an iFluor647-labled goat anti-mouse IgG (H + L) antibody (Genscript) at 4℃ in the dark for 40 minutes. The cells were washed twice with iced PBS, and then the fluorescence signal was detected by Calibur (BD Biosciences) flow cytometry, and according to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated. As shown in Table 1, the finally obtained hybridoma-derived antibody 125G4A4 has a high binding activity to CHO-K1 overexpressing human CD47 protein, with an EC ₅₀ of 0.22 nM.

1.2 Detection of binding activities of antibodies to CD47 on the surface of tumor cells by FACS

Human CD47 was endogenously expressed on the cell surface of human Burkitt lymphoma cell line Raji. The antibody 125G4A4 and the reference antibody Hu5F9 were serially diluted into PBS containing 2%fetal bovine serum (FBS, Gibco, Cat: 10100147) (the highest concentration being 46.3 nM, three fold dilution, 8 concentration points in total) . The diluted antibodies were mixed with and co-incubated with Raji cells (purchased from ATCC) (5*10 ⁵ cells /well) at 4℃ for 1 hour. After washing three times with PBS containing 2%fetal bovine serum (FBS) , a PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added and incubated with the cells at 4℃ in the dark for 1 hour. The cells were washed three times with PBS containing 2%fetal bovine serum (FBS) , and then the fluorescence signal was detected by CantoII (BD Biosciences) flow cytometry, and according to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated. As shown in Table 1, the hybridoma-derived antibody 125G4A4 has a binding activity to Raji cells, with an EC ₅₀ of 0.84 ± 0.02 nM.

1.3 Blockade of the interaction between human CD47 and SIRPα by anti-CD47 antibodies

ELISA assay was performed to detect the ability of 125G4A4 to block the interaction between human CD47 and SIRPα. The recombinant protein hCD47-Fc containing the extracellular domain of human CD47 fused with the Fc fragment of human IgG (ACROBiosystems, Cat: CD7-H5256) was coated onto a 96-well plate and incubated overnight at 4℃. After the plate was washed 3 times with PBST (PBS containing 0.5%Tween-20) , PBST containing 1%BSA was added for blocking the plate for 2 hours. After the plate was washed three times with PBST, the mixture of serially diluted antibody 125G4A4 or reference antibody Hu5F9 (the highest concentration being 66.7 nM, three fold dilution, 8 concentration points in total) together with SIRPα-His recombinant protein (ACROBiosystems, Cat: SIA-5225) with a final concentration of 2.5 μg/ml was added and incubated at room temperature for 1 hour. The plate was washed three times with PBST, and a horseradish peroxidase labeled goat anti-His-tag secondary antibody (CWBIO, Cat: CW0285M) was added to detect SIRPα captured by coated CD47 protein. After the 96-well plate was incubated at 37℃ for 30 minutes, the plate was washed 5 times with PBST, and a TMD (Surmodics, Cat: TMBW-1000-01) developing solution was added, and was incubated in the dark for 15 minutes. 2N H ₂SO ₄ was added to terminate the color-developing reaction. OD ₄₅₀ was read on a microplate reader. The absorbance value reflected the amount of SIRPα which bound to CD47. Graphpad was used for fitting a concentration dependent curve, and the IC ₅₀ of anti-CD47 antibody for blocking binding of CD47 to SIRPα was calculated. As shown in Table 1, 125G4A4 can effectively block the CD47/SIRPα interaction, with an IC ₅₀ of 3.06 nM.

1.4 Detection of activity of anti-CD47 antibodies in induction of human red blood cell agglutination

It is known that in the prior art, most of the anti-CD47 antibodies have the property of inducing red blood cell agglutination. It is widely believed that the property is closely related to clinical side effects such as anemia existing in the treatment by therapeutic anti-CD47 antibody. Therefore, we evaluate the anti-CD47 antibody in the present invention by a red blood cell agglutination experiment in vitro to screen the antibody without the property of inducing red blood cell agglutination. The method is as follows: collecting the healthy donor’s fresh human blood, washing the cells five times with PBS, and then diluting the cells to make a suspension containing 10%human red blood cells; mixing the red blood cell suspension with the experimental antibody (antibody 125G4A4 and reference antibody Hu5F9, the highest concentration being 667 nM, three fold dilution, 12 concentration points in total) , then adding the mixture into a round bottom 96-well plate; and incubating them at room temperature for 16 hours, then taking photos and determining the results according to the phenomenon of the cells in the well. If red blood cell agglutination occurs, cells are plated onto each well like a net, and a larger sheet-like cell layer will appear in the well with a diameter larger than that of the negative control well; on the contrary, if no hemagglutination occurs, the red blood cells will deposit at the bottom of the well, and smaller dot-like cell pellete precipitation will appear in the well. 125G4A4 shows no obvious phenomenon of inducing red blood cell agglutination in the experiment.

1.5 Determination of the pro-phagocytic effects of anti-CD47 antibodies on tumor cells by human macrophages

The ability of antibody 125G4A4 of the present invention to promote phagocytosis of tumor cells by macrophages was detected by assay based on flow cytometry. Human blood was freshly collected from healthy donors, and the peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) . Monocytes were further isolated and obtained by using the human total monocyte Isolation Kit (Miltenyi biotec, cat: 130-096-537) . To induce the monocytes to differentiate into macrophages, macrophage colony stimulating factor (M-CSF, R &D Systems, Cat: 216-MC) was added and the monocytes was subjected to adherent culture for 7 consecutive days. On the day of cellular phagocytosis experiment, the above-mentioned differentiated macrophages were starved in a serum-free medium for 2 hours. At the same time, target tumor cells Raji were flurorescent labeled with CFSE (eBioscience, Cat: 65-0850-85) according to the steps recommended by the instructions. The CFSE-labeled tumor cells and macrophages were mixed in a ratio of 4 : 1, and the experimental antibodies of a detected concentration were added and incubated at 37℃ for 2 hours. Then the cells were washed twice with PBS, and then digested with trypsin (Gibco, Cat: 25200072) ; an APC labeled anti-CD14 antibody (Biolegend, Cat: 325608) was added and incubated in the dark on ice in PBS containing 2%fetal bovine serum for 30 minutes. The cells were washed twice and analyzed by flow cytometry. The percentage of CFSE positive cells in CD14 positive macrophage populations was calculated. As shown in Table 1, 125G4A4 can effectively promote the phagocytic function of macrophages on tumor cells.

Table 1 Determination of the activity and function of hybridoma-derived antibody 125G4A4

Example 2 Humanization of hybridoma-derived antibody

2.1 Determination of variable region sequence of hybridoma-derived antibody

According to the method for hybridoma sequencing, the cells of hybridoma clone 125G4A4 were subjected to an expanded culture; total RNA was extracted with TRIzol (purchased from Ambio) and reverse transcribed into DNA with antibody-specific primers (Takara, PrimerScript 1 ^st Strand cDNA Synthesis Kit) ; and a gene fragment encoding mouse immunoglobulin V-region was subjected to amplification with antibody-specific primers. The variable region sequence of hybridoma-derived antibody was obtained by sequencing analysis. The amino acid sequences of the heavy chain variable region and the light chain variable region of the 125G4A4 antibody are as set forth in SEQ. ID Nos: 1 and 2, respectively, and the nucleotide sequences are as set forth in SEQ. ID Nos: 19 and 20, respectively.

2.2 Construction and expression of chimeric antibodies

According to the mechanism of action of CD47, in a specific embodiment of the present invention, the constant region of human IgG4 (S228P) is used as the heavy chain constant region of the antibody, and the human κ light chain constant region chain is used as the light chain constant region of the antibody. Mutation of serine at position 228 of IgG4 core hinge region to proline (S228P) can enhance the disulfide bond connection in the core hinge region and reduce the exchange of IgG4 Fab arm, and thereby greatly reduce the formation of half molecules. After the genes encoding heavy chain and light chain constant regions were synthesized, the heavy chain and light chain variable region genes were homologously recombined into a vector PTT5 with double enzyme digestion by EcoRI and BamHI. After sequenced to be correct, the heavy chain and light chain of an antibody at a molar ratio of 1.5: 1are co-transfected into HEK293 cells. After 120 hours of culture, the supernatant was collected by centrifugation and purified to obtain a chimeric antibody.

Before humanization design, it is necessary to mutate some post translational modification (PTM) sites in the CDR region to avoid affecting the protein conformation, thereby affecting the function thereof. According to PTM analysis, two PTM sites in the CDR of 125G4A4 were identified, including one NSS glycosylation site in the heavy chain and one DG isomerization site in the light chain. The NSS glycosylation site and DG isomerization sites were mutated into QSS and EG, respectively. The mutated chimeric antibody obtained by purification in this example is named as Ch-125G4-m35. The amino acid sequences of the heavy chain variable region and the light chain variable region of Ch-125G4-m35 antibody are as set forth in SEQ. ID Nos: 3 and 4, respectively.

2.3 Humanized design of chimeric antibodies

To select the human antibody backbone sequence with highest similarity to chimeric antibody 125G4A4m for humanization, the variable region sequence of chimeric antibody 125G4A4m was Blast aligned with the PDB Antibody database., The heavy chain variable region of 125G4A4 m has a higher sequence homology with human germline IGHV1-69, and the light chain variable region thereof has a higher sequence homology with human germline IGKV1-16. Then the amino acid sequence of the variable region CDR and the accurate boundary thereof are defined by the Kabat assignment system. Then, the CDR segments of the variable region of the murine antibody are grafted into the human backbone sequence to obtain the humanized antibody.

In order to maintain the activity of the humanized antibody, the framework amino acid sequences of the variable region and its surrounding region are analyzed with macromolecular docking analysis by using computer simulation technology to investigate their spatial stereoscopic binding mode. By calculating electrostatic force, van der Waals force, hydrophilicity and entropy, the key amino acid individuals that may interact with CD47 and maintain the spatial framework in the candidate antibody gene sequence are analyzed and grafted back to the selected human antibody gene framework. Meanwhile the amino acid positions in the framework region that must be reserved are marked. Based on the above process, the humanized antibody is synthesized. Some key sites in the antibody framework region were back mutated into the antibody framework region sequence of chimeric antibody Ch-125G4-m35. According to the number and arrangement of back mutations, a number of different humanized heavy chain variable regions (SEQ. ID No: 5, SEQ. ID No: 6, SEQ. ID No: 7) and light chain variable regions (SEQ. ID No: 8, SEQ. ID No: 9, SEQ. ID No: 10) were designed respectively (see Table 2) . The finally determined humanized antibody Hu-125G4A4-48 of the present invention was named as HMA02h14-48 hereafter. The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are as set forth in SEQ. ID Nos: 7 and 8, respectively.

Table 2 Number and arrangement of reverse mutations in 125G4A4m heavy chain and light chain

Table 3 Amino acid sequence of anti-CD47 antibody

Table 4 CDR amino acid sequences of anti-CD47 antibodies (Kabat definition)

2.4 Expression of humanized antibodies

The DNA fragments encoding the above-mentioned designed humanized heavy chain and light chain variable regions were amplified and cloned into a vector comprising a constant region expressing a human antibody to construct an antibody-expressing plasmid (pCDNA3.4, purchased from Thermo Cat#A14697) . The heavy and light chain expression vectors were co-transfected into Expire293 cells (Thermo Cat#A14525) . After culturing at 37℃ for 6 days, the supernatant was collected. According to the above-mentioned method, the recombinant antibody was obtained by protein A affinity purification for further characterization of the antibody. The humanized antibody is IgG4 S228P (IgG4P) subtype.

Example 3 Screening of humanized antibodies

Highly active humanized antibodies were screened by detecting the binding ability of humanized antibodies to cynomolgus monkey B cells, the ability of human macrophages to phagocytose tumor cells and the ability of induction of red blood cell agglutination.

Detection of the binding ability of the humanized antibody to cynomolgus monkey B cells: flow cytometry method was used to detect the binding of a series of humanized 125G4A4 antibodies to CD47 on the surface of cynomolgus monkey B cells. The method is as follows: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of cynomolgus monkeys (provided by Shanghai Yinuosi Bio-Technology Co., Ltd. ) by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) . PBMC was incubated with a series of humanized 125G4A4 antibodies or isotypes control (IgG4P) in PBS containing 2%fetal bovine serum at 4℃ for 30 minutes. Then the cells were washed three times and incubated with the secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2%fetal bovine serum at 4℃ in the dark for 30 minutes. The cells were washed three times and analyzed by flow cytometry. B cells were labeled with an anti-human CD20 antibody (Brilliant Violet 421 ^TM labeled anti-human CD20 Antibody, Biolegend, Cat: 302330) having cross-reactivity with cynomolgus monkeys, and detected by flow cytometry on Canto II (BD Biosciences) to obtain its average fluorescence intensity (MFI) .

According to the methods described in Examples 1.5 and 1.4, the ability of macrophages to phagocytose tumor cells and the ability of induction of red blood cell agglutination were detected respectively.

As shown in Table 5, a series of humanized 125G4A4 antibodies bind to CD47 expressed on cynomolgus monkey B cells under tested concentration. The antibodies promote phagocytosis of tumor cells Raji by macrophages, of which Hu-125G4A4m-48 displays the strongest phagocytic efficiency at 33 nM. The other activities of Hu-125G4A4m-48 are similar to those of chimeric antibody Ch-125G4m-m35. Moreover, the number of back mutations was smaller. Therefore, Hu-125G4A4m-48 was selected for further test, and was named as HMA02h14-48 hereafter.

Table 5 In vitro activity test of a series of anti-CD47 humanized antibodies

Example 4 Determination of binding activity of HMA02h14-48 to tumor cells by FACS

Human CD47 is endogenously expressed on the surface of human Burkitt lymphoma cell line Raji cells (Shanghai Institutes for Biological Sciences, SIBS, CCL-86 ^TM/ATCC) , human diffuse large cell lymphoma Toledo cells (

CRL-2631 ^TM) and human mantle cell lymphoma REC-1 cells (

CRL-3004 ^TM) . According to the detection method described in the preceding Example 1.2, flow cytometry was used to detect the binding of the humanized antibody HMA02h14-48 to CD47 on the surface of the above-mentioned tumor cell lines. The highest antibody concentration was 667 nM, the antibodies were serially diluted, and a total of 8 concentration points were tested.

As shown in figures 1-3. Both HMA02h14-48 and Hu5F9 bound to CD47 on the surface of tumor cells, including Raji, Toledo and REC-1. The maximum fluorescence intensity when HMA02h14-48 reaches a plateau is higher than that of Hu5F9, and the EC ₅₀ and the maximum fluorescence intensity are shown in Table 6.

Table 6 Binding activity of antibody HMA02h14-48 to CD47 on the surface of tumor cells

The negative isotype control antibody (isotype) used in this example and examples 5-11 was human IgG4P, which was purchased from Shanghai Chempartner Co., Ltd.

Example 5 Determination of binding affinity of antibody HMA02h14-48 to human CD47 by Biacore

Biacore was used to determine the binding kinetic parameters by measuring surface plasmon resonance (SPR) . This technology was used to detect the microscopic rate constants of the binding (Ka) and dissociation (Kd) of an antibody and an antigen. Based on the Ka and Kd values, the affinity value of the antibody and the antigen can be obtained. Both Biacore instrument (Biacore T200) and reagents were purchased from GE Healthcare. The anti-human Fc antibody was immobilized on sensor chip CM5. The purified antibodies (HMA02H14-48 and Hu5F9) were diluted in a mobile phase buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Tween-20, pH 7.4) , and flowed through a CM5 chip coated with anti-human Fc antibodies. Then the serially diluted human CD47-His (ACROBiosystems, Cat: CD7-H5227) fusion protein flowed through a detection chip to measure the binding of the antigen and the antibody, and then the mobile phase buffer flowed through the chip to detect the dissociation of the antigen from the antibody. The binding and dissociation signal data of the antigen and the antibody were collected at different concentrations, and fitted at 1: 1 by a Langmuir model to calculate the affinity of the antigen and the antibody.

As shown in Table 7, HMA02h14-48 binds to human CD47 with high affinity with a KD value of 7.77E-10 (M) .

Table 7 Determination of kinetic constants of humanized antibody binding to human CD47 by Biacore

Example 6 Detection of activity of HMA02h14-48 in blocking the interaction between human CD47 and SIRPα by ELISA

According to the method described in the preceding example 1.3, ELISA was used to detect the ability of HMA02h14-48 to block the interaction between human CD47 and SIRPα. The highest antibody concentration was 67 nM, the antibodies were serially diluted, and a total of 8 concentration points were tested.

As shown in figure 4, the antibody HMA02h14-48 in the present invention blocks the interaction between human CD47 and SIRPα with IC ₅₀ = 1.58 nM.

Example 7 Effects of HMA02h14-48 on phagocytosis of tumor cells by human macrophages

According to the method described in Example 1.5, the effect of HMA02h14-48 on promoting phagocytosis of human Burkitt lymphoma cell line Raji cells, human diffuse large cell lymphoma Toledo cells, human mantle cell lymphoma REC-1 cells and human promyelocytic leukemia cell line HL-60 cells by human macrophages was detected. The highest antibody concentration was 100 μg/mL, the antibodies were serially diluted, and a total of 8 concentration points were tested. The results were shown in Table 8 and Figures 5-8.

The results showed that compared with the reference antibodies Hu5F9 and SRF231, HMA02h14-48 could effectively promote the phagocytosis of human Burkitt lymphoma cell line Raji by macrophages. The highest phagocytic efficiency was up to 36.5%, and the phagocytosis rates at different concentrations from 0.1 to 100 μg/ml were higher than those of Hu5F9 and SRF231. The highest phagocytic efficiency of HMA02h14-48 for human mantle cell lymphoma REC-1 was up to 84.6%, and the phagocytosis rate could be maintained at about 70%even at a low concentration of 0.1 μg/ml, which was higher than those of Hu5F9 and SRF231. HMA02h14-48 promoted the phagocytosis of Toledo cells by macrophages, and the phagocytosis rate was up to 94.2%. HMA02h14-48 could promote the phagocytosis of tumor cell HL-60 by macrophages, and the highest phagocytic efficiency was up to 65%.

Table 8 Effect of antibody HMA02h14-48 for promoting phagocytosis of tumor cells by human MΦ

Example 8 Detection of effects of HMA02h14-48 on the induction of red blood cell agglutination in vitro

Human red blood cells were diluted to 10%in PBS, and incubated with CD47 antibody added in a round bottom 96-well plate for 16 hours at room temperature. The presence of non-precipitated red blood cells is evidence that proves red blood cell agglutination. Compared with the white dots formed by the precipitation of non-agglutinated red blood cells, the non-precipitated red blood cells could form a reticulated area which was larger than that of the negative isotype control antibody (see figure 9) . The results of the negative isotype control antibody (Isotype) were used as normal standards.

According to the method described in Example 1.4, the antibody HMA0214-48 was tested to see whether it induces red blood cell agglutination. The highest antibody concentration was 667 nM, the antibodies were serially diluted, and a total of 12 concentration points were tested.

As shown in figure 9, it showed that CD47 antibody Hu5F9 could significantly induce red blood cell agglutination when its concentration is 0.9 nM or above. By contrast, the antibody HMA02h14-48 in the present invention did not induce a significant hemagglutination of human red blood cells in vitro at different concentrations from 0.004 to 667 nM.

Example 9 Detection of binding activity of HMA02h14-48 to human red blood cells by FACS

It is known that in the prior art, when therapeutic anti-CD47 antibodies are used clinically, side effects such as anemia often occur. It is generally believed that anti-CD47 antibodies bind to CD47 on the surface of red blood cells, which would in turn cause the phagocytosis of red blood cells by macrophages. This could be another major cause of anemia. In the present invention, flow cytometry was used to detect the binding ability of HMA02h14-48 to human red blood cells to evaluate the risk of antibodies. Specifically, red blood cells from healthy donors were incubated with diluted HMA02h14-48 (the maximum concentration being 667 nM, 8 test concentration points in total) in PBS containing 2%fetal bovine serum at 4℃ for 30 minutes. Then the cells were washed three times and incubated with the secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2%fetal bovine serum at 4℃ in the dark for 30 minutes. The cells were washed three times with PBS containing 2%fetal bovine serum (FBS) , and then the fluorescence signal was detected by Canto II (BD Biosciences) flow cytometry. According to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated.

As shown in figure 10, the maximum mean fluorescence intensity of HMA02h14-48 bound to CD47 on the surface of human red blood cells was lower than that of control antibody Hu5F9. The maximum mean fluorescence intensity and EC ₅₀ thereof was shown in Table 9.

Table 9 Binding activity of antibody HMA02h14-48 to CD47 on the surface of human red blood cells

Example 10 Inhibition of Toledo Tumor Growth by Humanized Antibody HMA02h14-48

Objectives: A Toledo subcutaneous tumor model was established in NOD-Scid mice to study the anti-tumor activity of the antibody of the present invention.

Methods: human diffuse large B-cell lymphoma cells Toledo (

CRL-2631 ^TM) was cultured with RPMI1640 medium containing 10%fetal bovine serum. Tumor cells were suspended in RPMI1640 and implanted into male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd. ) subcutaneously in the right flank at a dose of 1 × 10 ⁷ cells/mouse.

15 days after tumor cell inoculation, mice were randomly divided into 6 groups according to tumor volume, Hu5F9 and HMA02h14-48 antibodies were diluted with PBS respectively, and the mice were administered at a dose of 10 mg/kg according to the schedule shown in Table 10. The negative isotype control antibody (isotype) IgG4P was purchased from Shanghai ChemPartner Co., Ltd.

Table 10. Dosage regimens of Hu5F9 and HMA02h14-48

Note: The grouping day is defined as day 0, and the next day for drug administration is day 1.

The tumor volumes (tumor volume = 0.5 × long diameter × short diameter ²) and body weights of the mice were measured regularly. The changes in tumor volume and body weight were statistically analyzed using student t-test in Excel software, wherein p < 0.05 indicates a significant statistical difference. The tumor regression rate of each antibody treatment group after administration was calculated.

The formula for calculating tumor regression rate in each treatment group is: [ (D ₀ average tumor volume-D _t average tumor volume) /D ₀ average tumor volume] × 100%.

The formula for calculating the relative weight of a mouse is: (weight of the mouse on the day of measurement/weight of the mouse at the time of grouping) × 100%.

Results:

The experimental results are shown in Table 11 and Figure 11.

The tumors in the isotype control antibody group grew well, while in therapeutic antibody treatment groups, the subcutaneous tumor volume gradually reduced compared with the initial volume until completely regressed. Groups with Hu5F9 and HMA02h14-48 antibodies administered at various doses achieved the effect of complete tumor regression (regression rate of 100%) when measured on day 11, compared with the control antibody in the control group, the tumor volume reduction were statistically significant. After the dosing discontinuation, the animals were observed until day 67, and there was still no sign of tumor regrowth. In addition, the animals in groups with HMA02h14-48 administered at various doses were in good status, and there was no significant difference in the body weights of the mice on day 21 compared with that before treatment. The body weight of mice in the group with a high dose of Hu5F9 on day 21 was reduced by about 5%compared with that on day 0, but there was no statistical difference compared with the initial weight (p > 0.05) ; however, there was no weight loss in the low-dose group of Hu5F9, suggesting a possible dose-related effect of Hu5F9 on body weight.

Based on the above-mentioned data, both Hu5F9 and HMA02h14-48 antibody treatments showed extremely significant anti-tumor effects. A single dose of either antibody at 10 mg/kg led to complete regression of the tumor and the duration is longa.

Table 11 Effect of Hu5F9 and HMA02h014-48 on Toledo subcutaneously transplanted tumor growth

Note: **is p < 0.01; The numbers in parentheses are tumor regression rates.

Example 11 Inhibition of REC-1 tumor growth by humanized antibody HMA02h14-48

Objectives: A REC-1 subcutaneous tumor model was established in NOD-Scid mice to study the anti-tumor activity of the antibody of the present invention.

Methods: human mantle cell lymphoma cells REC-1 (

CRL-3004 ^TM) was cultured in RPMI1640 medium containing 10%fetal bovine serum. Tumor cells were suspended in RPMI1640 and implanted into male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd. ) subcutaneously in the right flank at a dose of 5 × 10 ⁶ cells/mouse.

11 days after tumor cell inoculation, mice were randomly divided into 5 groups according to tumor volume, Hu5F9 and HMA02h14-48 antibodies were diluted with PBS, and the mice were administered according to the schedule shown in Table 12. The antibody Hu5F9 was prepared by GenScript, and the antibody HMA02h14-48 was prepared according to the method in Example 2. The isotype control antibody (isotype) IgG4P was purchased from Shanghai ChemPartner Co., Ltd.

Table12. Dosing regimens of Hu5F9 and HMA02h14-48

The tumor volumes (tumor volume = 0.5 × long diameter × short diameter ²) , and body weights of the mice were measured regularly. The tumor inhibition rate and regression rate of the antibody treatment group on day 12 after administration were calculated.

The formula for calculating tumor inhibition rate is as follows: [ (average tumor volume change in the control group-average tumor volume change in the treatment group) /average tumor volume change in the control group] × 100%. The changes in tumor volume and body weight were statistically analyzed using Student t-test in Excel software, wherein p < 0.05 indicates a significant statistical difference.

Results:

The experimental results were shown in Table 13 and Figure 12.

12 days after administration, compared with Isotype group, the tumor growth inhibition rate was 16.7% (p > 0.05) in the group treated with a single dose of Hu5F9 at 3 mg/kg; and the tumor growth inhibition rates were 3.8% (p > 0.05) , 54.7% (p < 0.01) and 107.2% (p < 0.001) , respectively, in the groups with a single dose of HMA02h14-48 at 1 mg/kg, 3 mg/kg and 10 mg/kg. Groups treated with a high dose of HMA02h14-48 antibody achieved complete tumor regression (regression rate of 100%) on day 10. In addition, there was no significant difference in the relative body weight of the mice in different treatment groups.

Taken together, HMA02h14-48 antibody showed dose-dependent effect in REC-1 model, and a single dose of 10 mg/kg led to complete tumor regression.

Table 13 Effect of Hu5F9 and HMA02h14-48 on REC-1 subcutaneously implanted tumor growth

Note: **is p < 0.01; The numbers in parentheses are tumor regression rates.

Example 12 Generation and production of anti-CD20/CD47 bispecific antibodies

Different bispecific antibodies capable of specifically binding to both human CD47 and human CD20 have been prepared in IgG-scFv (2+2) format or Crossmab (1+1) format.

IgG-scFv (2+2) format bispecific antibodies

In present invention, BsAb1, BsAb2, BsAb3, BsAb4, BsAb5, BsAb6 and BsAb8 are as examples of bispecific antibodies in IgG-scFv (2+2) format. Each of the two scFv fragements capable of specifically binding to hCD47 is connected via a peptide linker to the C-terminus of the two heavy chains of a whole antibody capable of specifically binding to hCD20. Each of the bispecific antibodies is composed of four polypeptide chains, two polypeptide chains are identical and in the format of VH (anti-CD20) -CH1-Fc domain subunit-peptide linker-scFv fragement (anti-CD47) , and the other two polypeptide chains are identical and in the format of VL (anti-CD20) -CL. As used herein, VH (anti-CD20) and VL (anti-CD20) refer respectively to the heavy and light chain variable domain of the antigen-binding domain capable of specifically binding to human CD20; scFv fragement (anti-CD47) refers to scFv fragement of the antigen-binding domain capable of specifically binding to human CD47, CL refers to the light chain constant region; "VL-CL" collectively refers to light chain (LC) and "VH -CH1-Fc domain subunit" collectively refers to heavy chain (HC) .

Crossmab (1+1) format bispecific antibodies

Anti-CD20/CD47bispecific antibodies in Crossmab (1+1) format (e.g. BsAb7) are IgG type with one Fab fragement capable of specifically binding to hCD47 and one Fab fragement capable of specifically binding to hCD20.

To generate the Crossmab (1+1) format bispecific antibody, the knob-into-hole technology was used to achieve heterodimerization. The S354C/T366W mutations have been introduced in one heavy chain (Fc knob heavy chain) and the Y349C/T366S/L368A/Y407V mutations are introduced in the other heavy chain (Fc hole heavy chain) . Furthermore, the CrossFab technology as described in WO WO2009/080252 A1 ensures correct light chain pairing. To be specific, the CH1 domain of the Fab arm capable of specifically binding to hCD20 was replaced by light constant region (CL) ; and light chain variable region of the Fab arm was linked to heavy chain CH1 domain. So, Crossmab (1+1) format bispecific antibody is composed of four different polypeptide chains. BsAb7 as an example, the first polypeptide chain is in the format of VH (anti-CD47) -CH1-Fc domain (S354C/T366W) , the second polypeptide chain is in the format of VL (anti-CD47) –CL, the third polypeptide chain is in the format of VH (anti-CD20) –CL–Fc domain (Y349C/T366S/L368A/Y407V) , and the forth polypeptide chain is in the format of VL (anti-CD20) –CH1.

In all these constructs, the antigen-binding domains capable of specifically binding to hCD47 are derived form HMA02h14-48 (as named as mAb1 hereafter) and the antigen-binding domains capable of specifically binding to hCD20 are derived from Rituximab (Rituximab, as named as mAb2, the amino acid sequence of heavy chain is as set forth in SEQ ID NO: 47, and the amino acid sequence of heavy chain is as set forth in SEQ ID NO: 50) or Obinutuzumab (the amino acid sequence of heavy chain as set forth in SEQ ID NO: 51, and the amino acid sequence of light chain as set forth in SEQ ID NO: 53) .

BsAb-ref is a reference antibody in IgG-scFv (2+2) format. The only difference between BsAb-ref and BsAb1 is the variable regions of Fab fragment. In BsAb-ref, the variable regions of Fab fragment are derived from Idarucizumab (amonoclonal antibody fragment (Fab) specific binding to Dabigatran (CAS ID: 211915-06-9) with very high affinity, INN Recommended 2014, 17) .

The amino acid sequences of the example antibodies are shown in Table 14-16 below.

Expression and purification

Construction of recombinant expression vectors was conducted using molecular biology protocol. In brief, DNA sequence encoding each polypeptide chain of an antibody was cloned into the ORF (Open Reading Frame) of a suitable expression vector, respectively. Recombinant expression vectors each comprising one coding DNA sequences were obtained and were used for subsequent expression after being verified by sequencing. Antibodies were expressed by transient transfection of Expi293 cells with recombinant expression vectors encoding the different peptide chains of each antibody. After culture cells for 5-7 days, antibodies were purified from cell culture supematant referring to standard protocols, such as protein A affinity chromatography, followed by a size exclusion chromatographic step.

Table 14: Amino acid sequences of antigen-binding domains of bispecific antibodies (BsAb1-8)

Table 15: Amino acid sequences of bispecific antibodies in IgG-scFv (2+2) format (BsAb1-6 and BsAb8)

Table 16: Amino acid sequences of bispecific antibodies in Crossmab (1+1) format (BsAb7)

Example 13: Detection of binding activities of anti-CD20/CD47 bispecific antibodies to both human CD20 and human CD47 proteins by ELISA assay

ELISA assay was performed to detect the binding activity of anti-CD20/CD47 bispecific antibodies to human CD47. Briefly, 1 μg/ml of human CD47-His (Acrobiosystems, Cat: CD47-H5227) was coated on 96-well plate (Corning, Cat: 9018) at 4℃ overnight. After washed with PBST (PBS + Tween 0.5%v/v) for 3 times, plates were blocked by PBST containing 1%BSA at 37℃ for 1 hour. After incubation, plates were washed again, then serial diluted antibodies were added at 100 μL/well and plates were incubated at 37℃ for 1 hour. After incubation, plates were washed for another three times, and anti-human IgG-HRP (Sigma, Cat: A0293) diluted in PBST was added for 1 hour at 37℃. Then, after washing three more times, 100 μL/well TMB substrates were added, then stopped by adding 1N HCL. OD at 450 nm was measured by Molecular Device Spectramax M2. The results were showed in Figure 13-1 and 13-2, all anti-CD20/CD47 bispecific antibodies have binding activities to human CD47 by ELISA with EC ₅₀ similar to mAb1. mAb2 and other negative controls showed negative in this test (Figure 13 and Table 17) .

ELISA assay was performed to test binding activities of anti-CD20/CD47 bispecific antibodies to human CD20. Briefly, serially diluted antibodies were coated on the 96-wells plate (corning, Cat: 9018) . After washing with PBST (PBS + Tween 0.5%v/v) for 3 times, plates were blocked by PBST containing 1%BSA at 37℃ for 1 hour. After incubation, plates were washed again, then 2ug/ml of human CD20 protein (Acrobiosystems, CD0-H52H3) were added at 100 μL/well and plated incubated at 37℃ for 1 hour. After incubation, plates were washed for another three times, and added anti-His-HRP (Sigma, A0293) diluted in PBST for 1 hour at 37℃. Then, after washing three more times, 100 μL/well TMB substrates were added, then stopped by adding 1N HCL. OD at 450 nm was measured by Molecular Device Spectramax M2. The results were showed in Figure 14, all anti-CD20/CD47 bispecific antibodies have binding activities to human CD20 by ELISA with EC ₅₀ similar to mAb2. mAb1 and other negative controls showed negative in this test (Figure 14 and Table 17) .

In order to test whether Anti-CD20/CD47 bispecific antibodies have binding activities to both human CD20 and human CD47, sandwich ELISA was performed. Briefly, 1 μg/mL of human CD47-Fc (Acrobiosystems, Cat: CD7-H5256) was coated on 96-well plate (Corning, Cat: 9018) at 4℃ overnight. After washing with PBST (PBS +Tween 0.5%v/v) for 3 times, plates were blocked by PBST containing 1%BSA at 37℃ for 1 hour. After incubation, plates were washed again, then serially diluted antibodies were added at 100 μL/well and incubated at 37℃ for 1 hour. After incubation, plates were washed for another three times, and then were incubated with 0.5 μg/mL of human CD20 protein (Acrobiosystems, CD0-H52H3) . Then, after washing, anti-human IgG-HRP (Sigma, Cat: A0293) was added and incubated for 1 hour at 37℃. Then, plates were washed for another three times, and was added with anti-His-HRP (Sigma, Cat: A0293) diluted in PBST for 1 hour at 37℃. Then, after washing three more times, 100 μL/well TMB substrates were added, then stopped by adding 1N HCL. OD at 450 nm was measured by Molecular Device Spectramax M2. As showed in Figure 15 and Table 17, anti-CD20/CD47 bispecific antibodies, captured by immobilized CD47 protein, have binding activities to human CD20 protein. It is demonstrated that those antibodies have binding activities to both human CD47 and human CD20 simultaneously. mAb1, mAb2 and other negative controls showed negative in this test. Herein and hereafter, the negative isotype control antibody hIgG4P was purchased from Shanghai ChemPartner Co., Ltd. and hIgG1was purchased from Biolgend (Cat#403502) .

Table 17 In vitro binding activity test of a series of Anti-CD20/CD47 bispecific antibodies

Note: *anti-Dabi refers to an antibody specific binding to Dabigratan (CAS ID: 211915-06-9) in the form of hIgG1, wherein the variable regions of the antibody are derived from Idarucizumab (INN Recommended 2014, 17) . It is used as an isotype control herein and hereafter.

Example14 Determination of binding affinity of anti-CD20/CD47 bispecific antibodies to both human CD20 and human CD47 in SPR

Biacore (SPR) was also performed to analyze the binding of anti-CD20/CD47 bispecific antibodies to human CD20 and/or human CD47.

To measure the binding affinity to human CD47, antibodies were captured by Chip pre-coated with anti-human Fc, and human CD47 protein (Acrobiosystems, Cat: CD47-H5227) diluted with running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Tween-20, pH 7.4) flowing over the chip. The interaction was measured by Biacore T200 (GE healthcare) . The association (Ka) , dissociation (Kd) rate and the affinity (KD, M) were calculated with 1: 1 Langmuir model.

To measure the binding affinity to human CD20, the CD20 protein was captured firstly by Chip pre-coated anti-His, then antibodies flow over the chip. The binding and release signals were recorded by Biacore T200.

The binding affinity of BsAb1, and mAb1 and mAb2 was listed in the table below.

Table 18 Affinity of BsAb1 binding to human CD47 and human CD20 protein measured by surface plasmon resonance (SPR) on Biacore

Example 15 Detection of binding activities of anti-CD20/CD47 bispecific antibodies on CD20 ⁺CD47 ⁺ tumor cells by FACS

Human diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-6 endogenously expresses CD20 and CD47. FACS analysis was performed to determine the binding activities of anti-CD20/CD47 bispecific antibody (BsAb1 and BsAb6) on SU-DHL-6. The test BsAb1 and BsAb6 as well as reference antibodies mAb1 and mAb2 were serially diluted by PBS containing 2%fetal bovine serum (FBS, Gibco, Cat: 10100147) (the highest concentration being 100 nM, 8 concentration points in total) . The diluted antibodies were incubated with SU-DHL-6 cells (ATCC, Cat: CRL-2959) (1*10 ⁵ cells /well) at 4℃ for 1 hour. After washing three times with PBS containing 2%FBS, a PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added and incubated with the cells at 4℃ in the dark for 1 hour. The cells were washed three times with PBS containing 2%FBS, and then the fluorescence signal was detected by CantoII (BD Biosciences) flow cytometry, and according to the mean fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated.

As shown in Figure 16, both BsAb1 and BsAb6 have binding activities to SU-DHL-6 cells. Moreover, the binding curve of BsAb1 was similar to that of anti-CD20 mAb (mAb2) . The difference between BsAb1 and BsAb6 was due to the different CD20 antigen epitopes they recognized.

A human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in hamster ovary cell line CHO-K1 to establish CHO-K1 cell line overexpressing human CD47 protein (CHO-K1/CD47 OE cell line) . To further validate that anti-CD47 scFv of BsAb1 and BsAb6 possesses the CD47 binding affinity, CHO-K1/CD47 OE cell line (CD20 ^-CD47 ⁺) was utilized and the detection method was according to that described above. It was observed that both bispecific antibodies could still bind to human CD47 expressing only cell line (Figure 17) . Based the above results, it could be concluded that BsAb1 and BsAb6 have binding activities to both human CD20 and CD47 on the cell surface.

Example 16 Detection the activities of anti-CD20/CD47 bispeific antibodies in blocking human CD47/SIRPα interaction

To determine the activities of anti-CD20/CD47 bispecific antibodies in blocking the interaction between human CD47 and SIRPα, the

Jurkat SIRPα Signaling Assay was employed. This assay utilizes the engineered Jurkat cells which co-express a ProLink ^TM (PK) tagged SIRPα (Receptor) and an Enzyme Acceptor (EA) tagged SH2 domain. Once CD47 (Ligand) engages, the receptor is activated and phosphorylated, and then recruits SH2-EA, which ultimately forces complementation of the two β-galactosidase enzyme fragment (EA and PK) . The resulting functional enzyme hydrolyzes substrate to generate a chemiluminescent signal. According to the manufacturer’s protocol, SU-DHL-6 cells (ATCC, Cat: CRL-2959) , which express high levels of CD20 and CD47, were pre-incubated with antibodies (BsAb1, or mAb1) (the highest concentration being 100 nM, 3-fold dilution, 12 concentration points in total) in an incubator at 37℃, with 5%CO ₂ for 1 hour. Jurkat SIRPα expressing cells (Eurofins, Cat: 93-1135C19) were then added and incubated in an incubator at 37℃, with 5%CO ₂ for 5 hours. After incubation, Bioassay PathHunter Bioassay Detection Kit (Eurofins, Cat: 93-0933) was used to develop a chemiluminescent signal. Read chemiluminescent signals with Envision. GraphPad was used for fitting a concentration dependent curve, and the IC ₅₀ was calculated.

As shown in Figure 18 and Table 19, both anti-CD20/CD47 bispecific antibody (BsAb1) and mAb1 could potently block the interaction between CD47 (expressing on SU-DHL-6) and SIRPα (on Jurkat SIRPα expressing cells) , and exhibited equivalent efficiencies (summarized in Table 19) .

To further investigate if anti-CD20/CD47 bispecific antibody (BsAb1 or BsAb6) possesses comparable blocking activity in CD47 expressing only cells, CHO-K1/CD47 OE cells were utilized and the detection method was according to that described above. It was observed that BsAb1and BsAb6 showed around 3-fold reduction in blocking activity when compared with anti-CD47 mAb (mAb1) in CHO-K1/CD47 OE cells (Figure 19 and Table 19) , which is consistent with the Biacore results. Taken together, BsAb1 maintains the similar blocking activity as mAb1 in CD20 ⁺CD47 ⁺ tumor cells, but lost partial blocking activity in CD47 expressing only cells.

Table 19 Activities of anti-CD20/CD47 bispecific antibody in blocking the CD47/SIRPα interaction by

Jurkat SIRPα Signaling Assay

Example 17 Determination of the pro-phagocytic effects of anti-CD20/CD47 bispecific antibody on tumor cells by human macrophages

To further investigate blockade of CD47/SIRPα interaction by anti-CD20/CD47 bispecific antibodies enables phagocytosis of tumor cells, ADCP assay was performed. Briefly, human blood was freshly collected from healthy donors, and the peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll- Paque PLUS (GE Healthcare, Cat: 17-1440-02) . Monocytes were further isolated by using the Human Pan Monocyte Isolation Kit (Miltenyi biotec, cat: 130-096-537) . To induce the differentiation of monocytes into macrophages, macrophage colony stimulating factor (M-CSF, R&D Systems, Cat: 216-MC) was added and the monocytes was subjected to culture for 7 consecutive days. On the day of cellular phagocytosis experiment, macrophages were starved in a serum-free medium for 2 hours. Target tumor cells (Daudi cells) were labeled with CFSE (eBioscience, Cat: 65-0850-85) according to the manufacturer’s protocol. The CFSE-labeled tumor cells (target cells, T) and macrophages (effector cells, E) were mixed with a ratio of 4: 1, and the test antibodies with serial dilution were added and incubated at 37℃ for 2 hours. Then the cells were washed twice with PBS, and then digested with trypsin (Gibco, Cat: 25200072) ; an APC labeled anti-CD14 antibody (Biolegend, Cat: 367118) was added and incubated in the dark on ice for 30 minutes. The cells were washed twice and analyzed by flow cytometry. FITC MFI values mean how many tumor cells were engulfed by human macrophages.

As shown in Figure 20, BsAb1 and BsAb8 showed strong phagocytosis activity on Daudi cells.

Example 18 Detection of activities of anti-CD20/CD47 bispecific antibodies in induction of human red blood cell hemagglutination

We futher determine if the antigen-binding domain capable of specifically binding to CD47 of the bispecific antibodies in the present invention induce red blood cell agglutination in vitro. The method is as follows: collecting fresh human blood from healthy, washing the cells five times with PBS, and then diluting the cells to make a suspension containing 10%human red blood cells; mixing the red blood cell suspension with serial diluted test antibodies BsAb1 and reference antibody Hu5F9 (the highest concentration being 10 μg/mL, 3-fold dilution, 10 concentration points in total) , then adding the mixture into a round bottom 96-well plate; and incubating them at room temperature for 16 hours, then taking photos and determining the results according to the phenomenon of the cells in the well. If red blood cell agglutination occurs, cells are plated onto each well like a net, and a larger sheet-like cell layer will appear in the well with a diameter larger than that of the negative control well. If no hemagglutination occurs, the red blood cells will deposit at the bottom of the well, and smaller dot-like cell pellet precipitation will appear in the well.

As shown in Figure 21, anti-CD47 antibody Hu5F9 could significantly induce red blood cell agglutination when its concentration is 0.12 μg/mL or above. By contrast, BsAb1 and BsAb4 did not induce hemagglutination in vitro.

Example 19 Detection of binding activity of anti-CD20/CD47 bispecific antibodies to human red blood cells by FACS

As the background described in example 9, flow cytometry was therefore performed to detect the binding activity of anti-CD20/CD47 bispecific antibodies to human red blood cells. Briefly, red blood cells from healthy donors were incubated with diluted BsAb1, BsAb6 or isotype control antibodies (the maximum concentration being 100 nM) in PBS containing 2%fetal bovine serum at 4℃ for 30 minutes. Then the cells were washed three times and incubated with the secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2%fetal bovine serum at 4℃ in the dark for 30 minutes. The cells were washed three times with PBS containing 2%fetal bovine serum (FBS) , and then the fluorescence signal was detected by Canto II (BD Biosciences) flow cytometry. According to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated.

As shown in Figure 22, BsAb1 and BsAb6 showed no binding activity on human red blood cells whereas Hu5F9 displayed strong binding affinity.

Example 20 Determination of the pro-phagocytic effects of anti-CD20/CD47 bispecific antibodies on human red blood cells

ADCP assay was performed to determine the pro-phagocytic effects of anti-CD20/CD47 bispecific antibodies on human red blood cells. The experimental procedure was described in example 17. The effector (E) cells (primary human macrophages) were co-incubated with target (T) cells (CFSE-labelled human red blood cells) with a ratio of 1: 4, for 2 hours in the presence of Hu5F9, BsAb1, BsAb6 and experimental controls (BsAb-ref and isotype controls) . FITC MFI means how many human red blood cells were engulfed by human macrophages.

As illustrated in Figure 23 and Figure 24, Hu5F9 promoted strong phagocytosis of human red blood cells. In contrast, no phagocytosis of human red blood cells was induced by BsAb1 and BsAb6 at all concentrations.

Example 21 Inhibition of SU-DHL-6 Tumor Growth by anti-CD20/CD47 bispecific antibodies

Objective 1: To evaluate the anti-tumor activity of BsAb1 in a SU-DHL-6 subcutaneous tumor model established in NOD-SCID mice.

Methods: Human diffuse large B-cell lymphoma cells SU-DHL-6 (

CRL-2959 ^TM) were cultured and expanded in RPMI1640 medium containing 10%fetal bovine serum. Tumor cells in exponential growth stage were harvested and implanted subcutaneously into the right flanks of NOD-SCID mice (Beijing Vital River Laboratory Animal Technology Co, Ltd. ) with 1 × 10 ⁷ cells/mouse.

11 days after tumor cell inoculation, mice were randomly divided into 6 groups according to tumor volume. BsAb-ref was diluted with PBS and BsAb1 was diluted with 50mM Citrate and 150mM NaCl (pH5.5) . The mice were administered with antibodies according to the schedule shown in Table 20. The control antibody Rituximab (mAb2) was commercially purchased from Roche.

Table 20 Dosing regimens for Rituximab, BsAb-ref and BsAb1

Data analysis:

Tumor volume = 0.5 × long diameter × short diameter ²

Tumor growth inhibition rate = [ (average tumor volume change in the control group-average tumor volume change in the treatment group) /average tumor volume change in the control group] × 100%.

Changes in tumor volume were analyzed using Student t-test by Excel software, wherein p < 0.05 indicates a statistically significant difference.

Results:

21 days after drug administration, the tumor growth inhibition rate of Rituximab (10 mg/kg) treated group was 44.1% (p < 0.05) . BsAb-ref at 13.3 mg/kg, as a control antibody, did not exhibit any anti-tumor activity with a TGI of -0.7% (p>0.05) . In the groups treated with BsAb1 at 6.6 mg/kg, 13.3 mg/kg and 26.6 mg/kg, the TGIs were 47.1% (p <0.01) , 85.8% (p < 0.01) and 87.0% (p < 0.01) , respectively (Table 21 and Figure 25) . Furthermore, in mice treated with 13.3 and 26.6 mg/kg of BsAb1, 3/8 and 4/8 mice achieved complete tumor regression..

Therefore, BsAb1 antibody showed dose-dependent anti-tumor effect in SU-DHL-6 model, although no further increase in efficacy beyond a dose level of 13.3 mg/kg.

Table 21 Effect of Rituximab, BsAb-ref and BsAb1 on tumor growth of subcutaneously implanted SU-DHL-6 xenograft in NOD-SCID mice

Note: *is p < 0.05; **is p < 0.01; The numbers in parentheses are tumor growth inhibition rates.

The day of grouping was defined as day 0, the day of the first drug administration was day 1.

Objective 2: To evaluate the anti-tumor activity of BsAb8 in a SU-DHL-6 subcutaneous tumor model established in nude mice.

Methods: Human diffuse large B-cell lymphoma cells SU-DHL-6 (

CRL-2959 ^TM) were cultured and expanded in RPMI1640 medium containing 10%fetal bovine serum. Tumor cells in exponential growth stage were harvested and implanted subcutaneously into the right flanks of nude mice (Lingchang Laboratory Animal Technology Co, Ltd) with 1 × 10 ⁷ cells/mouse.

Mice were randomly divided according to tumor volume. The human IgG control (anti-Dabi) was diluted with normal saline. The positive control antibody Rituximab (mAb2) was commercially purchased from Roche and was diluted with normal saline. BsAb8 was diluted with normal saline. The mice were administered with antibodies from Day 1 according to the schedule shown in Table 22.

Table 22 Dosing regimens for mAb2 and BsAb8

Data analysis:

Tumor volume = 0.5 × long diameter × short diameter ²

Tumor growth inhibition rate (TGI) = [1- (TV _t-TV ₀) _{drug treatment} / (TV _t-TV ₀) Human IgG control] × 100%. (TVt: tumor volume on the days post treatment, TV ₀: initial tumor volume before treatment. )

Comparisons of tumor volume change were analyzed using Student’s t-test by Excel software, wherein p < 0.05 indicates a statistically significant difference.

Results:

On Day 29, mAb2 (10 mg/kg) showed the TGI of 61.8% (p < 0.05) . BsAb8 at 4 mg/kg and 13.3 mg/kg displayed the TGIs of 82.8% (p <0.01) , and 88.5% (p < 0.01) , respectively (Table 23 and Figure 26) . Furthermore, in mice treated with 13.3 mg/kg of BsAb8, 1/6 mice achieved complete tumor regression.

Therefore, BsAb8 antibody showed dose-dependent anti-tumor effect in SU-DHL-6 model.

Table 23 Effect of mAb2, and BsAb8 on tumor growth of subcutaneously implanted SU-DHL-6 xenograft in in nude mice

Note: *is p < 0.05; **is p < 0.01 compared with Human IgG control by student’s t test.

Sequences of the present invention:

Claims

The bispecific antibody comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47, and

(b) at least one antigen-binding domain capable of specifically binding to CD20,

wherein the antigen-binding domain capable of specifically binding to CD47 comprises a VH and a VL, wherein the VH comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21, and the VL comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.
The bispecific antibody of claim 1, wherein

the VH of the antigen-binding domain capable of specifically binding to CD47 comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.
The bispecific antibody of claims 1 or 2, wherein

the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.
The bispecific antibody of any one of claims 1 to 3, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD47 are selected from

(1) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 2;

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 3, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 4;

(3) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 5, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10;

(4) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 6, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 9 or 10; or

(5) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.
The bispecific antibody of any one of claims 1 to 4, wherein

the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7, and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8.
The bispecific antibody of any one of claims 1 to 5, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a VH and a VL, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(1) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 25; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 28; or

(2) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 29, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 30, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 31; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 33, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 34.
The bispecific antibody of claim 6, wherein

the VH of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37, and the VL of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.
The bispecific antibody of any one of claims 1 to 7, wherein the bispecific antibody comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17, and the VL of the antigen-binding domain capable of specifically binding to CD47comprises LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16; and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(1) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 24, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 25; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 26, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 28; or

(2) a VH comprising HCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 29, HCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 30, and HCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 31; and a VL comprising LCDR1 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32, LCDR2 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 33, and LCDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 34.
The bispecific antibody of any one of claims 1 to 8, wherein the bispecific antibody comprises

(a) at least one antigen-binding domain capable of specifically binding to CD47 comprising a VH and a VL, wherein

the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7; and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10, and

(b) at least one antigen-binding domain capable of specifically binding to CD20 comprising a VH and a VL, wherein

the VH of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 35 or 37; and the VL of the antigen-binding domain capable of specifically binding to CD20 comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 36 or 38.
The bispecific antibody of claim 9, wherein

(a) the VH of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7; and the VL of the antigen-binding domain capable of specifically binding to CD47 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 8, and

(b) the VH and the VL of the antigen-binding domain capable of specifically binding to CD20 are selected from

(1) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 35 and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 36, or

(2) a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 37 and a VL comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 38.
The bispecific antibody of any one of claims 1 to 10, wherein any one of the antigen-binding domain is chimeric, fully human or humanized.
The bispecific antibody of any one of claims 1 to 11, wherein any one of the antigen-binding domain comprises a Fab fragement or a scFv fragement.
The bispecific antibody of any one of claims 1 to 12, wherein the antigen-binding domain capable of specifically binding to CD20 comprises a Fab fragement and the antigen-binding domain capable of specifically binding to CD47 comprises a scFv fragement or a Fab fragement.
The bispecific antibody of any one of claims 12 to 13, wherein the scFv fragement comprises a peptide linker connecting the C terminus of a VH to the N-terminus of a VL.
The bispecific antibody of claim 1 to 14, wherein the bispecific antibody is bivalent, trivalent or tetravalent.
The bispecific antibody of any one of claims 1 to 15, wherein the bispecific antibody comprises

(a) a first Fab fragement capable of specifically binding to CD47,

(b) a second Fab fragement capable of specifically binding to CD20, and

(c) an Fc domain composed of a first and a second subunit capable of stable association.
The bispecific antibody of claim 16, wherein the second Fab fragement is CrossFab (CL-CH1) .
The bispecific antibody of any one of claims 1 or 17, wherein the bispecific antibody comprises

(i) a first polypeptide chain in the format of: VH (anti-CD47) –CH1-Fc domain subunit,

(ii) a second polypeptide chain in the format of: VL (anti-CD47) –CL,

(iii) a third polypeptide chain in the format of: VH (anti-CD20) –CL–Fc domain subunit, and

(iv) a forth polypeptide chain in the format of: VL (anti-CD20) –CH1.
The bispecific antibody of any one of claims 1 to 18, wherein the bispecific antibody comprises

(i) a first polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 60,

(ii) a second polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61,

(iii) a third polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 62, and

(iv) a forth polypeptide chain comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 63.
The bispecific antibody of any one of claims 1 to 15, wherein the bispecific antibody comprises

(a) two scFv fragments capable of specifically binding to CD47, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments capable of specifically binding to CD20 and Fc domain,

wherein each of the two scFv fragments is connected to the whole antibody.
The bispecific antibody of claim 20, wherein each of the two scFv fragements is connected via a peptide linker to the whole antibody.
The bispecific antibody of any one of claims 20 to 21, wherein each of the two scFv fragements is connected via a peptide linker to the C-terminus of the two heavy chains of the whole antibody.
The bispecific antibody of any one of claims 14, 21 to 22, wherein the peptide linker comprises a GS linker.
The bispecific antibody of any one of claims 14, 21 to 23, wherein the peptide linker comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 39, 40, 41, 42, 43, or 44.
The bispecific antibody of any one of claims 20 to 24, wherein the scFv fragement comprises or consists of the amino acid sequence as set forth in SEQ ID NOs: 45 or 46.
The bispecific antibody of any of claims 20-25, wherein the two heavy chains and two light chains of a whole antibody are selected from:

(1) each of the heavy chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, 48, 49 or 78 and each of the light chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50; or

(2) each of the heavy chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51 or 52 and each of the light chains comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.
The bispecific antibody of any one of claims 1 to 15, wherein the bispecific antibody comprises an Fc domain.
The bispecific antibody of any one of claims 16 to 27, wherein the Fc domain is human IgG1Fc domain.
The bispecific antibody of any one of claims 16 to 28, wherein the Fc domain comprises one or more amino acid modification (s) promoting the association of the first and the second subunits of the Fc domain and/or one or more amino acid modification (s) increasing the binding affinity of the antibody to an FcRn.
The bispecific antibody of any one of claims 16-29, wherein the Fc domain comprises one or more amino acid substitution (s) at positions 428 and 434, preferably said amino acid substitutions are M428L and N434S (numbering according to Kabat EU index) .
The bispecific antibody of any one of claims 16 to 30, wherein each of the two subunits of the Fc domain comprises or consists of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 66, 67, 68, 69 or 77.
An isolated nucleic acid, encoding the bispecific antibody of any one of claims 1-31.
A recombinant vector or an expression vector, comprising one or more nucleic acids of claim 32, wherein the vector is suitable for the recombinant production of the bispecific antibody of any one of claims 1-31.
A host cell, comprising one or more recombinant vectors or expression vectors of claim 33.
An immunoconjugate, comprising the bispecific antibody of any one of claims 1-31.
A pharmaceutical composition, comprising the bispecific antibody of any one of claims 1-31, the nucleic acid of claim 32, the vector of claim 33, the host cell of claim 34, or the immunoconjugate of claim 35, and optionally comprising a pharmaceutically acceptable excipient.
A method for treating or preventing disease or condition in a subject, comprising administering to an individual an effective amount of the bispecific antibody of any one of claims 1-31, the nucleic acid of claim 32, the vector of claim 33, the host cell of claim 34, or the immunoconjugate of claim 35, or the pharmaceutical composition of claim 36.
The method of claim 37, wherein the disease or condition is CD47-related and/or CD20-related.
The method of any one of claims 37 -38, wherein the disease or condition is cancer.
The method of claim 39, wherein the cancer is hematological cancer, for example acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , Non-Hodgkin lymphoma (e.g., Burkitt’s lymphoma) , B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL) , chronic myelocytic leukemia (CML) , follicular lymphoma, small lymphotic lymphoma (SLL) , central nervous system (CNS) lymphoma, Richter’s Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and anaplastic large cell lymphoma. chronic lymphoid lymphoma (CLL) and non-Hodgkin’s lymphoma (NHL) .
Use of the bispecific antibody of any one of claims 1-31 , the nucleic acid of claim 32, the vector of claim 33, the host cell of claim 34, or the immunoconjugate of claim 35, or the pharmaceutical composition of claim 36 in the manufacture of a medicament for treating a disease or condition in a subject.