Antibodies targeting CD47 and uses thereof

Abstract

The present application provides antibodies or antigen-binding fragments thereof that specifically recognize CD47, and methods of making and using the same. The antibodies provided herein exhibit not only induction of phagocytosis on cancer cells, but also very low to no RBC hemagglutination and phagocytosis, showing an improved efficacy to toxicity ratio against cancer treatment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to international application No. PCT / CN2022 / 125317, filed on October 14, 2022, entitled “Antibodies targeting CD47 and uses thereof,” the contents of which are incorporated herein by reference in their entirety.

[0003] sequence declaration

[0004] The following content submitted as an XML file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: IEC232081PCT-seq1.xml, Record Date: October 12, 2023, Size: 136,200 bytes). Technical Field

[0005] The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically recognize CD47, and methods for making and using the same. Background Art

[0006] CD47 (differentiation cluster 47) (also known as integrin-associated protein (IAP)) is a 50-kDa membrane protein with an amino-terminal extracellular IgV domain, five transmembrane regions, and a short cytoplasmic tail that is alternatively spliced ​​(Brown EJ, Frazier WA 2001; Brown EJ 2001). It interacts with a variety of ligands, including but not limited to monoregulatory protein α (SIRPα), SIRPγ, integrins, and thrombospondin-1 (TSP-1) (Isenberg JS et al. 2009).

[0007] SIRPα is mainly expressed on myeloid cells (including macrophages, myeloid dendritic cells (DCs), granulocytes, mast cells) and their precursors (including hematopoietic stem cells). CD47 / SIRPα interaction transmits the "don't eat me" signal to prevent autophagy.

[0008] Analysis of patient tumors and matched adjacent normal (non-tumor) tissues revealed that CD47 protein is overexpressed on cancer cells, which efficiently helps them suppress phagocytic innate immune surveillance and elimination. Blocking the CD47-SIRPα interaction with anti-CD47 antibodies has been shown to effectively induce phagocytosis of tumor cells in vitro and inhibit the growth of various hematological and solid tumors in vivo. Therefore, CD47 is a validated target for cancer therapy, and its appropriate antagonists are needed for the preparation of human therapeutics.

[0009] Some CD47 blocking antibodies validated in clinical studies induce hemagglutination and anemia (Advani R et al. 2018). In addition, the therapeutic utility of CD47-SIRPα blocking monoclonal antibodies is largely impaired due to the ubiquitous expression of CD47 on aging red blood cells (RBCs), because the widespread expression of CD47 on normal cells leads to significant RBC toxicity and rapid target-mediated clearance. A priming strategy has been developed to avoid RBC depletion toxicity. Patients are first treated with low doses to remove aged RBCs, thereby inducing compensatory hematopoiesis (Advani R et al. 2018). To overcome these limitations, a new generation of CD47 antibodies is needed that efficiently target tumor cells while exerting minimal adverse effects on RBCs to avoid severe anemia. Summary of the invention

[0011] The present disclosure provides improved antibodies specific for CD47 (e.g., human CD47). The antibodies provided herein exhibit both induction of phagocytosis of cancer cells and very low to no hemagglutination and phagocytosis of RBCs, showing improved efficacy to toxicity ratios for cancer or proliferative disorders. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, vectors and host cells for preparing these antibodies, and methods of using these antibodies to treat subjects are also provided.

[0012] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to CD47, comprising: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 23, 22, 27, 47, 49, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50, 51 or 52; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO: 7 or 36.

[0013] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO:23, 22, 27, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:7; preferably, the HCDR1 is defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0014] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0015] (i) a HCDR1 comprising the sequence GYX1FTHHWX2H (SEQ ID NO: 116), wherein X1 is T or S, and X2 is M or I;

[0016] (ii) a HCDR2 comprising the sequence of MIDASDX3ETRYX4QKFX5G (SEQ ID NO: 117), wherein X3 is K, I or Y, X4 is A or S, and X5 is Q or K;

[0017] (iii) HCDR3 comprising the sequence of SEQ ID NO:90;

[0018] (iv) LCDR1 comprising the sequence of SEQ ID NO:93;

[0019] (v) LCDR2 comprising the sequence of SEQ ID NO:97; and

[0020] (vi) LCDR3 comprising the sequence of SEQ ID NO:99.

[0021] In certain embodiments, X1 is T, and / or X2 is M; preferably, the HCDR1 comprises the sequence of SEQ ID NO:57.

[0022] In certain embodiments, X4 is A, and / or X5 is Q; preferably, the HCDR2 comprises the sequence of SEQ ID NO:73, 72 or 77.

[0023] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0024] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 73 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively;

[0025] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 72 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively; or,

[0026] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 77 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0027] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0028] a HCDR1 comprising the amino acid sequence of SEQ ID NO:57, a HCDR2 comprising the amino acid sequence of SEQ ID NO:65, 66, 68, 69, 70, 71, 74, 75 or 76, a HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:93, 97 and 99, respectively; or,

[0029] HCDR1 comprising the amino acid sequence of SEQ ID NO:56, HCDR2 comprising the amino acid sequence of SEQ ID NO:65, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively; or,

[0030] HCDR1 comprising the amino acid sequence of SEQ ID NO:55, HCDR2 comprising the amino acid sequence of SEQ ID NO:62, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively; or,

[0031] HCDR1 comprising the amino acid sequence of SEQ ID NO:54, HCDR2 comprising the amino acid sequence of SEQ ID NO:62, 65, 66 or 67, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:93, 97 and 99, respectively; or,

[0032] A HCDR1 comprising the amino acid sequence of SEQ ID NO:53, a HCDR2 comprising the amino acid sequence of SEQ ID NO:63 or 64, a HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively.

[0033] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure further comprise: four heavy chain framework regions (HFR1, HFR2, HFR3 and HFR4) containing the amino acid sequences of SEQ ID NOs: 100, 101, 102 and 103, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3 and LFR4) containing the amino acid sequences of SEQ ID NOs: 108, 109, 110 and 111, respectively.

[0034] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0035] the VH comprises the sequence of SEQ ID NO:23, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0036] the VH comprises the sequence of SEQ ID NO:22, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0037] the VH comprises the sequence of SEQ ID NO: 27, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0038] The VH comprises the sequence of SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0039] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO:47, 49, 50, 51, 52, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:36; preferably, the HCDR1 is defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0040] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0041] (i) a HCDR1 comprising the sequence of GX1X2FX3HHWIH (SEQ ID NO: 118), wherein X1 is F or Y, X2 is T or S, and X3 is S or T;

[0042] (ii) a HCDR2 comprising the sequence of MIDASDSETRLX4X5X6X7KX8 (SEQ ID NO: 119), wherein X4 is S or V, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G;

[0043] (iii) HCDR3 comprising the sequence of SEQ ID NO:90;

[0044] (iv) LCDR1 comprising the sequence of SEQ ID NO:95;

[0045] (v) LCDR2 comprising the sequence of SEQ ID NO:98; and

[0046] (vi) LCDR3 comprising the sequence of SEQ ID NO:99.

[0047] In certain embodiments, X1 is F, X2 is T, and / or X3 is S; preferably, the HCDR1 comprises the sequence of SEQ ID NO:58.

[0048] In certain embodiments, X4 is S, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G.

[0049] In certain embodiments, X4 is S or V, X5 is D, X6 is S, X7 is F or V, and X8 is G; preferably, X4 is S, and / or X7 is F.

[0050] In certain embodiments, the HCDR2 comprises the sequence of SEQ ID NO:84, 86, 87, 88 or 89.

[0051] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0052] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 84 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or,

[0053] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 86 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or,

[0054] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 87 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or,

[0055] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 88 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or,

[0056] HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 89 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0057] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0058] a HCDR1 comprising the amino acid sequence of SEQ ID NO:58; a HCDR2 comprising the amino acid sequence of SEQ ID NO:61, 62, 81, 82, 83 or 85; a HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:95, 98 and 99, respectively; or

[0059] HCDR1 comprising the amino acid sequence of SEQ ID NO:53; HCDR2 comprising the amino acid sequence of SEQ ID NO:80; HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:95, 98 and 99, respectively; or

[0060] HCDR1 comprising the amino acid sequence of SEQ ID NO:59 or 60; HCDR2 comprising the amino acid sequence of SEQ ID NO:61; HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:95, 98 and 99, respectively; or

[0061] HCDR1 comprising the amino acid sequence of SEQ ID NO:53; HCDR2 comprising the amino acid sequence of SEQ ID NO:78 or 79; HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:95, 98 and 99, respectively.

[0062] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure further comprise: four heavy chain framework regions (HFR1, HFR2, HFR3, HFR4) containing the amino acid sequences of SEQ ID NOs: 104, 105, 106 and 107, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3, LFR4) containing the amino acid sequences of SEQ ID NOs: 112, 113, 114 and 115, respectively.

[0063] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0064] the VH comprises the sequence of SEQ ID NO:47, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0065] the VH comprises the sequence of SEQ ID NO:49, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0066] the VH comprises the sequence of SEQ ID NO:50, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0067] the VH comprises the sequence of SEQ ID NO:51, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0068] the VH comprises the sequence of SEQ ID NO:52, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; or

[0069] The VH comprises the sequence of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure as defined in any of the preceding embodiments may further comprise a heavy chain constant region (CH) comprising an amino acid sequence derived from a human immunoglobulin heavy chain constant region.

[0071] In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is an IgG4 heavy chain constant region, for example, an IgG4 heavy chain constant region with an S228P substitution. In certain embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 28.

[0072] In certain embodiments, the heavy chain constant region has effector function (eg, ADCC and / or ADCP) or enhanced effector function (eg, ADCC and / or ADCP).

[0073] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure as defined in any of the preceding embodiments may further comprise a light chain constant region (CL) comprising an amino acid sequence derived from a human immunoglobulin light chain constant region.

[0074] In certain embodiments, the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO:29.

[0075] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein:

[0076] The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 23 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or,

[0077] The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 22 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or,

[0078] The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 27 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or,

[0079] The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 47 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 36 and a CL comprising the sequence of SEQ ID NO: 29; or,

[0080] The heavy chain comprises a VH comprising the sequence of SEQ ID NO:49 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:36 and a CL comprising the sequence of SEQ ID NO:29.

[0081] In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure as defined in any of the preceding embodiments is selected from the group consisting of scFv, Fab, Fab', (Fab')2, Fv fragment, diabody, bispecific antibody, multispecific antibody and humanized antibody.

[0082] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are present in an amount of about 10 -6 M to about 10 -10 M, for example, about 10 -7 M to about 10 -9 M, about 10 -6 M to about 10 -8 M or 10 -7 M to about 10 -8 M or about 10 -8 M to about 10 -9 M of K D Binds wild-type human CD47.

[0083] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are present in an amount of about 10 -6 M to about 10 -10 M, for example, about 10 -7 M to about 10 -9 M, about 10-6 M to about 10 -8 M or about 10 -7 M to about 10 -8 M or about 10 -8 M to about 10 -9 M of K D Binds to a human CD47 mutant protein having a C33G mutation (eg, as shown in SEQ ID NO: 1).

[0084] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signaling; induce phagocytosis of tumor cells; and inhibit the growth of various hematological tumors and solid tumors.

[0085] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure do not induce significant hemagglutination and phagocytosis of RBCs.

[0086] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure have an improved efficacy to toxicity ratio.

[0087] In another aspect, the present disclosure provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the present disclosure and an effector molecule. In certain embodiments, the effector molecule is a therapeutic agent. In certain embodiments, the therapeutic agent is selected from the group consisting of: a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid.

[0088] On the other hand, the present disclosure provides a bispecific or multispecific antibody comprising an antibody or antigen-binding fragment thereof of the present disclosure. In certain embodiments, the bispecific or multispecific antibody specifically binds to CD47 (e.g., human CD47) and a second target. In certain embodiments, the bispecific or multispecific antibody comprises a first antigen-binding domain from an antibody or antigen-binding fragment thereof of the present disclosure and a second antigen-binding domain from an antibody directed against a second target. In certain embodiments, the second target is an immunomodulatory receptor or a tumor-associated antigen.

[0089] In another aspect, the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, or a bispecific or multispecific antibody of the disclosure.

[0090] In another aspect, the present disclosure provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present disclosure.

[0091] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid molecule of the present disclosure or the vector of the present disclosure.

[0092] In another aspect, a method for producing an antibody or antigen-binding fragment thereof, or a bispecific or multispecific antibody of the present disclosure is provided, the method comprising culturing a host cell comprising an isolated nucleic acid molecule of the present disclosure or a vector of the present disclosure under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody, and recovering the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody from a culture of the cultured host cell.

[0093] On the other hand, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, or an immunoconjugate disclosed herein, or a bispecific or multispecific antibody disclosed herein, or an isolated nucleic acid molecule, vector, or host cell disclosed herein, and a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical composition disclosed herein may further comprise an additional therapeutic agent.

[0094] In certain embodiments, the additional therapeutic agent is an anti-tumor agent.

[0095] In certain embodiments, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, e.g., a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity. In certain embodiments, the additional therapeutic antibody binds to a target other than CD47. In certain embodiments, the target is selected from an immunomodulatory receptor or a tumor-associated antigen.

[0096] In certain embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0097] In another aspect, provided is a use of an antibody or antigen-binding fragment thereof, or an immunoconjugate, or a bispecific or multispecific antibody, or an isolated nucleic acid molecule, vector or host cell, or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for use in treating cancer or a proliferative disorder in a subject.

[0098] In another aspect, provided is an antibody or antigen-binding fragment thereof of the present disclosure, or an immunoconjugate of the present disclosure, or a bispecific or multispecific antibody of the present disclosure, or an isolated nucleic acid molecule, vector or host cell of the present disclosure, or a pharmaceutical composition of the present disclosure for use in treating cancer or a proliferative disorder in a subject.

[0099] In another aspect, a method of treating cancer or a proliferative disorder in a subject is provided; wherein the method comprises administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the disclosure, or an immunoconjugate of the disclosure, or a bispecific or multispecific antibody of the disclosure, or an isolated nucleic acid molecule, vector or host cell of the disclosure, or a pharmaceutical composition of the disclosure.

[0100] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof of the present disclosure inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signaling; induce phagocytosis of tumor cells; and inhibit the growth of various hematological tumors and solid tumors.

[0101] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof of the present disclosure do not induce significant hemagglutination and phagocytosis of RBCs.

[0102] In certain embodiments, in any of the therapeutic methods or uses described herein, the antibodies or antigen-binding fragments thereof of the present disclosure have an improved efficacy to toxicity ratio.

[0103] In certain embodiments, in any of the methods or uses of treatment described herein, the cancer or proliferative disorder is associated with CD47 and / or SIRPα expression.

[0104] In certain embodiments, in any of the methods or uses for the treatment described herein, cancer or proliferative disorders are blood cancers, such as leukemia, lymphoma or myeloma. In certain embodiments, the blood cancer is a leukemia selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / tumors (MPDS) and myelodysplastic syndrome (MDS). In certain embodiments, the blood cancer is a lymphoma selected from the group consisting of Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma and follicular lymphoma (small cells and large cells). In certain embodiments, the blood cancer is a myeloma selected from the group consisting of multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones protein myeloma.

[0105] In certain embodiments, in any of the methods of treatment or uses described herein, the cancer or proliferative disorder is a solid tumor, such as breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumor, ovarian tumor, lung tumor, pancreatic tumor, prostate tumor, melanoma tumor, colorectal tumor, lung tumor, head and neck tumor, bladder tumor, esophageal tumor, liver tumor and kidney tumor.

[0106] In certain embodiments, in any of the methods or uses of treatment described herein, the subject is a mammal, such as a human.

[0107] In certain embodiments, in any of the methods or uses of treatment described herein, the antibody or antigen-binding fragment thereof, or the immunoconjugate of the disclosure, or the bispecific or multispecific antibody of the disclosure, or the pharmaceutical composition of the disclosure is used in combination with an additional therapeutic agent or an additional therapy.

[0108] In certain embodiments, the additional therapeutic agent is an anti-tumor agent.

[0109] In certain embodiments, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, e.g., a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity. In certain embodiments, the additional therapeutic antibody binds to a target other than CD47. In certain embodiments, the target is selected from an immunomodulatory receptor or a tumor-associated antigen.

[0110] In certain embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0111] In certain embodiments, the additional therapy is a standard cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0112] Further features and advantages of certain embodiments of the present disclosure will become more fully apparent from the following description of the embodiments thereof and the accompanying drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 . Anti-CD47 antibody M6 ( Figure 1 a) M11( Figure 1 b) M15( Figure 1 c) M16( Figure 1 d) M21( Figure 1 e) M22( Figure 1f) M23( Figure 1 g)、M24( Figure 1 h)、M25( Figure 1 i) M27( Figure 1 j), Mk( Figure 1 k), Mo( Figure 1 l)、Mp( Figure 1 m), Mq( Figure 1 n) and Mr( Figure 1 o) Binding to the SHP-77 tumor line. M4, Hu5F9, TJC4 antibodies, TTI-622 and an isotype control antibody were used for comparison in each graph.

[0114] Figure 2 . Anti-CD47 antibody M6 ( Figure 2 a) M11( Figure 2 b) M15( Figure 2 c) M16( Figure 2 d) M21( Figure 2 e) M22( Figure 2 f) M23( Figure 2 g)、M24( Figure 2 h)、M25( Figure 2 i) M27( Figure 2 j), Mk( Figure 2 k), Mo( Figure 2 l)、Mp( Figure 2 m), Mq( Figure 2 n) and Mr( Figure 2 o) Binding to human erythrocytes. M4, Hu5F9, TJC4 antibodies, TTI-622 and an isotype control antibody were used for comparison in each graph.

[0115] Figure 3 . Anti-CD47 antibody M4 ( Figure 3 a) M6( Figure 3 b) M16( Figure 3 c) M22 ( Figure 3 d) M23( Figure 3 e) M27( Figure 3 f), Mk( Figure 3 g), Mq( Figure 3 h) Mr( Figure 3 i) Hu5F9( Figure 3 j) TJC4( Figure 3 k) and TTI-622( Figure 3 l) Binding to human and cynomolgus CD47 CHO-K1 stable cell lines.

[0116] Figure 4 . Anti-CD47 antibody M21 ( Figure 4 a) M22( Figure 4 b) M23( Figure 4 c) M27 ( Figure 4 d) Mk( Figure 4 e) Mo( Figure 4 f), Mp( Figure 4 g), Mq( Figure 4 h) and Mr( Figure 4 i) Induced phagocytosis of CCRF-CEM tumor cells. In each figure, comparisons were made using Hu5F9 and TJC4 antibodies, TTI-622 and an isotype control antibody.

[0117] Figure 5 Phagocytosis of human erythrocytes by anti-CD47 antibodies Phagocytosis of RBCs induced by 6.4 pM and 32 pM of Hu5F9, and by 32 pM and 160 pM of the remaining antibodies were performed at a 1:1 effector to target cell ratio.

[0118] Figure 6 Phagocytosis of CCRF-CEM tumor cells induced by selected variants of the humanized 108C10A6 antibody. Hu5F9 and isotype control antibodies were used as positive and negative controls, respectively.

[0119] Figure 7 Comparison of phagocytosis of human erythrocytes and tumor cells by selected anti-CD47 antibodies at fixed concentrations (Hu5F9 at 6.4 pM and 32 pM, other antibodies at 32 pM and 160 pM, and isotype control antibody at 6.4 pM, 32 pM and 160 pM).

[0120] Figure 8 .Hemagglutination of human erythrocytes induced by anti-CD47 antibodies.

[0121] Fig. 9 . In vivo efficacy of humanized anti-CD47 antibodies. Fig. 9 a shows the change in mean tumor volume when mice were given anti-CD47 antibody, benchmark (TJC4 and TTI-622) and isotype control. Fig. 9 b- Fig. 9 m is a spider graph showing the changes in tumor volume of each tumor-bearing mouse after treatment. Fig. 9 n shows the changes in mean body weight of tumor-bearing mice after treatment. DETAILED DESCRIPTION

[0122] definition

[0123] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. For a better understanding of the present disclosure, the definitions and explanations of the terms are provided below.

[0124] As used herein, the term "CD47" refers to cluster of differentiation 47, also known as integrin-associated protein (IAP). CD47 is a 50-kDa membrane protein with an amino-terminal extracellular IgV domain, five transmembrane regions, and a short cytoplasmic tail region that is alternatively spliced. An exemplary amino acid sequence of human CD47 protein is provided in NCBI reference sequence: NP_942088.1. In the sequence of the IgV domain of CD47, it was confirmed that the first cysteine ​​residue Cys33 forms a disulfide bond with Cys263 of the multi-spanning membrane (MMS) domain. The presence of free Cys33 in the IgV domain can induce the formation of CD47 IgV domain homodimers. Therefore, in addition to wild-type CD47, the term "CD47" also includes CD47 mutant proteins with a C33G mutation. This mutant protein can be used to determine the true monovalent binding affinity of anti-CD47 antibodies.

[0125] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Unless otherwise specified or clear from the context, the term "antibody" as used herein may include a complete antibody and any antigen binding fragment thereof (i.e., "antigen binding portion") or a single chain. In one embodiment, an "antibody" is typically composed of two pairs of polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ light chains and λ light chains. The heavy chain can be classified as μ, δ, γ, α or ε, and the isotype of the antibody can be defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L Composition. H and V LThe V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are relatively conserved, termed framework regions (FRs). H and V L Each of the heavy chain / light chain is composed of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable region (V H and V L ) form antibody binding sites, respectively. The term "antibody" is not limited to any particular method for producing antibodies, for example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies.

[0126] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the AbM numbering system (Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art can easily identify the CDRs defined by each numbering system. In addition, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). In certain embodiments, the CDRs of the antibodies disclosed herein are defined according to the Kabat, AbM, IMGT or Chothia numbering systems or any combination thereof. Unless otherwise specified or clear from the context, the HCDR1 of the antibodies disclosed herein is preferably defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0127] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0128] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, and the polypeptide is also referred to as an "antigen-binding portion". Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In certain embodiments, antigen-binding fragments include fragments of Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementary determining regions (CDRs), single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody (the at least a portion being sufficient to confer specific antigen-binding ability to the polypeptide).

[0129] As used herein, the term "Fd fragment" refers to a fragment consisting of V H and C H 1 domain; the term "dAb fragment" refers to an antibody fragment consisting of V H The term "Fab fragment" refers to an antibody fragment consisting of V L 、V H , C L and C H The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments, which are connected by a disulfide bridge at the hinge region.

[0130] As used herein, the term "Fv fragment" refers to a fragment consisting of the V fragment of a single arm of an antibody. L and V H The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) is able to recognize and bind to an antigen, but the affinity is less than that of the entire binding site.

[0131] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, having a general structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having an amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0132] As used herein, the term "diabody" refers to a dimer of scFv, which is composed of VH and VL domains connected by a short peptide linker. The linker is too short to form intrachain pairing of VH and VL domains. Instead, two such scFv fragments are co-expressed to form multimers through interchain pairing (cross pairing) of VH and VL domains.

[0133] Each antigen-binding fragment maintains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen. Antigen-binding fragments can be obtained from a given antibody (e.g., a complete antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments can be screened for specificity in the same manner as the complete antibody.

[0134] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology to the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from human immunoglobulin (recipient antibody).

[0135] In order to prepare humanized antibodies, mouse CDR regions can be transplanted onto human framework sequences by using any method known in the art. Two strategies have been used to select human receptor frameworks in the present application, including using the most closely related human germline genes of the parent mouse antibody or using a "fixed framework" that performs well. Some CDR residues are mutated into their human counterparts to increase the humanization of the antibody or are mutated into some other residues to change the binding affinity of the antibody to a certain level, so that the toxicity to cancer cells is maintained, while minimizing the toxicity to RBC.

[0136] As used herein, the term "bispecific antibody" refers to an artificial hybrid antibody having two different heavy chain / light chain pairs, resulting in two antigen binding sites specific for different antigens. Bispecific antibodies can be produced by a variety of methods, including connecting a first antibody or a fragment thereof to a second antibody or a fragment thereof, for example, by chemical coupling, genetic fusion, non-covalent association or other means. "Multispecific antibody" refers to an artificial hybrid antibody having more than two different binding specificities, including, for example, a trispecific antibody or a tetraspecific antibody.

[0137] As used herein, the term "specifically bind" or "specific for" refers to the binding of two molecules in a non-random manner, such as the reaction between an antibody and an antigen to which it is directed. In some embodiments, an antibody that specifically binds to an antigen (or is specific for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M (e.g., less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less) D )Antibodies that bind to an antigen.

[0138] As used herein, the term "K D "" refers to the dissociation constant of a specific antibody-antigen interaction and is used to describe the binding affinity of an antibody to an antigen. The smaller the dissociation constant, the tighter the antibody binds and the higher the affinity between the antibody and the antigen. Typically, an antibody (e.g., an antibody of the present disclosure) has a dissociation constant of less than about 10 -5 M (e.g., less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller)K D Binding antigen (e.g., human CD47), the K D The antibodies disclosed herein are measured by surface plasmon resonance (SPR), for example, in a BIACORE device. -6 M to about 10 -10 M, for example, about 10 -7 M to about 10 -9 M, about 10 -6 M to about 10 -8 M, or about 10 -7 M to about 10 -8 M, or about 10 -8 M to about 10 -9 M of K D Binds human CD47.

[0139] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, phages, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)); phages (such as lambda phages or M13 phages) and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may include multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may include an origin of replication.

[0140] As used herein, the term "host cell" refers to a cell into which a vector can be introduced or transformed, including but not limited to prokaryotic cells (such as Escherichia coli or Bacillus subtilis), and eukaryotic cells (such as mammalian cells (e.g., mouse cells or human cells), insect cells or yeast cells). Suitable eukaryotic cells include but are not limited to NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells or MDCKII cells.

[0141] As used herein, the term "identity" refers to the degree of matching between two polypeptides or between two nucleic acids. When the two sequences used for comparison have the same base or amino acid monomer subunit at a certain site (for example, each of the two DNA molecules has adenine at a certain site, or each of the two polypeptides has lysine at a certain site), the two molecules are identical at that site. The percentage of identity between two sequences is a function of the number of identical sites shared by the two sequences relative to the total number of sites for comparison × 100. For example, if 6 of the 10 sites of the two sequences are matched, the two sequences have 60% identity. For example, the DNA sequences: CTGACT and CAGGTT have 50% identity (3 of the 6 sites are matched). Generally, the comparison of the two sequences is carried out in a manner that produces maximum identity. This comparison can be carried out using a computer program (such as the Align program (DNAstar, Inc.) based on the method of Needleman et al. (J.Mol.Biol.48:443-453,1970)). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0142] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Gennaro AR, ed., 19th edition Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffered saline. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, and can stabilize the desired activity of active ingredients in drugs (including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc.). In certain exemplary embodiments, pharmaceutically acceptable carriers or excipients include sterile injectable liquids (e.g., aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, the sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0143] As used herein, the term "treatment / treating" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, relieving symptoms, reducing the scope of the disease, stabilizing (i.e., not aggravating) the disease state, delaying or slowing disease progression, and relieving symptoms (partially or completely), whether detectable or undetectable. In addition, "treatment" also refers to a survival period that is extended compared to the expected survival period (if not receiving treatment). In certain embodiments, the beneficial or desired clinical outcomes described herein include, but are not limited to, slowing tumor progression, cancer regression, reducing tumor growth or size, reducing the severity of at least one disease symptom, increasing the frequency and duration of the disease asymptomatic period, preventing damage or disability caused by disease torture, or otherwise improving the patient's disease symptoms.

[0144] As used herein, the term "subject" refers to any human or non-human animal receiving prophylactic or therapeutic treatment. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0145] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for treating a disease refers to an amount that effectively cures or at least partially blocks the disease and its complications in a patient suffering from the disease. The determination of such an effective amount is within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's immune system, the patient's overall condition (such as age, weight and sex), the route of drug administration, other therapies used simultaneously, etc.

[0146] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (the Fc region of a native sequence or amino acid sequence variant), which vary with the isotype of the antibody. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods for altering the effector functions of antibodies are known in the art, for example, by introducing mutations in the Fc region.

[0147] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which cytotoxic effector cells specifically bind to target cells to which antigens are attached through the binding of Ig to Fc receptors (FcRs) presented on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), and then kill the target cells by secreting cytotoxins. Methods for detecting ADCC activity of antibodies are known in the art.

[0148] As used herein, the term "antibody-dependent cellular phagocytosis (ADCP)" refers to a form of phagocytosis in which antibody-opsonized target cells activate FcγR on the surface of effector cells (such as monocytes and macrophages) with phagocytic potential to induce phagocytosis, thereby leading to internalization and degradation of target cells by phagosome acidification. Methods for detecting the ADCP activity of antibodies are known in the art.

[0149] As used herein, the terms "cancer," "tumor," and "proliferative disorder" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases.

[0150] The terms "cancer", "tumor" or "proliferative disorder" may include, but are not limited to, solid cancers. "Solid cancers" include, but are not limited to, breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.

[0151] The term "cancer", "tumor" or "proliferative disorder" also includes, but is not limited to, hematological malignancies."Hematological malignancies" can be considered to refer to at least blood cancers. Such cancers originate from other cells of blood-forming tissues (such as bone marrow) or immune systems. Hematological malignancies include, but are not limited to, leukemias (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / tumors (MPDS) and myelodysplastic syndrome (MDS)), lymphomas (e.g., Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cells and large cells)), myeloma (e.g., multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones protein myeloma).

[0152] As used herein, the term "about" when used to modify a numerical value or numerical range means that deviations of up to 10% (e.g., up to 5% above, up to 2% above, or up to 1% above) and up to 10% (e.g., up to 5% below, up to 2% below, or up to 1% below) above the stated value or range are still within the intended meaning of the stated value or range.

[0153] Antibodies of the Disclosure

[0154] The inventors found that by reducing the binding affinity of the anti-CD47 antibody, the toxicity to RBCs was significantly reduced. Therefore, the inventors adjusted the binding affinity of the anti-CD47 antibody to a certain level so that the toxicity to cancer cells was maintained while minimizing the toxicity to RBCs, thereby generating the improved anti-CD47 antibodies provided herein that show better tumor growth inhibition but without significant hemagglutination and phagocytosis of RBCs.

[0155] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds CD47 (e.g., human CD47), comprising: HCDR1, HCDR2, and HCDR3 of VH as shown in SEQ ID NO: 23, 22, 27, 47, 49, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50, 51 or 52; and LCDR1, LCDR2, and LCDR3 of VL as shown in SEQ ID NO: 7 or 36.

[0156] In certain embodiments, the CDRs are defined according to the Kabat, AbM, IMGT or Chothia numbering systems, or any combination thereof. In certain embodiments, the HCDR1 is defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0157] VH5 Series

[0158] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO:23, 22, 27, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:7.

[0159] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0160] (i) a HCDR1 comprising the sequence GYX1FTHHWX2H (SEQ ID NO: 116), wherein X1 is T or S, and X2 is M or I;

[0161] (ii) a HCDR2 comprising the sequence of MIDASDX3ETRYX4QKFX5G (SEQ ID NO: 117), wherein X3 is K, I or Y, X4 is A or S, and X5 is Q or K;

[0162] (iii) HCDR3 comprising the sequence of SEQ ID NO:90;

[0163] (iv) LCDR1 comprising the sequence of SEQ ID NO:93;

[0164] (v) LCDR2 comprising the sequence of SEQ ID NO:97; and

[0165] (vi) LCDR3 comprising the sequence of SEQ ID NO:99.

[0166] In certain embodiments, X1 is T, and / or X2 is M. In certain embodiments, the HCDR1 comprises the sequence of SEQ ID NO:57.

[0167] In certain embodiments, X4 is A, and / or X5 is Q; preferably, the HCDR2 comprises the sequence of SEQ ID NO:73, 72 or 77.

[0168] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 73 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0169] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 72 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0170] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 77 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0171] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 65 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0172] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 66 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0173] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 68 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0174] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 69 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0175] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 70 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0176] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 71 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0177] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 74 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0178] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 75 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0179] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 57, 76 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively. In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 56, 65 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0180] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 56, 65 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0181] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 55, 62 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0182] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 54, 62 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0183] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 54, 65 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0184] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 54, 66 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0185] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 54, 67 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0186] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 53, 63 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0187] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 53, 64 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

[0188] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure further comprise: four heavy chain framework regions (HFR1, HFR2, HFR3 and HFR4) containing the amino acid sequences of SEQ ID NOs: 100, 101, 102 and 103, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3 and LFR4) containing the amino acid sequences of SEQ ID NOs: 108, 109, 110 and 111, respectively.

[0189] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 23, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0190] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 22, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0191] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 27, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0192] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0193] VH10 Series

[0194] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO:47, 49, 50, 51, 52, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:36.

[0195] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0196] (i) a HCDR1 comprising the sequence of GX1X2FX3HHWIH (SEQ ID NO: 118), wherein X1 is F or Y, X2 is T or S, and X3 is S or T;

[0197] (ii) a HCDR2 comprising the sequence of MIDASDSETRLX4X5X6X7KX8 (SEQ ID NO: 119), wherein X4 is S or V, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G;

[0198] (iii) HCDR3 comprising the sequence of SEQ ID NO:90;

[0199] (iv) LCDR1 comprising the sequence of SEQ ID NO:95;

[0200] (v) LCDR2 comprising the sequence of SEQ ID NO:98; and

[0201] (vi) LCDR3 comprising the sequence of SEQ ID NO:99.

[0202] In certain embodiments, X1 is F, X2 is T, and / or X3 is S. In certain embodiments, the HCDR1 comprises the sequence of SEQ ID NO:58.

[0203] In certain embodiments, X4 is S, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G.

[0204] In certain embodiments, X4 is S or V, X5 is D, X6 is S, X7 is F or V, and X8 is G; preferably, X4 is S, and / or X7 is F.

[0205] In certain embodiments, the HCDR2 comprises the sequence of SEQ ID NO:84, 86, 87, 88 or 89.

[0206] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 84 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0207] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 86 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0208] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 87 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0209] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 88 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0210] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 89 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0211] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 61 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0212] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 62 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0213] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 81 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0214] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 82 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0215] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 83 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0216] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 58, 85 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0217] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 53, 80 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0218] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 53, 78 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0219] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 53, 79 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0220] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 59, 61 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0221] In certain embodiments, the HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NOs: 60, 61 and 90, respectively; and the LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

[0222] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure further comprise: four heavy chain framework regions (HFR1, HFR2, HFR3, HFR4) containing the amino acid sequences of SEQ ID NOs: 104, 105, 106 and 107, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3, LFR4) containing the amino acid sequences of SEQ ID NOs: 112, 113, 114 and 115, respectively.

[0223] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 47, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0224] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 49, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0225] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 50, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0226] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 51, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0227] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 52, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0228] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: a VH comprising the sequence of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto; and / or a VL comprising the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0229] Constant region

[0230] The antibodies or antigen-binding fragments thereof of the present disclosure may further comprise a constant region sequence derived from a mammalian (eg, murine or human) immunoglobulin.

[0231] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a constant region sequence derived from a human immunoglobulin.

[0232] Any immunoglobulin constant region can be used in the antibodies disclosed herein. In certain embodiments, the heavy chain constant region can be a heavy chain constant region of human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. The light chain constant region can be λ or κ. In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, the light chain constant region is κ.

[0233] The selection of constant regions depends in part on whether effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC)) are required. In certain embodiments, heavy chain constant regions with less or reduced effector functions are preferred. In certain embodiments, heavy chain constant regions with effector functions or enhanced effector functions are preferred. In certain embodiments, heavy chain constant regions with ADCC and / or ADCP or enhanced ADCC and / or ADCP are preferred.

[0234] In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain constant region (CH) comprising an amino acid sequence derived from a human immunoglobulin heavy chain constant region.

[0235] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain constant region that is a wild-type heavy chain constant region.

[0236] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise a heavy chain constant region that is a variant of a wild-type heavy chain constant region. The constant region may be altered (e.g., mutated) to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: stability, Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, and / or effector functions).

[0237] In certain embodiments, one or more mutations (eg, one or more amino acid substitutions) can be introduced into the Fc region of the constant region to increase the stability of the antibody or extend the half-life of the antibody.

[0238] In certain embodiments, one or more mutations (e.g., one or more amino acid substitutions) can be introduced into the Fc region of the constant region to alter (increase, decrease or eliminate) one or more effector functions of the antibody, such as ADCC, CDC or ADCP, compared to an antibody without the one or more mutations.

[0239] In certain embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises an IgG4 heavy chain constant region. In one embodiment, the IgG4 constant region is a wild-type constant region. In another embodiment, the IgG4 constant region comprises a mutation (e.g., S228P) that changes one or more effector functions to reduce Fab arm exchange. In certain embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0240] In certain embodiments, the light chain of an antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain constant region (CL) comprising an amino acid sequence derived from a human immunoglobulin light chain constant region.

[0241] In certain embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:29.

[0242] Full-length Antibodies

[0243] The antibodies of the present disclosure may be antibodies comprising two heavy chains and two light chains having a general "Y" shaped structure.

[0244] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein: the heavy chain comprises a VH comprising the sequence of SEQ ID NO:23 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:7 and a CL comprising the sequence of SEQ ID NO:29.

[0245] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein: the heavy chain comprises a VH comprising the sequence of SEQ ID NO:22 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:7 and a CL comprising the sequence of SEQ ID NO:29.

[0246] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein: the heavy chain comprises a VH comprising the sequence of SEQ ID NO:27 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:7 and a CL comprising the sequence of SEQ ID NO:29.

[0247] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein: the heavy chain comprises a VH comprising the sequence of SEQ ID NO:47 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:36 and a CL comprising the sequence of SEQ ID NO:29.

[0248] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain, wherein: the heavy chain comprises a VH comprising the sequence of SEQ ID NO:49 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:36 and a CL comprising the sequence of SEQ ID NO:29.

[0249] Fab

[0250] The antibodies disclosed herein may also be Fab fragments, Fab', F(ab)2, Fv, scFv or any other type of fragment of an antibody having a general "Y"-shaped structure, which substantially retains the ability to specifically bind to CD47 (e.g., human CD47), and the ability to inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signaling, induce phagocytosis of tumor cells and inhibit tumor growth.

[0251] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are selected from the group consisting of scFv, Fab, Fab', (Fab')2, Fv fragments, diabodies, bispecific antibodies, multispecific antibodies, or humanized antibodies.

[0252] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are present in an amount of about 10 -6 M to about 10 -10 M, for example, about 10 -7 M to about 10 -9 M, about 10 -6 M to about 10 -8 M, or about 10 -7 M to about 10 -8 M, or about 10 -8 M to about 10 -9 M of K D Binds wild-type human CD47.

[0253] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are present in an amount of about 10 -6 M to about 10 -10 M, for example, about 10 -7 M to about 10 -9 M, about 10 -6 M to about 10 -8 M, or about 10 -7 M to about 10 -8 M, or about 10 -8 M to about 10 -9 M of K D Binds to a human CD47 mutant protein having a C33G mutation (eg, as shown in SEQ ID NO: 1).

[0254] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signaling; induce phagocytosis of tumor cells; and inhibit the growth of various hematological tumors and solid tumors.

[0255] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure do not induce significant hemagglutination and phagocytosis of RBCs.

[0256] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure have an improved efficacy to toxicity ratio.

[0257] Also provided are "conservative sequence modifications" to the antibody sequences provided herein, i.e., nucleotide sequence modifications and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. For example, modifications can be introduced by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). The resulting modified antibodies can be screened for their binding activity.

[0258] Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Examples of conservative substitutions are shown in the table below. Therefore, the non-essential amino acid residues predicted in the antibodies disclosed herein can preferably be replaced by another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art. See, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0259] Derivatized Antibodies

[0260] The antibodies or antigen-binding fragments thereof disclosed herein can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Typically, the derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to CD47. Therefore, the antibodies or antigen-binding fragments thereof disclosed herein are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof disclosed herein can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide capable of mediating the association of an antibody or antigen-binding fragment thereof with another molecule (e.g., an avidin or polyhistidine tag).

[0261] Labeled Antibodies

[0262] In certain embodiments, the antibody or its Fab of the present disclosure is labeled, such as a detectable marker. A detectable marker can be any substance that can be detected directly or indirectly (for example, by enzymatic reaction or molecular interaction). In certain embodiments, the marker can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. In certain embodiments, the detectable marker can be suitable for immunoassay (for example, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the marker is selected from enzymes, radionuclides, fluorescent dyes, luminescent materials (for example, chemiluminescent materials) or biotin. In certain embodiments, the marker can be connected to the antibody or its Fab of the present disclosure via joints of different lengths to reduce potential steric hindrance.

[0263] In certain embodiments, the labeled antibodies or antigen-binding fragments thereof as described herein can be used to detect the presence of CD47 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

[0264] In certain embodiments, a method for detecting CD47 in a cell is provided, the method comprising contacting the cell with a labeled antibody or antigen binding fragment thereof as described herein. In certain embodiments, a method for detecting the presence of CD47 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of CD47 protein in a biological sample. In certain embodiments, the CD47 is human CD47. In certain embodiments, the method comprises contacting a biological sample with a labeled antibody or antigen binding fragment thereof as described herein under conditions that allow the antibody or antigen binding fragment thereof to bind to CD47, and detecting a signal from the label. This method can be an in vitro or in vivo method. In certain embodiments, a method for diagnosing a disease associated with CD47 expression in an individual (e.g., cancer) is provided, the method comprising administering a labeled antibody or antigen binding fragment thereof as described herein to an individual, and detecting a label in the individual. In certain embodiments, a labeled antibody or antigen binding fragment thereof as described herein is used to select a subject eligible for therapy with any anti-CD47 therapeutic agent (e.g., an antibody or antigen binding fragment thereof, an immunoconjugate, or a bispecific or multispecific antibody as described herein), wherein CD47 is a biomarker for selecting patients.

[0265] Immunoconjugates

[0266] In another aspect, an immunoconjugate is provided, comprising an antibody or antigen-binding fragment thereof as described herein and an effector molecule. Exemplary effector molecules include, but are not limited to, drugs, toxins, radioisotopes, proteins, peptides, and nucleic acids. In certain embodiments, the effector molecule is a therapeutic agent.

[0267] In certain embodiments, the immunoconjugates of the present disclosure comprise an antibody or antigen-binding fragment thereof as described herein, 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), or radioactive isotopes.

[0268] In certain embodiments, the immunoconjugates of the present disclosure are antibody-drug conjugates (ADCs) in which an antibody or antigen-binding fragment thereof as described herein is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588 and 7,498,298); calicheamicin or a derivative thereof (see U.S. Pat. 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)); and anthracyclines 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. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0269] In certain embodiments, the immunoconjugates of the disclosure comprise an antibody or antigen-binding fragment thereof as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modicin A chain, alpha-sarcin, Aleurites fordii proteins, Dianthus dianthus proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

[0270] In certain embodiments, the immunoconjugates disclosed herein comprise an antibody or antigen-binding fragment thereof as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as tc99m or I123 for scintigraphic studies, or a spin label such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, "MRI").

[0271] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0272] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0273] Bispecific Antibodies

[0274] The antibody or its antigen binding fragment of the present disclosure can be used to form a bispecific or multispecific antibody. The antibody or its antigen binding fragment of the present disclosure can be a part of a bispecific or multispecific antibody, which includes a second functional module (e.g., a second antibody) with a binding specificity different from the binding specificity of the antibody or its antigen binding fragment of the present disclosure, so that it can bind to at least two different binding sites and / or target molecules. For example, the antibody or its antigen binding fragment of the present disclosure can be connected to a second antibody or its antigen binding fragment, which specifically binds to any protein that can be used as a potential target for combination therapy. In order to produce bispecific or multispecific antibodies, the antibody or its antigen binding fragment of the present disclosure can be connected to one or more other binding molecules (e.g., other antibodies, antibody fragments, peptides or binding mimetics) (e.g., by chemical coupling, gene fusion, non-covalent association or other means).

[0275] Thus, in another aspect, the present disclosure provides bispecific or multispecific antibodies comprising an antibody or antigen-binding fragment thereof of the present disclosure.

[0276] In certain embodiments, the bispecific or multispecific antibody specifically binds to CD47 (e.g., human CD47) and a second target. In certain embodiments, the bispecific or multispecific antibody comprises a first antigen binding domain from an antibody or antigen binding fragment thereof of the disclosure and a second antigen binding domain from an antibody against a second target.

[0277] In certain embodiments, the second target is an immunomodulatory receptor or a tumor-associated antigen.

[0278] Antibody production

[0279] The antibodies disclosed herein can be obtained by genetic engineering recombinant technology. For example, the DNA molecules encoding the genes of the heavy and light chains of the antibodies disclosed herein can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells, such as HEK293 cells, CHO cells or other cells that do not produce immunoglobulins. Then, the transfected host cells are cultured under specific conditions, and these transfected host cells express the antibodies disclosed herein.

[0280] The antigen-binding fragments disclosed herein can be obtained by hydrolysis of intact antibody molecules. Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from recombinant host cells; and Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab')2 fragments can also be directly isolated from recombinant host cell cultures. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

[0281] In another aspect, the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, or a bispecific or multispecific antibody of the disclosure.

[0282] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain variable region and the light chain variable region, respectively, of an antibody or antigen-binding fragment thereof of the disclosure.

[0283] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain and the light chain, respectively, of an antibody or antigen-binding fragment thereof of the disclosure.

[0284] In certain embodiments, the isolated nucleic acid molecules comprise different nucleotide sequences encoding different polypeptide chains of a bispecific or multispecific antibody, respectively.

[0285] In another aspect, the present disclosure provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present disclosure.

[0286] In certain embodiments, the vector of the present disclosure is, for example, a plasmid, a cosmid, a phage, etc. In certain embodiments, the vector can express the antibody or antigen-binding fragment thereof of the present disclosure in a subject (eg, a mammal such as a human).

[0287] In certain embodiments, the vector comprises a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the present disclosure, respectively. The first nucleotide sequence and the second nucleotide sequence may be located on the same or different vectors.

[0288] In certain embodiments, the vector comprises a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain and light chain of the antibody or antigen-binding fragment thereof of the present disclosure, respectively. The first nucleotide sequence and the second nucleotide sequence may be located on the same or different vectors.

[0289] In certain embodiments, the vector comprises different nucleotide sequences encoding different polypeptide chains of a bispecific or multispecific antibody, respectively. The different nucleotide sequences can be located on the same or different vectors.

[0290] On the other hand, the present disclosure provides a host cell comprising or transformed with the following: an isolated nucleic acid molecule of the present disclosure or a vector of the present disclosure. Such host cells include, but are not limited to, prokaryotic cells (such as E. coli cells) and eukaryotic cells (such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells and human cells)).

[0291] In another aspect, a method for producing an antibody or antigen-binding fragment thereof, or a bispecific or multispecific antibody of the present disclosure is provided, the method comprising culturing a host cell comprising an isolated nucleic acid molecule of the present disclosure or a vector of the present disclosure under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody, and recovering the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody from a culture of the cultured host cell.

[0292] Pharmaceutical composition

[0293] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure, or an immunoconjugate of the present disclosure, a bispecific or multispecific antibody of the present disclosure, or an isolated nucleic acid molecule, vector, or host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0294] In certain embodiments, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof of the present disclosure. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding fragment thereof of the present disclosure. In certain embodiments, the antibody or antigen-binding fragment thereof is the only active ingredient contained in the pharmaceutical composition.

[0295] In certain embodiments, the pharmaceutical composition comprises an immunoconjugate of the present disclosure. In certain embodiments, the pharmaceutical composition comprises an effective amount of an immunoconjugate of the present disclosure. In certain embodiments, the immunoconjugate is the only active ingredient contained in the pharmaceutical composition.

[0296] In certain embodiments, the pharmaceutical composition comprises a bispecific or multispecific antibody of the present disclosure. In certain embodiments, the pharmaceutical composition comprises an effective amount of a bispecific or multispecific antibody of the present disclosure. In certain embodiments, the bispecific or multispecific antibody is the only active ingredient contained in the pharmaceutical composition.

[0297] In certain embodiments, the pharmaceutical composition of the present disclosure may further comprise an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-tumor agent.

[0298] In certain embodiments, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, e.g., a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity. In certain embodiments, the additional therapeutic antibody binds to a target other than CD47. In certain embodiments, the target is selected from an immunomodulatory receptor or a tumor-associated antigen.

[0299] In certain embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0300] In certain embodiments, the antibody or antigen-binding fragment thereof or the immunoconjugate or the bispecific or multispecific antibody of the present disclosure and the additional therapeutic agent can be provided as separate components or as components of a single composition. The antibody or antigen-binding fragment thereof or the immunoconjugate or the bispecific or multispecific antibody of the present disclosure can be used in combination with other agents simultaneously, separately or sequentially.

[0301] Pharmaceutical compositions may be provided in unit dosage form (ie, a dose for a single administration).

[0302] The pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers and / or excipients. The formulation depends on the selected route of administration. For parenteral administration, the pharmaceutical composition is preferably sterile and substantially isotonic and manufactured under GMP conditions. For example, the antibodies disclosed herein can be formulated in aqueous solutions for injection, preferably in physiologically compatible buffers, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solutions. The solution may contain formulation agents, such as suspending agents, stabilizers and / or dispersants. Alternatively, the antibody may be in lyophilized form for construction with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

[0303] The pharmaceutical compositions described herein can be used to induce phagocytosis of cancer cells and treat cancer or proliferative disorders without inducing significant hemagglutination and phagocytosis of RBCs.

[0304] Uses and methods

[0305] The antibodies or antigen-binding fragments thereof disclosed herein can specifically bind to CD47 (e.g., human CD47), inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signal transduction, thereby inducing phagocytosis of tumor cells and inhibiting the growth of various blood tumors and solid tumors. At the same time, the antibodies or antigen-binding fragments thereof disclosed herein do not induce significant hemagglutination and phagocytosis of RBCs, exerting minimal adverse effects on RBCs to avoid severe anemia. Therefore, the antibodies described herein can be used for treatment in a variety of therapeutic applications, including the treatment of cancer or proliferative disorders.

[0306] In one aspect, the disclosure provides a method of treating cancer or a proliferative disorder in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, immunoconjugate, bispecific or multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition disclosed herein.

[0307] In another aspect, the disclosure provides use of an antibody or antigen-binding fragment thereof, an immunoconjugate, a bispecific or multispecific antibody, an isolated nucleic acid molecule, a vector, a host cell, or a pharmaceutical composition disclosed herein in the manufacture of a medicament for use in treating cancer or a proliferative disorder in a subject.

[0308] In another aspect, the disclosure relates to an antibody or antigen-binding fragment thereof, immunoconjugate, bispecific or multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition disclosed herein for use in treating cancer or a proliferative disorder in a subject.

[0309] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof of the present disclosure inhibit, block, antagonize, neutralize or otherwise interfere with CD47 expression, activity and / or signaling; induce phagocytosis of tumor cells; and inhibit the growth of various hematological tumors and solid tumors.

[0310] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof of the present disclosure do not induce significant hemagglutination and phagocytosis of RBCs.

[0311] In certain embodiments, in any of the therapeutic methods or uses described herein, the antibodies or antigen-binding fragments thereof of the present disclosure have an improved efficacy to toxicity ratio.

[0312] In certain embodiments, the cancer or proliferative disorder involved in the methods or uses described herein is associated with CD47 and / or SIRPα expression. In certain embodiments, the cancer or proliferative disorder is associated with CD47 expression.

[0313] In certain embodiments, the cancers or proliferative disorders involved in the methods or uses of treatment described herein include, but are not limited to, solid tumors and hematological malignancies.

[0314] In certain embodiments, the cancer or proliferative disorder is a blood cancer, such as a leukemia, a lymphoma, or a myeloma. In certain embodiments, the blood cancer is a leukemia selected from the group consisting of: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / tumors (MPDS) and myelodysplastic syndrome (MDS). In certain embodiments, the blood cancer is a lymphoma selected from the group consisting of: Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma and follicular lymphoma (small cells and large cells). In certain embodiments, the blood cancer is a myeloma selected from the group consisting of: multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones protein myeloma.

[0315] In certain embodiments, the cancer or proliferative disorder is a solid tumor, e.g., breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.

[0316] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies, immunoconjugates, or bispecific or multispecific antibodies of the present disclosure can be used alone. Alternatively, the antibodies, immunoconjugates, or bispecific or multispecific antibodies of the present disclosure can be used in combination with another therapeutic agent. In certain embodiments, the other therapeutic agent is an anti-tumor agent.

[0317] In certain embodiments, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, e.g., a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity. In certain embodiments, the additional therapeutic antibody binds to a target other than CD47. In certain embodiments, the target is selected from an immunomodulatory receptor or a tumor-associated antigen.

[0318] In certain embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0319] In certain embodiments, in any of the methods or uses of treatment described herein, an antibody, immunoconjugate, or bispecific or multispecific antibody of the disclosure is used in combination with an additional therapy (e.g., standard cancer treatment such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care).

[0320] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof, immunoconjugates, bispecific or multispecific antibodies or pharmaceutical compositions disclosed herein can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. A preferred dosage form is an injection. This injection can be a sterile injectable solution. Alternatively, for ease of storage and use, a sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying).

[0321] In certain embodiments, in any of the methods or uses of treatment described herein, the antibodies or antigen-binding fragments thereof, immunoconjugates, bispecific or multispecific antibodies or pharmaceutical compositions disclosed herein can be administered by any suitable method known in the art (including but not limited to oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops) or nasal routes). However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person will understand that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies disclosed herein are administered by intravenous injection or bolus.

[0322] In certain embodiments, in any of the methods or uses of treatment described herein, the subject is a human. In certain embodiments, the subject has cancer.

[0323] Examples

[0324] The following examples discuss the humanization and optimization of monoclonal antibodies against human CD47, and also provide exemplary methods by which the binding, blocking, induction of RBC phagocytosis and RBC hemagglutination, and tumor growth inhibition activities of the antibodies described in this application can be determined.

[0325] Unless otherwise specified, the examples provided below are for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0326] Example 1: Optimization of anti-human CD47 antibody

[0327] Humanization pioneered by Winter and colleagues (Winter G, Harris WJ 1993) takes advantage of the conservative nature of antibody frameworks, which allows the mouse CDRs to be grafted onto human acceptor frameworks. Strategies for selecting human acceptor frameworks include using the human germline genes that are most closely related to the parental mouse antibody or using a well-performing "fixed framework". We used two strategies for the humanization and optimization of mouse anti-human CD47 antibodies.

[0328] Closely related human germline genes as sequence receptors

[0329] The VH (hereinafter referred to as 108VH, SEQ ID NO: 2) and VL (hereinafter referred to as 108VL, SEQ ID NO: 3) sequences of the mouse anti-CD47 antibody 108C10A6 (WO 2019 / 144895 A1) were analyzed. The humanization of 108VL was based on the humanized VL sequence of 108VL1.M1 (SEQ ID NO: 5). Germline was performed so that more mouse CDR residues became their human germline counterparts, resulting in the sequence of 108VL1.M5 (SEQ ID NO: 7). The most appropriate human VH framework on which the CDR-grafted heavy chain was constructed was identified as those of GenBank accession number ABM67212. The 108VH CDR was directly transplanted onto the ABM67212 human framework to produce the humanized VH sequence of 108VH5 (SEQ ID NO: 6). No back mutations were performed in the framework region. In order to remove the potential acid-labile Asp52-Pro53 motif in HCDR2, the P53A mutation was introduced into the humanized VH sequence. In addition, the mouse HCDR residues were gradually mutated to their human germline counterparts to increase the humanization of the sequence, resulting in the sequences of 108VH5.M5 (SEQ ID NO: 8), 108VH5.M6 (SEQ ID NO: 9), 108VH5.M8-108VH5.M12 (SEQ ID NO: 10-14), 108VH5.M14-108VH5.M16 (SEQ ID NO: 15-17). In order to remove the potential isomerization of the Asp55-Gly56 motif in the HCDR2 of 108VH5.M16, mutations of Asp55 and Gly56 were performed to generate sequences of 108VH5.M17 (SEQ ID NO: 18), 108VH5.M18 (SEQ ID NO: 19), 108VH5.M20-108VH5.M27 (SEQ ID NO: 20-27). These humanized VH and VL sequences were connected to the human IgG4 constant region sequence (SEQ ID NO: 28) and the human kappa light chain constant region sequence (SEQ ID NO: 29) with S228P mutations to construct full-length IgG. Heavy chain and light chain plasmids were prepared, paired and used to produce IgG by HEK293 cells. The supernatant of transfected HEK293 cells was collected and surface plasmon resonance (SPR) was performed on a BIAcore T200 instrument (GE Healthcare).

[0330] In the sequence of the IgV domain of CD47, it was confirmed that the first cysteine ​​residue Cys33 forms a disulfide bond with Cys263 of the multi-span membrane (MMS) domain (Rebres RA 2001). The presence of free Cys33 in the IgV domain can induce the formation of CD47 IgV domain homodimers. Therefore, in addition to wild-type human CD47 (Acro Biosystems, catalog number CD7-H5227), a CD47 mutant protein with a C33G mutation (hereinafter referred to as CD47-C33G) is also used to determine the true monovalent binding affinity. The SPR assay is performed as follows: the antibodies secreted into the culture medium are captured by the interaction between the polyclonal antibody and the human Fc onto a sensor chip pre-coated with goat anti-human pAb (Jackson ImmunoResearch, catalog number 109-005-098). Increasing concentrations (ranging from 2.5 nM to 1.28 μM) of wild-type CD47 or CD47-C33G were flowed over the sensor chip surface and allowed to bind the captured antibody for 100 s, followed by injection of running buffer to allow antigen protein dissociation. The association rate (k) was calculated based on the association and dissociation curves. a ) and dissociation rate (k d ), and these rates were used to estimate the equilibrium dissociation constant (K D The binding affinities of the chimeric antibody 108C10A6 and the previous humanized antibody 108VH4.M4-VL1.M1 (WO 2019 / 144895A1, variable domain SEQ ID NOs: 373 and 377) and the TJC4 positive control antibody (US 2020 / 0140565A1, variable domain SEQ ID NOs: 31 and 32) were also measured for comparison.

[0331] As might be expected, for antibodies with lower monovalent binding affinities (particularly for antibodies with μM or sub-μM D The difference between the binding affinities measured using wild-type CD47 and CD47-C33G was significant. After humanization, a series of humanized antibodies with binding affinities ranging from 1.3 μM to 3.4 nM were generated (Table 1).

[0332] Table 1. Humanization of 108C10A6 using the framework of the closest related human germline sequence.

[0333] Humanized 4D5 framework as sequence acceptor

[0334] The humanized 4D5 framework has been optimized using a consensus approach, and the robust stability of the 4D5 scFv has been demonstrated (and Plückthun, 1999), and was therefore used as a sequence acceptor, on which the CDRs of 108VL1.M1 and 108VH were directly grafted, resulting in the sequences of 108VL10.M1 (SEQ ID NO: 30) and 108VH10 (SEQ ID NO: 31), respectively. SPR affinity measurements were performed on the directly grafted antibodies as described above. The monovalent binding affinity decreased by 34-fold (Table 2, Experiment 1). 108VL10.M1 was germlined, and more mouse CDR residues were changed to their human counterparts in the sequence of its closest human Vκ germline IGKV1-39*01, resulting in the sequences of 108VL10.Ma-108VL10.Md (SEQ ID NO: 32-35). Similarly, the mouse VH CDR residues were changed to their human counterparts in the sequence of its closest human VH germline IGHV3-7*01, resulting in sequences of 108VH10.Ma-108VH10.Mf (SEQ ID NOs: 37-42). From SPR experiment 2, it can be seen that except for 108VL10.Mb, which reduced the binding affinity by 7.7 times, none of the VL germline mutations significantly reduced the monovalent binding affinity of the directly transplanted antibody. Therefore, the germline mutations of 108VL10.Ma, 108VL10.Mc and 108VL10.Md were combined to further increase the humanization of VL, resulting in the sequence of 108VL10.Me (SEQ ID NO: 36). For VH germline, 108VH10.Ma did not affect the binding affinity, so it was kept in the humanized sequence. 108VH10.Mb also did not affect the binding affinity, but it may increase the risk of oxidation. 108VH10.Mc, 108VH10.Md and 108VH10.Me significantly reduced or completely abolished binding to human CD47-C33G and were therefore not incorporated into the humanized VH sequence. 108VH10.Mf reduced the monovalent binding affinity by 6.7-fold compared to 108VH10 (Table 2, Experiment 2). Since attempts to remove the potentially acid-labile Asp52-Pro53 motif in HCDR2 by replacing Asp52 with Lys (108VH10.Md) or Pro53 with Gln (108VH10.Me) were unsuccessful, the P53A mutation was introduced into the humanized 108VH10.Ma sequence, resulting in the sequence of 108VH10.Mg (SEQ ID NO: 43). Additional single-site germline mutations were incorporated into the 108VH10.Mg sequence to generate sequences of 108VH10.Mh-108VH10.Ml (SEQ ID NOs: 44-48). 108VH10.Mg-108VH10.Ml was paired with 108VL10.Me to generate more humanized antibodies. The binding affinities of these antibodies are shown in Experiment 3 of Table 2.Compared to 108VH10.Mg, 108VH10.Mi, 108VH10.Mj and 108VH10.Ml did not affect the binding affinity at all, so they were combined to generate the sequence of 108VH10.Mo (SEQ ID NO: 49). Two germline mutations S61V, F64V and their combination were incorporated into 108VH10.Mo, generating the sequences of 108VH10.Mp (SEQ ID NO: 50), 108VH10.Mq (SEQ ID NO: 51) and 108VH10.Mr (SEQ ID NO: 52), respectively. With these mutations, the monovalent binding affinity was further reduced to about 170nM (experiment 4).

[0335] Table 2. Humanization of 108C10A6 using the humanized 4D5 framework as sequence acceptor.

[0336] Example 2: Generation of humanized anti-CD47 antibodies

[0337] In brief, a plasmid of a humanized antibody was prepared and used to transfect CHO cells. Supernatants were collected 8 days after transfection. Secreted antibodies were purified by protein A affinity chromatography and size exclusion chromatography. Two anti-CD47 antibodies TJC4 and Hu5F9 (US2015 / 0183874 A1, variable domain SEQ ID NOs: 37 and 42) and TTI-622 fusion protein (US10906954 B2, SEQ ID NO: 26) were also produced in-house according to the sequences in the published patents as positive controls.

[0338] Example 3: Binding to human cancer cell line SHP-77 and human erythrocytes

[0339] Briefly, SHP-77 tumor cells (ATCC, catalog number CRL-2195) were cultured and cultured at 2×10 5Cells / well were plated in 96-well plates and incubated with various concentrations of anti-CD47 antibodies, the positive control molecules mentioned in Example 2, and isotype control IgG4, κ (Sino biological, catalog number HG4K) at 37°C for 30 minutes. The cells were washed three times and then incubated with Alexa Fluor 647 fluorescently labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch Inc., catalog number 109-605-098) at 37°C for 30 minutes. The cells were washed three times and resuspended in Dulbecco's phosphate buffered saline (DPBS) buffer. Samples were acquired on a BD FACSCelesta flow cytometer (BD Biosciences) to determine the geometric mean fluorescence intensity (MFI) at each antibody concentration. Raw data were analyzed using FlowJo software. The half-maximal effective concentration (EC50) values ​​were calculated using the sigmoidal dose-response curve in GraphPad Prism software (Tables 3 and Figure 1 ). Overall, as the binding affinity of the antibodies decreased, the binding activity to SHP-77 cells decreased, as indicated by increasing EC50 values ​​and decreasing maximum MFI values. Among all variants, M6, M11, and M15 bound to the tumor line with the lowest binding activity (even lower than that of TTI-622).

[0340] Similarly, the same antibodies were also used for binding to human red blood cells (RBC) (Tables 3 and Figure 2 ). A similar trend was observed for RBC binding, only the differences in EC50 and maximum MFI values ​​between antibodies were more significant.

[0341] Table 3. Binding activity of humanized antibodies.

[0342] Example 4: Binding to human and cynomolgus CD47 overexpressing cell lines

[0343] To determine the binding activity of anti-CD47 antibodies to cells expressing human and cynomolgus CD47, CHO-K1 cells overexpressing human and cynomolgus CD47 were incubated with gradient concentrations of several selected antibodies produced in Example 2, followed by incubation with Alexa Fluor 647 fluorescently labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch Inc., catalog number 109-605-098) at 37°C for 30 minutes. The samples were analyzed by flow cytometry. The binding activity of anti-CD47 antibodies is shown in Figure 3and Table 4. Most anti-CD47 antibody variants bound to human and cynomolgus CD47 overexpressing cell lines with similar EC50 values ​​(Table 4), however, the maximum binding signal of cynomolgus CD47 cells was much lower than that of human CD47 cells ( Figure 3 ), which may be due to differences in the levels of CD47 expression on the cells (2.7 log units change in human CD47-CHO-K1 compared with controls; in contrast, 1.8 log units change in cynomolgus monkey CD47-CHO-K1 compared with controls).

[0344] Table 4. Binding activity of anti-CD47 antibodies to human and cynomolgus CD47 CHO-K1 stable cell lines.

[0345] Example 5: In vitro functional study of anti-CD47 antibodies

[0346] Blocking the CD47-SIRPα interaction with anti-CD47 antibodies induced phagocytosis of CD47-expressing tumor cells and RBCs by macrophages in vitro. Therefore, this assay was used for functional and safety studies. Monocytes were purified from peripheral blood mononuclear cells (PBMCs, HemaCare, catalog number PB009C-3) by negative selection using a whole monocyte isolation kit (Miltenyi Biotech, catalog number 130-096-537) according to the manufacturer's instructions. The cells were isolated by centrifugation at 1 × 10 6 The cells / mL were inoculated in RPMI-1640 medium (Gibco, catalog number 22400-089) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, catalog number 10099-141C) and 100ng / mL M-CSF (GenScript, catalog number Z02924-50) to induce monocyte-derived macrophages. The cells were cultured for 8 days to differentiate monocytes into macrophages. CFSE-labeled CCRF-CEM tumor cells (ATCC, catalog number CCL-119) were treated with various concentrations of anti-CD47 antibodies or human IgG4 isotype controls at 37°C for 1 hour, and then co-cultured with purple-labeled macrophages at a 1:1 effector to target cell ratio at 37°C for 1 hour. After co-culture, cells were collected and analyzed by flow cytometry. The percentage of CFSE+ target cells was recorded. The phagocytosis of target cells induced by antibodies was calculated as follows:

[0347] EC50 values ​​were calculated using sigmoidal dose-response curves in GraphPad Prism software (Table 5 and Figure 4). The functional activities of M22, M23, M27, Mk, and Mo were inferior to those of Hu5F9, similar to those of TJC4, and superior to those of TTI-622; whereas the functional activities of Mp, Mq, and Mr were inferior to those of Hu5F9 and TJC4, and similar to those of TTI-622. Overall, the functional activities of the anti-CD47 variants derived from 108C10A6 were consistent with the monovalent binding affinities of the variants, with the exception of M21.

[0348] Table 5. Phagocytosis of tumor cells induced by anti-CD47 antibodies.

[0349] Example 6: Phagocytosis of RBCs induced by CD47 targeting molecules

[0350] Phagocytosis of RBCs induced by fixed concentrations of antibodies (6.4 pM, 32 pM, and 160 pM) was performed at a 1:1 effector to target cell ratio. Figure 5 As can be seen in the , Hu5F9 induced the greatest phagocytosis of RBCs, and this was induced at lower antibody concentrations (6.4pM and 32pM). The original humanized 108C10A6 antibody (i.e., M4) with high CD47 binding affinity also showed 20% phagocytosis of RBCs at 160pM, whereas humanized antibody variants with reduced affinity (i.e., M22, M23, M27, Mo, and Mk) and benchmark molecules (such as TJC4 and TTI-622) showed little to no phagocytosis ( Figure 5 ), suggesting that these antibodies may have a better safety profile with regard to anemia.

[0351] Example 7: Comparison of efficacy and toxicity of humanized 108C10A6 antibody variants

[0352] To determine which variant or variants of the humanized 108C10A6 antibody may have the largest therapeutic window in clinical practice, CCRF-CEM cancer cell phagocytosis was performed as described in Example 5 using M4, M23, Mk, Mo along with positive and negative control antibodies. Figure 6As can be seen, all 4 selected humanized variants showed comparable tumor cell efficacy and potency. The phagocytosis of tumor cells at fixed concentrations (Hu5F9 at 6.4pM and 32pM, and the other antibodies at 32pM and 160pM) was plotted together with the phagocytosis of RBCs shown in Example 6. For Hu5F9, the phagocytosis of RBCs was more significant than the phagocytosis of tumor cells. At the specified concentrations, M4, M23, Mk, and Mo showed similar tumor cell phagocytosis; in other respects, M23, Mk, and Mo showed little to no RBC phagocytosis, while M4 showed significant RBC phagocytosis ( Figure 7 ). The results showed that M23, Mk and Mo had better efficacy to toxicity ratio than M4 and Hu5F9.

[0353] Example 8: RBC hemagglutination induced by anti-CD47 antibodies

[0354] Some CD47 blocking antibodies validated in clinical studies (such as Hu5F9) induce hemagglutination and anemia (Advani R et al. 2018). To overcome this limitation, therapeutic agents with limited hemagglutination are needed. RBC hemagglutination induced by anti-CD47 antibodies was performed with selected variants of humanized anti-CD47 antibodies. Human RBCs were washed three times with DPBS. Increasing concentrations of anti-CD47 antibodies (up to 100 μg / mL) were added to the wells containing RBCs, and the plates were incubated at 37°C for 2 hours. The plates were then imaged. For Hu5F9 above concentrations of 0.41 μg / mL and for TJC4 above concentrations of 3.70 μg / mL, a diffuse fuzzy pattern was observed, indicating hemagglutination; while for all concentrations of other anti-CD47 antibodies, small dotted circles were observed, indicating that no obvious hemagglutination was observed ( Figure 8 ).

[0355] Example 9: In vivo efficacy of humanized anti-CD47 antibodies

[0356] The in vivo efficacy of anti-CD47 antibodies was investigated using a tumor cell-derived xenograft model. Briefly, SHP-77 tumor cells were cultured and inoculated in female NCG (NOD / ShiLtJGpt-Prkdc em26Cd52 Iq em26Cd22 / Gpt, GemPharmatech) mice (6-7 weeks old) were injected subcutaneously in the flank with 1×10 7 The size of the tumor was measured using a caliper, and the tumor volume was calculated as length × width × width / 2. When the tumor was palpable and reached an average volume of approximately 140 mm 3At 4 pm, mice were randomized into groups of 4 mice each and treated with anti-CD47 antibodies. CD47-targeting agents were administered intraperitoneally 3 times per week at 3 mg / kg and 10 mg / kg for the first 5 doses and twice per week for the next 7 doses. Body weights were measured throughout the study. When tumor volume reached 2000 mm 3 Animals were sacrificed at the end of the study (35 days after treatment). Fig. 9 As shown in the , benchmark molecules (such as TJC4 and TTI-622) showed very little tumor growth inhibitory activity, while variants of the humanized 108C10A6 antibody showed better tumor growth inhibition. Among all variants, M4 and M23 showed complete tumor growth inhibition even at a low dose of 3 mg / kg. In 1 of 4 mice in the 3 mg / kg Mo group, the tumor recurred, indicating that the in vivo efficacy of Mo may be slightly worse than that of M4 and M23. Among the 4 humanized variants, Mk showed the least tumor growth inhibition. There was no sign of significant weight loss throughout the study ( Fig. 9 n).

[0357] References

[0358] Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol., 11 (2001), pp.130-135.

[0359] Brown EJ. Integrin-associated protein (CD47): an unusual activator of Gprotein signaling. J. Clin. Invest., 107 (2001), pp. 1499-1500.

[0360] Isenberg JS, Annis DS, Pendrak ML, Ptaszynska M, Frazier WA, Mosher DF, Roberts DD. Differential interactions of thrombospondin-1,-2, and-4 with CD47and effects on cGMP signaling and ischemic injury responses. J Biol Chem. 2009;284:1116-1125.

[0361] Advani R, Flinn I, Popplewell L, Forero A, Bartlett NL, Ghosh N, et al. CD47 blockade by Hu5F9-G4 and rituximab in non-Hodgkin’s lymphoma. N Engl J Med. 2018;379(18):1711-21.

[0362] Winter G, Harris WJ. Humanized antibodies. Trends Pharmacol Sci 1993;14:139-43.

[0363] Robert A. Rebres, Louise E. Vaz, Jennifer M. Green, and Eric J. Brown Normal ligand binding and signaling by CD47 (integrin-associated protein) requires a long range disulfide bond between the extracellular and membrane-spanning domains. J Biol Chem. 2001 Sep 14;276(37):34607-16.

[0364] A. A. Plückthun Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering. Biochemistry, 38(1999), pp. 8739-8750.

[0365] Sequence

[0366] Table 6

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD47, comprising: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 23, 22, 27, 47, 49, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 50, 51 or 52; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO: 7 or 36.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 23, 22, 27, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO: 7; Preferably, the HCDR1 is defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof comprises: (i) a HCDR1 comprising the sequence GYX1FTHHWX2H (SEQ ID NO: 116), wherein X1 is T or S, and X2 is M or I; (ii) a HCDR2 comprising the sequence of MIDASDX3ETRYX4QKFX5G (SEQ ID NO: 117), wherein X3 is K, I or Y, X4 is A or S, and X5 is Q or K; (iii) HCDR3 comprising the sequence of SEQ ID NO:90; (iv) LCDR1 comprising the sequence of SEQ ID NO:93; (v) LCDR2 comprising the sequence of SEQ ID NO:97; and (vi) LCDR3 comprising the sequence of SEQ ID NO:99; Preferably, X1 is T, and / or X2 is M; Preferably, the HCDR1 comprises the sequence of SEQ ID NO: 57; Preferably, X4 is A, and / or X5 is Q; preferably, the HCDR2 comprises the sequence of SEQ ID NO: 73, 72 or 77.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 73 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively; HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 72 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively; or, HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 57, 77 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 97 and 99, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 57, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 65, 66, 68, 69, 70, 71, 74, 75 or 76, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 93, 97 and 99, respectively; or, HCDR1 comprising the amino acid sequence of SEQ ID NO:56, HCDR2 comprising the amino acid sequence of SEQ ID NO:65, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively; or, HCDR1 comprising the amino acid sequence of SEQ ID NO:55, HCDR2 comprising the amino acid sequence of SEQ ID NO:62, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively; or, HCDR1 comprising the amino acid sequence of SEQ ID NO:54, HCDR2 comprising the amino acid sequence of SEQ ID NO:62, 65, 66 or 67, HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:93, 97 and 99, respectively; or, A HCDR1 comprising the amino acid sequence of SEQ ID NO:53, a HCDR2 comprising the amino acid sequence of SEQ ID NO:63 or 64, a HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:93, 97 and 99, respectively.

6. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 5, further comprising: four heavy chain framework regions (HFR1, HFR2, HFR3 and HFR4) containing the amino acid sequences of SEQ ID NOs: 100, 101, 102 and 103, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3 and LFR4) containing the amino acid sequences of SEQ ID NOs: 108, 109, 110 and 111, respectively.

7. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 6, comprising: a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises the sequence of SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 22, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 27, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25 or 26, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% sequence identity thereto.

8. The antibody or antigen-binding fragment thereof of claim 1, comprising: HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO:47, 49, 50, 51, 52, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48; and LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:36; Preferably, the HCDR1 is defined according to the AbM numbering system, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein: (i) a HCDR1 comprising the sequence of GX1X2FX3HHWIH (SEQ ID NO: 118), wherein X1 is F or Y, X2 is T or S, and X3 is S or T; (ii) a HCDR2 comprising the sequence of MIDASDSETRLX4X5X6X7KX8 (SEQ ID NO: 119), wherein X4 is S or V, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G; (iii) HCDR3 comprising the sequence of SEQ ID NO:90; (iv) LCDR1 comprising the sequence of SEQ ID NO:95; (v) LCDR2 comprising the sequence of SEQ ID NO:98; and (vi) LCDR3 comprising the sequence of SEQ ID NO:99; Preferably, X1 is F, X2 is T, and / or X3 is S; Preferably, the HCDR1 comprises the sequence of SEQ ID NO: 58; Preferably, X4 is S, X5 is D or Q, X6 is K or S, X7 is F or V, and X8 is D or G; Preferably, X4 is S or V, X5 is D, X6 is S, X7 is F or V, and X8 is G; preferably, X4 is S, and / or X7 is F; Preferably, the HCDR2 comprises the sequence of SEQ ID NO:84, 86, 87, 88 or 89.

10. The antibody or antigen-binding fragment thereof according to claim 9, comprising: HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 84 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or, HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 86 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or, HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 87 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or, HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 88 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively; or, HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 89 and 90, respectively; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 95, 98 and 99, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO:58; a HCDR2 comprising the amino acid sequence of SEQ ID NO:61, 62, 81, 82, 83 or 85; a HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:95, 98 and 99, respectively; or HCDR1 comprising the amino acid sequence of SEQ ID NO:53; HCDR2 comprising the amino acid sequence of SEQ ID NO:80; HCDR3 comprising the amino acid sequence of SEQ ID NO:90; and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs:95, 98 and 99, respectively.

12. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 11, further comprising: four heavy chain framework regions (HFR1, HFR2, HFR3, HFR4) containing the amino acid sequences of SEQ ID NOs: 104, 105, 106 and 107, respectively; and / or four light chain framework regions (LFR1, LFR2, LFR3, LFR4) containing the amino acid sequences of SEQ ID NOs: 112, 113, 114 and 115, respectively.

13. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 12, comprising: a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises the sequence of SEQ ID NO: 47, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 51, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO: 52, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% sequence identity thereto; or The VH comprises the sequence of SEQ ID NO:37, 42, 43, 44, 45, 46 or 48, or an amino acid sequence having at least 80% sequence identity thereto; and / or the VL comprises the sequence of SEQ ID NO:36, or an amino acid sequence having at least 80% sequence identity thereto.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, further comprising: a heavy chain constant region (CH) comprising an amino acid sequence derived from a heavy chain constant region of a human immunoglobulin; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the heavy chain constant region is an IgG4 heavy chain constant region, for example, an IgG4 heavy chain constant region having an S228P substitution; preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 28; Preferably, the heavy chain constant region has effector function (e.g., ADCC and / or ADCP) or enhanced effector function (e.g., ADCC and / or ADCP); Preferably, the antibody or antigen-binding fragment thereof further comprises: a light chain constant region (CL) comprising an amino acid sequence derived from a human immunoglobulin light chain constant region; Preferably, the light chain constant region is a kappa light chain constant region; preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO:

29.

15. The antibody or antigen-binding fragment thereof according to claim 14, comprising a heavy chain and a light chain, wherein: The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 23 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or, The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 22 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or, The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 27 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 7 and a CL comprising the sequence of SEQ ID NO: 29; or, The heavy chain comprises a VH comprising the sequence of SEQ ID NO: 47 and a CH comprising the sequence of SEQ ID NO: 28; and the light chain comprises a VL comprising the sequence of SEQ ID NO: 36 and a CL comprising the sequence of SEQ ID NO: 29; or, The heavy chain comprises a VH comprising the sequence of SEQ ID NO:49 and a CH comprising the sequence of SEQ ID NO:28; and the light chain comprises a VL comprising the sequence of SEQ ID NO:36 and a CL comprising the sequence of SEQ ID NO:

29.

16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, Fab, Fab', (Fab')2, Fv fragment, diabody, bispecific antibody, multispecific antibody and humanized antibody.

17. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and an effector molecule.

18. The immunoconjugate of claim 17, wherein the effector molecule is a therapeutic agent selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid.

19. A bispecific or multispecific antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16; Preferably, the bispecific or multispecific antibody specifically binds CD47 (e.g., human CD47) and a second target; Preferably, the second target is an immunomodulatory receptor or a tumor-associated antigen.

20. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof, or the bispecific or multispecific antibody of claim 19.

21. A vector comprising the isolated nucleic acid molecule of claim 20.

22. A host cell comprising the isolated nucleic acid molecule of claim 20 or the vector of claim 21.

23. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 or the bispecific or multispecific antibody according to claim 19, the method comprising: Cultivating a host cell comprising the isolated nucleic acid molecule of claim 20 or the vector of claim 21, or a host cell of claim 22, under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody; and recovering the antibody or antigen-binding fragment thereof, or the bispecific or multispecific antibody from the culture of the cultured host cells.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16, the immunoconjugate as described in claim 17 or 18, the bispecific or multispecific antibody as described in claim 19, the isolated nucleic acid molecule as described in claim 20, the vector as described in claim 21, or the host cell as described in claim 22, and a pharmaceutically acceptable carrier and / or excipient.

25. The pharmaceutical composition of claim 24, further comprising an additional therapeutic agent; Preferably, the additional therapeutic agent is an anti-tumor agent; Preferably, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, for example, a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity; Preferably, the additional therapeutic antibody binds a target other than CD47; Preferably, the target is selected from an immunomodulatory receptor or a tumor-associated antigen; Preferably, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor or a radionuclide.

26. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the bispecific or multispecific antibody of claim 19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, or the host cell of claim 22, or the pharmaceutical composition of claim 24 or 25 in the manufacture of a medicament for use in treating cancer or a proliferative disorder in a subject; Preferably, the cancer or proliferative disorder is associated with CD47 expression; Preferably, the cancer or proliferative disorder is a hematological cancer, such as a leukemia, lymphoma or myeloma; Preferably, the hematological cancer is a leukemia selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / neoplasms (MPDS) and myelodysplastic syndrome (MDS); Preferably, the hematological cancer is a lymphoma selected from the group consisting of Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cell and large cell); Preferably, the hematological cancer is a myeloma selected from the group consisting of multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones myeloma; Preferably, the cancer or proliferative disorder is a solid tumor, such as breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumor, ovarian tumor, lung tumor, pancreatic tumor, prostate tumor, melanoma tumor, colorectal tumor, lung tumor, head and neck tumor, bladder tumor, esophageal tumor, liver tumor and kidney tumor; Preferably, the subject is a mammal, such as a human.

27. The use of claim 26, wherein the antibody or antigen-binding fragment thereof, the immunoconjugate, the bispecific or multispecific antibody, or the pharmaceutical composition is used in combination with another therapeutic agent or another therapy; Preferably, the additional therapeutic agent is an anti-tumor agent; Preferably, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, for example, a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity; Preferably, the additional therapeutic antibody binds a target other than CD47; Preferably, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide; Preferably, the additional therapy is a standard cancer treatment such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative care.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the bispecific or multispecific antibody of claim 19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, or the host cell of claim 22, or the pharmaceutical composition of claim 24 or 25, for use in treating cancer or a proliferative disorder in a subject; Preferably, the cancer or proliferative disorder is associated with CD47 expression; Preferably, the cancer or proliferative disorder is a hematological cancer, such as a leukemia, lymphoma or myeloma; Preferably, the hematological cancer is a leukemia selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / neoplasms (MPDS) and myelodysplastic syndrome (MDS); Preferably, the hematological cancer is a lymphoma selected from the group consisting of Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cell and large cell); Preferably, the hematological cancer is a myeloma selected from the group consisting of multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones myeloma; Preferably, the cancer or proliferative disorder is a solid tumor, such as breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumor, ovarian tumor, lung tumor, pancreatic tumor, prostate tumor, melanoma tumor, colorectal tumor, lung tumor, head and neck tumor, bladder tumor, esophageal tumor, liver tumor and kidney tumor; Preferably, the subject is a mammal, such as a human.

29. The antibody or antigen-binding fragment thereof, immunoconjugate, bispecific or multispecific antibody, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition for use according to claim 28, wherein the antibody or antigen-binding fragment thereof, the immunoconjugate, the bispecific or multispecific antibody, or the pharmaceutical composition is used in combination with another therapeutic agent or another therapy; Preferably, the additional therapeutic agent is an anti-tumor agent; Preferably, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, for example, a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity; Preferably, the additional therapeutic antibody binds a target other than CD47; Preferably, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide; Preferably, the additional therapy is a standard cancer treatment such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative care.

30. A method of treating cancer or a proliferative disorder in a subject, wherein the method comprises administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the bispecific or multispecific antibody of claim 19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, or the host cell of claim 22, or the pharmaceutical composition of claim 24 or 25; Preferably, the cancer or proliferative disorder is associated with CD47 expression; Preferably, the cancer or proliferative disorder is a hematological cancer, such as a leukemia, lymphoma or myeloma; Preferably, the hematological cancer is a leukemia selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / neoplasms (MPDS) and myelodysplastic syndrome (MDS); Preferably, the hematological cancer is a lymphoma selected from the group consisting of Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cell and large cell); Preferably, the hematological cancer is a myeloma selected from the group consisting of multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence Jones myeloma; Preferably, the cancer or proliferative disorder is a solid tumor, such as breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast tumor, ovarian tumor, lung tumor, pancreatic tumor, prostate tumor, melanoma tumor, colorectal tumor, lung tumor, head and neck tumor, bladder tumor, esophageal tumor, liver tumor and kidney tumor; Preferably, the subject is a mammal, such as a human.

31. The method of claim 30, wherein the antibody or antigen-binding fragment thereof, the immunoconjugate, the bispecific or multispecific antibody, or the pharmaceutical composition is used in combination with an additional therapeutic agent or an additional therapy; Preferably, the additional therapeutic agent is an anti-tumor agent; Preferably, the additional therapeutic agent is an additional therapeutic antibody for cancer treatment, for example, a therapeutic antibody having ADCC and / or ADCP activity or enhanced ADCC and / or ADCP activity; Preferably, the additional therapeutic antibody binds a target other than CD47; Preferably, the target is selected from an immunomodulatory receptor or a tumor-associated antigen; Preferably, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide; Preferably, the additional therapy is a standard cancer treatment such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative care.

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