CD47-specific humanized antibodies and pharmaceutical compositions containing them for the prevention or treatment of CD47-related diseases.

Abstract

The present invention relates to a humanized antibody specific to CD47 and a pharmaceutical composition comprising the same for preventing or treating a CD47-related disease, and more specifically, to a humanized antibody specific to CD47 and a pharmaceutical composition comprising the humanized antibody for preventing or treating a disease mediated by cells that overexpress CD47. In the present invention, 16 types of humanized antibodies were produced using an antibody (3A5 antibody) that binds to CD47, and it was confirmed that the above humanized anti-CD47 antibodies specifically bind to the CD47 antigen. In addition, the humanized anti-CD47 antibody of the present invention not only blocks CD47-SIRPα binding, but also binds to cells that overexpress CD47, promoting macrophage function by macrophages and suppressing the growth of tumors that express CD47, and can therefore be used in the prevention or treatment of diseases or tumors in which immune responses are suppressed due to overexpression of CD47.

Description

Detailed description of the invention

[0001] [Technical field] The present invention relates to a CD47-specific humanized antibody and a pharmaceutical composition containing the same for the prevention or treatment of CD47-related diseases, and more particularly to a CD47-specific humanized antibody and a pharmaceutical composition containing the humanized antibody for the prevention or treatment of diseases mediated by cells overexpressing CD47.

[0002] [Background technology] CD47, also known as an integrin-binding protein (IAP), is a transmembrane glycoprotein widely expressed on the cell surface. It belongs to the immunoglobulin superfamily and interacts with various ligands such as integrins, SIRPα (signal regulatory protein α), SIRPγ, and thrombospondin.

[0003] SIRPα is primarily expressed in myeloid cells, including macrophages, granule cells, myeloid dendritic cells (DCs), mast cells, and hematopoietic stem cells (HSCs), as well as their precursors. SIRPα suppresses macrophage-mediated phagocytosis of host cells, and ligation of SIRPα on macrophages by CD47 expressed on host target cells generates an inhibitory signal mediated by SHP-1, negatively regulating phagocytosis.

[0004] In the innate immune system, CD47 exerts its function through binding to SIRPα expressed on myeloid cells, and under physiological conditions, widespread expression of CD47 prevents healthy cells from being eliminated by the innate immune system.

[0005] However, because tumor cells can effectively evade immune surveillance by overexpressing CD47, CD47 and the CD47-SIRPα signaling system have recently attracted significant attention as potential drug targets in tumor therapy. Existing studies have shown that CD47 expression is upregulated in most human cancers (e.g., NHL, AML, breast cancer, colon cancer, glioblastoma, glioma, ovarian cancer, bladder cancer, prostate cancer), and elevated CD47 expression levels have been shown to be associated with invasive disease and low survival rates.

[0006] The therapeutic effects of anti-CD47 antibodies on tumors are associated with various mechanisms. First, anti-CD47 antibodies block the binding of CD47 on tumor cells to SIRPα on macrophages, causing phagocytosis of tumor cells. Second, anti-CD47 antibodies can induce cytotoxicity of tumor cells involving NK cells, directly inducing cell death and eliminating tumor cells. Finally, anti-CD47 antibodies activate CD8+ T cells, triggering an acquired T cell immune response, which can further kill tumor cells.

[0007] Antibodies specific to CD47 are being developed for the treatment or diagnosis of tumor cells that overexpress CD47 or diseases associated with CD47 overexpression. Various anti-CD47 antibodies are disclosed in international publications WO2018-075857, WO2017-121771, and WO2013-119714.

[0008] As mentioned above, monoclonal antibodies are primarily produced using mice for therapeutic purposes. However, non-human antibodies, such as mouse-derived monoclonal antibodies, are recognized as foreign antigens in the human body, thus inducing an immune response. Furthermore, their short half-life limits their therapeutic effect.

[0009] To address the above issues, humanized antibodies were developed in which only the CDR site that binds to the antigen is replaced with human antibodies. The current method for replacing mouse antibodies with humanized antibodies involves selecting the human antibody gene that is most similar to the antibody to be replaced, and then replacing only the CDR site of the mouse antibody with the CDR site of the human antibody using a method called CDR transplantation. Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the gene has been humanized.

[0010] [Overview of the prefecture] [Problems the invention aims to solve] In this invention, an antibody (3A5) that binds to CD47 to reduce the immune response in the human body was selected, and a humanized anti-CD47 antibody was produced using this antibody. The humanized anti-CD47 antibody produced in this invention was confirmed to specifically bind to the CD47 antigen, effectively block CD47-SIRPα binding, induce cell death (apoptosis) in peripheral leukemia-derived T cells (Jurkat cells), and effectively suppress tumor growth, thus completing the invention.

[0011] Therefore, the object of the present invention is to provide a humanized antibody that specifically binds to CD47, a polynucleotide encoding the antibody, a vector expressing the antibody, and recombinant cells transformed with the vector.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diseases mediated by cells that overexpress CD47, comprising a humanized antibody that specifically binds to the above-mentioned CD47.

[0013] [Means for solving the problem] In order to achieve the above objectives, The present invention relates to (1) a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; (2) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 17 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 18; (3) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 23 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 24; (4) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 29 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 30; (5) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 35 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 36; (6) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 41 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 42; (7) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 47 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 48; (8) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 53 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 54; (9) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 59 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 60; (10) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 65 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 66; (11) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 71 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 72; (12) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 77 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 78; (13) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 83 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 84; (14) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 89 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 90; (15) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 95 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 96; or (16) A humanized antibody or fragment thereof that specifically binds to CD47, comprising a heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 101 and a light chain variable region indicated by the amino acid sequence of SEQ ID NO: 102.

[0014] In a preferred embodiment of the present invention, the antibody can be a monoclonal antibody, preferably scFv (Single-chain variable fragment). The present invention also provides a polynucleotide encoding a humanized antibody or a fragment thereof that specifically binds to the above CD47.

[0015] The present invention also provides a vector containing a polynucleotide encoding a humanized antibody or a fragment thereof that specifically binds to the above CD47. The present invention also provides a recombinant cell that produces a humanized antibody or a fragment thereof that specifically binds to CD47 and is transformed with the above vector.

[0016] To achieve other purposes, The present invention provides a pharmaceutical composition for preventing or treating a disease mediated by cells overexpressing CD47, which contains a humanized antibody or a fragment thereof that specifically binds to the above CD47.

[0017] In a preferred embodiment of the present invention, the composition can further contain an immune checkpoint inhibitor, and the immune checkpoint inhibitor can preferably be an anti-PD-1 antibody. In another preferred embodiment of the present invention, the disease mediated by cells overexpressing CD47 can be cancer or a tumor overexpressing CD47.

[0018] In another preferred embodiment of the present invention, the cancer or tumor can be selected from the group consisting of hematological cancer, ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblast-induced CNS tumor, monocytic leukemia, B-cell-induced leukemia, T-cell-induced leukemia, B-cell-induced lymphoma, T-cell-induced lymphoma, and mast cell-induced tumor.

[0019] [Advantages of the Invention] In this invention, 16 types of humanized antibodies were produced using an antibody that binds to CD47 (3A5 antibody), and it was confirmed that the above-mentioned humanized anti-CD47 antibody specifically binds to the CD47 antigen.

[0020] Furthermore, the humanized anti-CD47 antibody of the present invention not only blocks CD47-SIRPα binding, but also binds to cells that overexpress CD47, promoting macrophage activity by macrophages and suppressing the growth of tumors that express CD47. Therefore, it can be applied to the prevention or treatment of diseases or tumors in which the immune response is suppressed due to CD47 overexpression. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows data obtained by flow cytometer analysis of the binding affinity of the 3A5 (mouse) antibody selected in this invention to CD47-overexpressing tumor cells (MCF-7). [Figure 2a] Figure 2 shows data obtained by flow cytometry, confirming the binding affinity of 16 humanized 3A5 antibodies of the present invention to CD47-overexpressing tumor cells (MCF-7). [Figure 2b] Figure 2 shows data obtained by flow cytometry confirming the binding affinity of 16 humanized 3A5 antibodies of the present invention to CD47-overexpressing tumor cells (MCF-7). [Figure 3] Figure 3 shows data confirming the CD47-SIRPα binding blocking ability of the humanized Hu3A5(V10) antibody of the present invention. [Figure 4] Figure 4 shows data confirming that the humanized Hu3A5(V10) antibody of the present invention promotes macrophage phagocytosis by binding to CD47 on the surface of peripheral leukemia-derived T cells (Jurkat cells). [Figure 5] Figure 5 shows data confirming (A) the presence or absence of red blood cell and platelet binding, and (B) the degree of red blood cell agglutination, for the humanized Hu3A5(V10) antibody of the present invention and a commercially available CD47 antibody (clone#CC2C6). [Figure 6]Figure 6 shows (A) a schematic diagram of the animal experiment method and (B) data confirming tumor size after administration of the 3A5 (mouse) antibody of the present invention to mice transplanted with human CD47-expressing mouse colon adenocarcinoma cells. A commercially available mouse PD-1 antibody (clone#RMP1-14) was used. [Figure 7] Figure 7 shows (A) a schematic diagram of the animal experiment method and (B) data confirming tumor size after administration of the Hu3A5(V10) antibody when the humanized Hu3A5(V10) antibody of the present invention was administered to C57BL / 6-hCD47 / hSIRPα knock-in mice (hCD47 KI) transplanted with rat colon adenocarcinoma cells expressing human CD47. [Figure 8] Figure 8 shows the data obtained by immunohistochemistry (IHC) of the major organs of mice that were administered Hu3A5(V10) antibody as shown in Figure 7, followed by C57BL / 6-hCD47 / hSIRPα lysis. [Modes for carrying out the invention]

[0022] The present invention will be described in detail below. Humanized antibody that specifically binds to CD47 In one embodiment, the present invention includes (1) a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; (2) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 17 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 18; (3) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 23 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 24; (4) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 29 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 30; (5) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 35 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 36; (6) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 41 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 42; (7) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 47 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 48; (8) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 53 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 54; (9) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 59 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 60; (10) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 65 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 66; (11) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 71 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 72; (12) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 77 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 78; (13) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 83 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 84; (14) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 89 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 90; (15) The heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 95 and the light chain variable region indicated by the amino acid sequence of SEQ ID NO: 96; or (16) A humanized antibody or fragment thereof that specifically binds to CD47, comprising a heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 101 and a light chain variable region indicated by the amino acid sequence of SEQ ID NO: 102.

[0023] In this invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of the remaining portion, after removing the CDR site which is the core part for antigen binding, corresponds to that of an antibody produced by a human, thereby increasing its similarity to a human antibody. The most common method for humanizing an antibody (non-human antibody) is the CDR-grafting method, in which the CDR site of an animal antibody is transplanted into a human antibody. However, humanized antibodies can be produced using methods known in the industry, without being limited to this method.

[0024] In the present invention, the antibody may be a monoclonal antibody. In the present invention, the term "monoclonal antibody" refers to an antibody produced by a single antibody-forming cell, characterized by a uniform primary structure (amino acid sequence). It recognizes only one antigenic determinant and is generally produced by culturing hybridoma cells, which are fused cancer cells and antibody-producing cells.

[0025] In this invention, the term "CDR," or "complementarity-determining region," refers to a non-contiguous antigen-binding site found within the variable regions of both the heavy chain and light chain. In this invention, the term "antibody" can refer not only to the complete form having two full-length light chains and two full-length heavy chains, but also to fragments of the antibody molecule. A fragment of the antibody molecule means a fragment that possesses at least peptide tag (epitope) binding function, and includes scFv, Fab, F(ab'), F(ab')2, single domain, etc.

[0026] Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, an invariant region of the light chain, and the first invariant region (CH1) of the heavy chain, and possesses one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced as the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is a minimal antibody fragment that has only a heavy chain variable region and a light chain variable region. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by disulfide bonds, while in short-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally covalently linked via a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes or, preferably, produced through genetic engineering.

[0027] The monoclonal antibody that specifically binds to CD47 according to the present invention can be produced using the whole or partial CD47 protein peptide as an immunogen (or antigen). More specifically, first, immunosensitization is performed by injecting CD47, a fusion protein containing the CD47 protein, or a carrier containing the CD47 protein into the subcutaneous, intramuscular, vein, foot protuberance, or abdominal cavity of a mammal other than a human, together with an adjuvant (e.g., Freund adjuvant) as an immunoenhancing agent, one or more times. The mammal other than a human is preferably a mouse, rat, hamster, guinea pig, chicken, rabbit, cat, dog, pig, goat, sheep, donkey, horse, or cattle (including transgenic animals engineered to produce antibodies derived from other animals, such as transgenic mice that produce human antibodies), and more preferably a mouse, rat, hamster, guinea pig, chicken, or rabbit. Immunization can be performed 1 to 4 times at intervals of approximately 1 to 21 days from the initial immunization, and antibody-producing cells can be obtained from the immunosensitized mammals approximately 1 to 10 days after the final immunization. The number of immunizations and the time intervals can be appropriately changed depending on the characteristics of the immunogen used.

[0028] The production of monoclonal antibody-secreting hybridomas can be carried out according to the method of Kayla and Milstein et al. (Nature, 1975, Vol. 256, pp. 495-497) and similar methods. Hybridomas can be produced by fusing antibody-producing cells, preferably contained in the spleen, with myeloma cells of mammalian origin that lack the ability to produce autoantibodies, selected from the group consisting of spleen, lymph nodes, bone marrow, or tonsils taken from an animal other than a human immunosensitized as described above. The mammal may be a mouse, rat, guinea pig, hamster, chicken, rabbit, or human, and is preferably a mouse, rat, chicken, or human.

[0029] Cell fusion can be performed using methods such as fusion promoters containing polyethylene glycol or Sendai virus, or by using electrical pulses. For example, antibody-producing cells and mammalian cells capable of unlimited proliferation are suspended in a fusion medium containing a fusion promoter in a ratio of approximately 1:1 to 1:10, and incubated at approximately 30 to 40°C for approximately 1 to 5 minutes. Common fusion media such as MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium are commonly used, and it is preferable to remove any serum such as bovine serum.

[0030] The method for screening hybridoma clones that produce the monoclonal antibody described above involves first transferring the fusion cells obtained as described above to a selective medium such as HAT medium and culturing them at approximately 30-40°C for approximately 3 days to 3 weeks to kill cells other than hybridomas. Subsequently, after culturing the hybridomas in a microtiter plate or the like, the region where the reactivity between the immunogen used in the immune response of non-human animals described above and the culture supernatant has increased can be identified using an immunoassay method such as RIA (radioactive substance-marked immuno antibody) or ELISA (enzyme-LiNKed immunosorbent assay). The clones that produce the monoclonal antibody found in this way will then exhibit specific binding affinity to the immunogen described above. The monoclonal antibodies of the present invention can be obtained by culturing such hybridomas in vivo or in vitro. Conventional methods for culturing mammalian-derived cells are used for culturing, and conventional methods in this field for purifying antibodies in general are used to collect the monoclonal antibodies from the culture. Examples of such methods include salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography, gel electrophoresis, and isoelectric focusing, which may be applied in combination as needed. The purified monoclonal antibodies are then concentrated and dried, and prepared as liquid or solid phase depending on the application.

[0031] In one specific embodiment of the present invention, in order to produce an antibody that specifically binds to CD47, a hybridoma that produces an anti-CD47 antibody was manufactured and screened, and an antibody that specifically binds to CD47 (scFv) was screened and named 3A5.

[0032] It was confirmed that the above 3A5 antibody consists of a heavy chain variable region including the CDR1 region (GYTFTSYW) represented by the amino acids of SEQ ID NO: 1, the CDR2 region (IDPSDSYT) represented by the amino acids of SEQ ID NO: 2, and the CDR3 region (ARGGKRAMDY) represented by the amino acids of SEQ ID NO: 3, and a light chain variable region including the CDR1 region (QSLVHSNGNTY) represented by the amino acids of SEQ ID NO: 4, the CDR2 region (KVS) represented by the amino acids of SEQ ID NO: 5, and the CDR3 region (SQSTHVPFT) represented by the amino acids of SEQ ID NO: 6.

[0033] Specifically, it was confirmed that the 3A5 antibody consists of a heavy chain variable region represented by the amino acids in SEQ ID NO: 7 and a light chain variable region represented by the amino acids in SEQ ID NO: 8, with the heavy chain variable region being encoded by the nucleotide sequence of SEQ ID NO: 9 and the light chain variable region being encoded by the nucleotide sequence of SEQ ID NO: 10.

[0034] In another specific embodiment of the present invention, 16 humanized antibodies were produced by modifying the anti-CD47 antibody 3A5 to a human-compatible structure, and these were named Hu3A5(V1), Hu3A5(V2), Hu3A5(V3), Hu3A5(V4), Hu3A5(V5), Hu3A5(V6), Hu3A5(V7), Hu3A5(V8), Hu3A5(V9), Hu3A5(V10), Hu3A5(V11), Hu3A5(V12), Hu3A5(V13), Hu3A5(V14), Hu3A5(V15), and Hu3A5(V16).

[0035] The heavy chain variable region (CDR) and light chain variable region (CDR) of the 16 humanized anti-CD47 antibodies listed above are the same as those of 3A5, and the remaining portion after removing the CDR portion has been humanized. Preferably, the Hu3A5(V1) antibody consists of a heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region indicated by the amino acid sequence of SEQ ID NO: 12, wherein the heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 13 and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 14.

[0036] The Hu3A5(V2) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 17 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 18. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 19, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 20.

[0037] The HU3A5(V3) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 23 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 24. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 25, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 26.

[0038] The Hu3A5(V4) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 29 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 30. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 31, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 32.

[0039] The Hu3A5(V5) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 35 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 36. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 37, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 38.

[0040] The Hu3A5(V6) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 41 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 42. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 43, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 44.

[0041] The Hu3A5(V7) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 47 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 48. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 49, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 50.

[0042] The Hu3A5(V8) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 53 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 54. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 55, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 56.

[0043] The Hu3A5(V9) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 59 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 60. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 61, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 62.

[0044] The Hu3A5(V10) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 65 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 66. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 67, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 68.

[0045] The Hu3A5(V11) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 71 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 72. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 73, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 74.

[0046] The Hu3A5(V12) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 77 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 78. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 79, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 80.

[0047] The Hu3A5(V13) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 83 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 84. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 85, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 86.

[0048] The Hu3A5(V14) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 89 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 90. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 91, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 92.

[0049] The Hu3A5(V15) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 95 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 96. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 97, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 98.

[0050] The Hu3A5(V16) antibody consists of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 101 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 102. The heavy chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 103, and the light chain variable region can be coded by the nucleotide sequence of SEQ ID NO: 104.

[0051] The CD47-specific antibody of the present invention is preferably an scFv (single chain variable fragment) and can be produced by genetic recombination technology such that the heavy chain variable region and the light chain variable region are linked by a linker. The linker can preferably be represented by the amino acid sequence of SEQ ID NO: 107 or encoded by the nucleotide sequences of SEQ ID NOs: 108 to 123, but is not limited thereto.

[0052] When linked via light chain variable site-linker-heavy chain variable site, Hu3A5(V1) antibody is represented by the amino acid sequence of SEQ ID NO: 15 or the nucleotide sequence of SEQ ID NO: 16, Hu3A5(V2) antibody by the amino acid sequence of SEQ ID NO: 21 or the nucleotide sequence of SEQ ID NO: 22, 3A5(V3) antibody by the amino acid sequence of SEQ ID NO: 27 or the nucleotide sequence of SEQ ID NO: 28, Hu3A5(V4) antibody by the amino acid sequence of SEQ ID NO: 33 or the nucleotide sequence of SEQ ID NO: 34, Hu3A5(V5) antibody by the amino acid sequence of SEQ ID NO: 39 or the nucleotide sequence of SEQ ID NO: 40, Hu3A5(V6) antibody by the amino acid sequence of SEQ ID NO: 45 or the nucleotide sequence of SEQ ID NO: 46, Hu3A5(V7) antibody by the amino acid sequence of SEQ ID NO: 51 or the nucleotide sequence of SEQ ID NO: 52, and 3A5(V8) antibody by the amino acid sequence of SEQ ID NO: 57 or the nucleotide sequence of SEQ ID NO: 58 The nucleotide sequences of Hu3A5(V9) and Hu3A5(V10) can be represented by the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64, Hu3A5(V11) can be represented by the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76, Hu3A5(V12) can be represented by the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82, Hu3A5(V13) can be represented by the amino acid sequence of SEQ ID NO: 87 or SEQ ID NO: 88, Hu3A5(V14) can be represented by the amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94, Hu3A5(V15) can be represented by the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 100, and Hu3A5(V16) can be represented by the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 106.

[0053] In the present invention, the antibody can prevent CD47 from interacting with signal-regulating proteins (SIRPα) or promote macrophage-mediated phagocytosis of CD47-expressing cells.

[0054] In one specific embodiment of the present invention, it was confirmed that all 3A5 antibodies selected in the present invention and the 16 humanized Hu3A5 antibodies thereof specifically bind to cells that overexpress CD47 (Figures 1 and 2). Furthermore, when it was confirmed whether the humanized Hu3A5(V10) antibody of the present invention prevents the interaction between CD47 and SIRPα, it was confirmed that it effectively blocks CD47-SIRPα binding, as shown in Figure 3.

[0055] In another specific embodiment of the present invention, in order to confirm whether humanized 3A5 antibodies can actually be used as antibody therapeutic agents, Hu3A5(V10) antibody was used to treat T cells (Jurkat cells) derived from peripheral leukemia. The results showed that Hu3A5(V10) effectively induced phagocytosis of Jurkat cells by macrophages.

[0056] CD47 is also present in large quantities on the surface of red blood cells. When administered anti-CD47 antibodies attach to red blood cells, macrophages engulf the red blood cells, leading to anemia and hemagglutination (red blood cell entanglement). Side effects due to red blood cell phagocytosis have been reported with some commercially available or clinically ongoing CD47 antibodies.

[0057] In another specific embodiment of the present invention, to confirm whether the humanized 3A5 antibody induces hemagglutination, the presence or absence of red blood cell / platelet binding and the presence or absence of hemagglutination reaction were examined for the humanized Hu3A5(V10) antibody and a commercially available anti-CD47 antibody (clone#CC2C6). As shown in Figure 5, the commercially available anti-CD47 antibody (clone#CC2C6) reacted with red blood cells and induced hemagglutination, whereas the humanized Hu3A5(V10) antibody of the present invention did not bind to red blood cells or platelets (Figure 5A) and did not induce hemagglutination (Figure 5B).

[0058] In other words, the humanized anti-CD47 antibody of the present invention not only specifically recognizes cells that overexpress CD47 and blocks CD47-SIRPα binding, thereby suppressing immune evasion by cancer or tumor cells, but also effectively promotes macrophage activity by macrophages on cancer cells that overexpress CD47. Furthermore, since the humanized anti-CD47 antibody does not induce hemagglutination, it can be used more safely and effectively as an antibody therapeutic agent for the prevention or treatment of cancer or tumors that overexpress CD47.

[0059] From another perspective, the present invention relates to a polynucleotide encoding an antibody that specifically binds to the above-mentioned CD47. In the present invention, the term "polynucleotide" generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, separated to any length. In some embodiments, the polynucleotides of the present invention can be produced by (1) in-vitro amplification such as polymerase chain reaction (PCR) amplification, (2) cloning and recombination, (3) purification such as cleavage and gel electrophoresis separation, or (4) synthesis such as chemical synthesis, preferably the separated polynucleotides are produced by recombinant DNA technology. In the present invention, nucleic acids for encoding antibodies or their antigen-binding fragments can be produced by various methods known to the art, including but not limited to the application of restriction fragment operation of synthetic oligonucleotides or SOE PCR.

[0060] In another aspect, the present invention relates to a vector comprising a polynucleotide encoding an antibody that specifically binds to the above-mentioned CD47, and recombinant cells transformed with the above-mentioned vector.

[0061] In this invention, the term "expression vector" refers to a gene preparation containing essential regulatory elements, such as a promoter, to enable the expression of a target gene in a suitable host cell. The vector can be selected from one or more plasmids, retroviral vectors, and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, be incorporated into the genome itself.

[0062] Furthermore, vectors may contain expression regulatory elements that enable the coding region to be accurately expressed in a suitable host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements for regulating gene transcription or mRNA translation. The specific structure of the expression regulatory sequence may vary depending on the function of the species or cell type, but typically includes a 5' non-transcriptional sequence and a 5' or 3' non-translating sequence that are involved in transcription initiation and translation initiation, respectively, such as a TATA box, cap sequence, or CAAT sequence. For example, the 5' non-transcriptional expression regulatory sequence may include a promoter region that may contain a promoter sequence for transcribing and regulating a functionally linked nucleic acid.

[0063] In this invention, the term “promoter” means the smallest sequence sufficient to direct transcription. Furthermore, it may include a promoter configuration sufficient to express a regulated promoter-dependent gene that is induced by cell type-specific or external signals or sanctions, and such configuration may be located in the 5' or 3' portion of the gene. Both conservative and inducible promoters are included. Promoter sequences may be derived from prokaryotes, eukaryotes, or viruses.

[0064] In the present invention, the term "transformed organism" means a cell transformed by introducing a vector having polynucleotides encoding one or more target proteins into a host cell. Methods for producing transformed organisms by introducing an expression vector into a host cell include the calcium phosphate method or the cyanthium chloride / rubidium chloride method described in the literature (Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1.74, 1989), electroporation, electroinjection, chemical treatment methods such as PEG, and methods using a gene gun.

[0065] When transformants expressing the above vector are cultured in a nutrient medium, large quantities of antibody proteins can be produced and isolated. The culture medium and conditions can be appropriately selected and used based on those commonly used for host cells. Conditions such as temperature, pH of the medium, and culture time must be appropriately adjusted to suit cell growth and large-scale protein production during culture.

[0066] The vector according to the present invention can be used to transform host cells, preferably mammalian cells, for antibody production. Suitable host cells capable of expressing fully glycated proteins include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Agl4,293 cells, HeLa cells, etc., and these cells are readily available, for example, from ATCC (American Type Culture Collection, USA).

[0067] Compositions for the prevention or treatment of diseases mediated by CD47 overexpression From another perspective, the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease mediated by CD47 overexpression, comprising a humanized antibody that specifically binds to CD47.

[0068] In the present invention, the disease mediated by the overexpression of CD47 may be a cancer or tumor that overexpresses CD47, and preferably, the cancer or tumor that overexpresses CD47 may be selected from the group consisting of hematological cancers, ovarian cancers, colon cancers, breast cancers, lung cancers, myelomas, neuroblast-induced CNS tumors, monocytic leukemias, B-cell-induced leukemias, T-cell-induced leukemias, B-cell-induced lymphomas, T-cell-induced lymphomas, and mast cell-induced tumors.

[0069] In the present invention, the composition may further include a therapeutic agent for diseases mediated by cells overexpressing CD47, the therapeutic agent existing covalently bound to the heavy and / or light chain of an antibody that specifically binds to CD47, or can be administered in combination with the CD47-specific humanized antibody of the present invention.

[0070] The above-mentioned therapeutic agents include small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), etc. The above-mentioned therapeutic agents may also be anticancer agents. Anticancer agents include non-peptidic (i.e., non-protein) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell proliferation inhibitors. Non-limiting examples of anticancer agents include alkylating agents, nitrosourea, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds may also be used.

[0071] Furthermore, the therapeutic agent added to the above composition may preferably be an immune checkpoint inhibitor, and more preferably an anti-PD-1 antibody.

[0072] In a preferred embodiment of the present invention, to confirm whether the 3A5 antibody inhibits the growth of tumors expressing CD47, mouse murine colon adenocarcinoma cells (MC38-hCD47) expressing CD47 were transplanted into mice as shown in the schematic diagram in Figure 6A. Then, a control antibody (Rat IgG), an anti-PD-1 antibody, an anti-CD47 antibody (3A5 antibody), and an anti-PD-1 antibody (clone#RMP1-14) + anti-CD47 antibody (3A5 antibody) were administered, respectively. As a result, as shown in Figure 6B, it was confirmed that the growth of tumors overexpressing CD47 was suppressed in the group administered with 3A5 antibody alone, and that the tumor growth inhibitory efficacy was particularly superior in the group administered with both 3A5 antibody and anti-PD-1 antibody. This indicates that when anti-PD-1 antibody, an immune barrier inhibitor, is administered in combination with the 3A5 antibody of the present invention, a synergistic effect is observed in tumor treatment.

[0073] In another preferred embodiment of the present invention, to confirm the tumor growth inhibitory efficacy of humanized 3A5 antibody, rat colon adenocarcinoma cells expressing human CD47 were transplanted into C57BL / 6-hCD47 / hSIRPα-lysed mice in which the mouse CD47 and SIRPα genes were replaced with human CD47 and human SIRPα genes. Subsequently, control antibody (Rat IgG), anti-PD-1 antibody (clone#RMP1-14), anti-CD47 antibody (Hu3A5(V10) antibody), and anti-PD-1 antibody (clone#RMP1-14) + anti-CD47 antibody (Hu3A5(V10) antibody) were administered, respectively (Figure 7A). As a result, it was confirmed that tumor growth was suppressed in the group administered with Hu3A5(V10) antibody alone, and in particular, the tumor growth inhibitory efficacy was confirmed to be most superior in the group administered with both Hu3A5(V10) antibody and anti-PD-1 antibody.

[0074] Furthermore, after administration of Hu3A5(V10) antibody, immunohistochemistry (IHC) of the major organs of C57BL / 6-hCD47 / hSIRPα-lysed mice revealed no major tissue damage due to inflammation.

[0075] In other words, the humanized Hu3A5 antibody of the present invention can target cancers or tumors that overexpress CD47 without damaging other tissues and suppress tumor growth, thus confirming that it can be applied as an antibody therapeutic agent for the prevention or treatment of cancers or tumors that overexpress CD47.

[0076] The above pharmaceutical composition preferably contains a therapeutically effective amount of the antibody of the present invention. As used herein, “therapeutic effective amount” means the amount of therapeutic agent required to treat, improve, or prevent a target disease or condition, or the amount of therapeutic agent required to exhibit a detectable therapeutic or preventive effect. For a given antibody, the therapeutic effective amount can be initially determined using a cell culture assay or, typically, an animal model such as rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can be used to determine useful doses and routes of administration for human administration.

[0077] The precise effective dose for human patients depends on the severity of the disease, the patient's general health status, age, weight and sex, diet, administration time, frequency of administration, drug manufacturability, responsiveness, and tolerance / response to treatment. The above dose can be determined by standard experiments and is within the scope of clinician discretion. Generally, the effective dose is 0.01 to 50 mg / kg, preferably 0.1 to 20 mg / kg, and more preferably about 15 mg / kg.

[0078] The composition can be administered to the patient individually or in combination with other formulations, drugs, or hormones. The dosage of the antibody of the present invention varies depending on the nature of the condition being treated, the grade of malignant lymphoma or leukemia, and whether the antibody is used for disease prevention or to treat an existing condition.

[0079] The frequency of administration depends on the half-life of the antibody molecule and the duration of the drug's effect. If the antibody molecule has a short half-life (e.g., 2-10 hours), it is necessary to provide a dose once a day or more frequently. Alternatively, if the antibody molecule has a long half-life (e.g., 2-15 days), it is necessary to provide a dose once a day, once a week, or once every one or two months.

[0080] Furthermore, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for antibody administration. The carrier must be toxic and must not cause the production of antibodies harmful to the individual to whom the composition is administered. Suitable carriers include gradually metabolized polymers such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, and inactive virus particles.

[0081] Pharmaceutically acceptable salts include mineral salts such as hydrochlorides, hydrobroms, phosphates, and sulfates, or salts of organic acids such as acetic acid, propionic acid, malonic acid, and benzoic acid.

[0082] The pharmaceutically acceptable carrier in the therapeutic composition may further include liquids such as water, saline, glycerol, and ethanol. Furthermore, auxiliary substances such as wetting agents, emulsifiers, or pH buffers may be present in such compositions. The carrier may be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions for the patient to take the drug composition.

[0083] Preferred forms for administration include, for example, forms suitable for parenteral administration by injection or infusion. If the product is for infusion or injection, it may take the form of a suspension, solution, or emulsion in an oil or water-soluble excipient, containing formulations such as suspensions, preservatives, stabilizers, and / or dispersants. Alternatively, the antibody molecule may be in an anhydrous form and can be reconstituted in a suitable sterile solution before use.

[0084] Once formulated, the composition of the present invention can be administered directly to a patient. The patient being treated may be an animal. However, it is preferable to adapt the composition for administration to a human patient.

[0085] The pharmaceutical compositions of the present invention can be administered by routes including, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intra-arterial, intra-bone marrow, intraspinal cavity, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, vaginal, or rectal routes. Typically, the therapeutic compositions can be prepared as injectable substances in the form of liquid solutions or suspensions. Alternatively, a solid form suitable for liquid excipient-containing solutions or suspensions can be prepared before injection.

[0086] The composition can generally be delivered directly by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or into the interstitial space of tissues. The composition can also be administered to the wound site. Dosage processing may be a single or multiple dosing schedule.

[0087] Diagnosis or monitoring of diseases mediated by cells expressing CD47 From another perspective, the present invention relates to a composition for the diagnosis or monitoring of diseases mediated by cells expressing CD47, comprising an antibody that specifically binds to CD47.

[0088] The antibody that specifically binds to CD47 can be labeled directly or indirectly. Indirect labeling involves a secondary antibody containing a detectable label, where the secondary antibody binds to the antibody that specifically binds to CD47. Other indirect labeling involves biotin, where the antibody that specifically binds to biotinylated CD47 can be detected using avidin or streptavidin containing a detectable label.

[0089] Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels are not limited to magnetic beads, fluorescent dyes (e.g., fluorescein sothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32 P) includes enzymes (e.g., mustard deperoxide, mustard peroxide, mustard oxidase, etc., mustard deperoxide, alkaline phosphatase, luciferase, and those commonly used in enzyme-linked immunosorbent assays (ELISA)) and labeling systems such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0090] Furthermore, for diagnostic or monitoring purposes, the antibodies described above can be labeled with fluorescent proteins and may contain contrast agents or radioisotopes. When the antibody that specifically binds to CD47 according to the present invention is used in a diagnostic kit, the antibody is immobilized on a support, which can be a microplate, microarray, chip, glass, beads or particles, or a membrane.

[0091] The following are preferred embodiments to aid in understanding the present invention. However, these embodiments are provided to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0092] Example 1: Production and screening of antibodies that specifically bind to CD47 To screen for CD47 peptide-specific antibodies, hybridomas that produce antibodies that bind to CD47 were fabricated and the antibodies were screened.

[0093] First, spleen cells were extracted after immunization with the CD47 protein (Acrobiosystems, cat#CD7-HA2E9), and hybridoma cells were created through cell fusion with mouse myeloma cells. Mouse myeloma cells used for cell fusion do not possess HGPRT (Hypoxanthine Guanidine-Phosphoribosyl-Transferase) and therefore cannot survive in HAT medium. However, hybridomas can survive in HAT medium by fusing with spleen cells. This allows for the proliferation of hybridomas only, so they are usually grown in HAT medium until hybridomas are established.

[0094] The limiting dilution method was used to select hybridomas that produce antibodies that bind to CD47 from among the proliferated hybridomas. First, the number of cells was reduced to less than one per 96 wells, and the antibody obtained from a clone proliferated from a single cell was confirmed by ELISA to see if it bound to CD47, and clones that bound to CD47 were selected. The above process was repeated three times to select hybridomas that produce antibodies that bind to CD47. Antibodies that bind to CD47 were obtained by this method.

[0095] The above antibody was named 3A5, and its base sequence and amino acid sequence were analyzed. The sequence information for the heavy chain variable region and light chain variable region of each antibody, based on the sequence analysis results, is shown in Table 1 below. The underlined parts in Table 1 represent complementary determining regions (CDRs).

[0096] [Table 1]

[0097] Example 2: Confirmation of the specificity of selected antibodies against CD47 - ELISA and flow cytometer 2-1: ELISA analysis In this invention, ELISA analysis was performed to confirm the specificity of the 3A5 antibody established in Example 1 for CD47.

[0098] First, to encode the CD47 peptide, the CD47 protein (Acrobiosystems, cat#CD7-HA2E9) was aliquoted into a 96-well plate at 100 ng / well and reacted overnight at 4°C. Then, it was treated with 1 X PBST containing 3% BSA and blocked at room temperature for 30 minutes.

[0099] Each well was treated with 3 μl of hybridoma cell culture medium from each clone producing the 3A5 antibody, reacted at room temperature for 2 hours, and then washed three times with 1 X PBST. The wells were then treated with the secondary antibody (anti-HRP, 1:10,000) and reacted at room temperature for 30 minutes, washed three times with 1 X PBST, and then treated with TMB for color development and reacted at room temperature for 5 minutes. Finally, the reaction was terminated by treating with a 1N H2SO4 stop solution, and the absorbance was measured at 450 nm.

[0100] [Table 2]

[0101] [Table 3]

[0102] As a result, as shown in Table 3, it was confirmed that the 3A5 antibody selected in this invention specifically binds to CD47. 2-2: Analysis using a flow cytometer In this invention, flow cytometry was performed to confirm the specificity of the 3A5 antibody established in Example 1 for CD47.

[0103] First, the breast cancer cell line MCF-7 (1 x 10) overexpresses CD47. 7After reacting the sample with 3A5 antibody (1 μg) for 30 minutes, the surface was stained with a secondary antibody and then measured using a flow cytometer.

[0104] A CD47 antibody (Biolegend PE anti-human CD47, cat# 323108, 5 μl) was used as a positive control, and a PE-conjugated goat anti-mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA, 5 μl) was used as a secondary antibody.

[0105] [Table 4]

[0106] As a result, as shown in Figure 1 and Table 4, we confirmed that the 3A5 antibody specifically binds to cells that overexpress CD47. Example 3: Production of humanized antibodies based on 3A5 antibody Humanized antibodies were produced by modifying the 3A5 antibody selected in Example 1 above to a structure corresponding to humans.

[0107] Specifically, using the germline sequence of a human antibody as a frame, we produced 16 types of humanized mouse 3A5 antibodies using the CDR grafting method, which involves replacing the CD47-binding CDR of a mouse antibody with that of a human antibody. The humanized antibodies were named Hu3A5(V1), Hu3A5(V2), Hu3A5(V3), Hu3A5(V4), Hu3A5(V5), Hu3A5(V6), Hu3A5(V7), Hu3A5(V8), Hu3A5(V9), Hu3A5(V10), Hu3A5(V11), Hu3A5(V12), Hu3A5(V13), Hu3A5(V14), Hu3A5(V15), and Hu3A5(V16), and their amino acid sequences were analyzed.

[0108] The sequence information for the heavy chain and light chain variable regions of the antibodies, based on sequence analysis results, is shown in Tables 5 to 20 below. The underlined parts indicate complementary determining regions (CDRs), and the CDRs of all 16 humanized 3A5 antibodies are the same as those of the mouse 3A5 antibodies (Table 1) mentioned above.

[0109] In Tables 5 through 20, scFv refers to an antibody consisting of a light chain variable site-linker-heavy chain variable site structure, and the underlined portion in the scFv amino acid sequence and scFv base sequence represents the linker portion.

[0110] [Table 5] JPEG0007860629000006.jpg63170

[0111] [Table 6] JPEG0007860629000008.jpg61170

[0112] [Table 7] JPEG0007860629000010.jpg63170

[0113] [Table 8] JPEG0007860629000012.jpg65170

[0114] [Table 9] JPEG0007860629000014.jpg61170

[0115] [Table 10] JPEG0007860629000016.jpg61170

[0116] Table 11 JPEG0007860629000018.jpg64170

[0117] Table 12 JPEG0007860629000020.jpg65170

[0118] Table 13 JPEG0007860629000022.jpg66170

[0119] Table 14 JPEG0007860629000024.jpg65170

[0120] Table 15 JPEG0007860629000026.jpg64170

[0121] Table 16 JPEG0007860629000028.jpg65170

[0122] Table 17 JPEG0007860629000030.jpg64170

[0123] Table 18 JPEG0007860629000032.jpg63170

[0124] [Table 19] JPEG0007860629000034.jpg64170

[0125] [Table 20] JPEG0007860629000036.jpg60170

[0126] Example 4: Confirmation of the specificity of humanized 3A5 antibody against CD47 - ELISA and flow cytometer 4-1: ELISA analysis In this invention, the 16 humanized antibodies established in Example 3 were subjected to ELISA analysis to confirm their specificity for CD47.

[0127] First, to encode the CD47 peptide, the CD47 protein (Acrobiosystems, cat#CD7-HA2E9) was aliquoted into a 96-well plate at a concentration of 100 ng / well and incubated overnight at 4°C. Then, it was treated with 1 X PBST containing 3% BSA and blocked at room temperature for 30 minutes.

[0128] After treating each well with 0.8 μg of purified humanized antibody, the wells were reacted at room temperature for 2 hours and then washed three times with 1 X PBST. Secondary antibody (anti-HRP, 1:5,000) was then treated at room temperature for 30 minutes, followed by three washes with 1 X PBST. TMB was then treated for color development and reacted at room temperature for 5 minutes. Finally, the reaction was terminated by treating with a 1N H2SO4 stop solution, and the absorbance was measured at 450 nm.

[0129] [Table 21]

[0130] [Table 22]

[0131] As a result, as shown in Table 22, it was confirmed that the Hu3A5(V1), Hu3A5(V2), Hu3A5(V3), Hu3A5(V4), Hu3A5(V5), Hu3A5(V1), Hu3A5(V6), Hu3A5(V7), Hu3A5(V8), Hu3A5(V9), Hu3A5(V10), Hu3A5(V11), Hu3A5(V12), Hu3A5(V13), Hu3A5(V14), Hu3A5(V15), and Hu3A5(V16) antibodies selected in the present invention specifically bind to CD47.

[0132] 4-2: Analysis using flow cytometry In this invention, flow cytometry was performed to confirm the specificity of the 16 humanized 3A5 antibodies established in Example 3 for CD47.

[0133] First, we examined the breast cancer cell line MCF-7 (1 x 10⁶) which overexpresses CD47. 7 After reacting each of the individual 3A5 cells with 16 types of humanized 3A5 antibodies (1 μl each) for 30 minutes, the surface was stained with a secondary antibody and then measured using a flow cytometer.

[0134] A CD47 antibody (Biolegend PE anti-human CD47, cat# 323108, 5 μl) was used as the positive control, and a PE-conjugated goat anti-mouse IgG antibody (Biolegend Inc., cat# 405307, USA, 5 μl) was used as the secondary antibody.

[0135] As a result, as shown in Figure 2, it was confirmed that all 16 types of humanized 3A5 antibodies specifically bind to cells that overexpress CD47. Example 5: Confirmation of the ability of the humanized 3A5 antibody to block CD47-SIRPα binding The binding of CD47 expressed on cancer cells to SIRPα on macrophages negatively regulates phagocytosis and induces immune escape of cancer cells. In the present invention, it was attempted to confirm whether the humanized Hu3A5 (V10) antibody blocks the interaction of CD47-SIRPα.

[0136] MCF-7 cells expressing CD47 were treated with Hu3A5 (V10) antibodies at concentrations of 10 -1 、10 0 、10 1 、10 2 、10 3 、10 4 、10 5 、10 6 and 10 7 ng / ml respectively, so that the Hu3A5 (V10) antibody binds to the MCF-7 cells. Next, after reacting the MCF-7 cells attached with the above Hu3A5 (V10) antibody with SIRPα protein labeled with PE (Acrobiosystems, cat#SIA-HP252), the degree of binding of SIRPα to CD47 on the cell surface was analyzed by flow cytometry.

[0137] As a result, as shown in Figure 3, it was confirmed that the humanized 3A5 (V10) antibody of the present invention effectively blocks CD47-SIRPα binding. Example 6: The effect of improving the phagocytic activity of macrophages by the humanized 3A5 antibody was confirmed.

[0138] Peripheral blood mononuclear cells (PBMCs) were separated from the blood of normal humans with Ficoll-Paque and placed in a 24-well plate containing AIM-V medium. After waiting for monocytes to attach to the bottom, they were cultured in AIM-V medium for 7 days so that monocytes differentiated into macrophages.

[0139] Peripheral blood mononuclear cells (PBMCs) were co-cultured with low-level feline cells coated with CFSE, and then added to a co-culture plate containing differentiated macrophages along with Hu3A5(V10) antibody. The phagocytic action of the macrophages, which digests the low-level feline cells, was then performed using CFSE. + CD14 + Cellular analysis was performed. Hu3A5(V10) antibody was added to concentrations of 0.01, 0.1, 1, and 10 μg / ml, respectively, and human IgG (hlIgG) at a concentration of 10 μg / ml was used as the control group.

[0140] As a result, as shown in Figure 4, it was confirmed that the humanized Hu3A5(V10) antibody of the present invention binds to cancer cells that overexpress CD47 and promotes the phagocytic activity of macrophages against cancer cells. Example 7: Confirmation of the presence or absence of hemagglutination by humanized 3A5 antibody In this invention, to confirm whether the humanized 3A5 antibody induces hemagglutination, the presence or absence of red blood cell / platelet binding and the presence or absence of hemagglutination reaction were examined for Hu3A5(V10) antibody and commercially available anti-CD47 antibody (clone#CC2C6).

[0141] Blood collected from healthy volunteers was washed three times with PBS containing 1 mmol / l EDTA (ethylenediaminetetraacetic acid). The washed blood was then diluted in PBS containing 1 mmol / l EDTA at a ratio of 1:400 to prepare human red blood cells (RBCs). The washed RBCs were dispensed into 96-well round culture plates at a rate of 100 μl / well and treated with anti-human CD47 antibody (anti-human CD47 mAb, CC2C6 clone) and the humanized Hu3A5(V10) antibody of the present invention at concentrations of 0, 0.1, 0.5, 1, 5, 10, and 25 μg / ml, respectively. The 96-well plates with added antibodies were incubated in a 37°C incubator for 2 hours to confirm RBC hemagglutination.

[0142] As shown in Figure 5, the commercially available anti-CD47 antibody (clone#CC2C6) reacted with red blood cells to induce hemagglutination, whereas the humanized Hu3A5(V10) antibody of the present invention did not bind to red blood cells or platelets (Figure 5A) and did not induce hemagglutination (Figure 5B).

[0143] Example 8: Confirmation of the efficacy of 3A5 antibody in inhibiting the growth of CD47-overexpressing tumors. In this invention, animal experiments were conducted as shown in the schematic diagram in Figure 6A to confirm whether the 3A5 antibody inhibits the growth of tumors expressing CD47.

[0144] First, we gave 2.5 × 10⁶ mice (6 weeks old, female) to C57BL / 6 mice. 6 Human CD47-expressing mouse colon adenocarcinoma cells (MC38-hCD47, Biocytogen) were transplanted subcutaneously. Ten days after cancer cell transplantation, the size of the cancerous tissue in each mouse was measured and divided into four groups as follows to ensure uniformity (n=5 per group).

[0145] 1) Rat IgG administration group (control group) 2) Anti-PD-1 antibody administration group. 3) Anti-CD47 antibody (3A5 antibody) administration group 4) Group receiving combination therapy with anti-PD-1 antibody and anti-CD47 antibody (3A5 antibody) Each experimental group received a 200 μg concentration of antibody intraperitoneally, administered three times at 5-day intervals. Cancer tissue size was measured regularly during antibody administration to assess cancer growth.

[0146] As a result, as shown in Figure 6B, we confirmed that the growth of tumors overexpressing CD47 was suppressed in the group administered with 3A5 antibody alone, and that the tumor growth inhibitory efficacy was particularly superior in the group administered with both 3A5 antibody and anti-PD-1 antibody. This means that when anti-PD-1 antibody, an immune barrier inhibitor, is administered in combination with the 3A5 antibody of the present invention, a synergistic effect is observed in tumor treatment.

[0147] Example 9: Confirmation of the efficacy of humanized 3A5 antibody in inhibiting the growth of CD47-overexpressing tumors. To confirm the tumor growth inhibitory efficacy of the humanized 3A5 antibody, C57BL / 6-hCD47 / hSIRPα knock-in mice (hCD47 KI) were prepared in which the mouse CD47 and SIRPα genes were replaced with human CD47 and human SIRPα genes.

[0148] 25 C57BL / 6-hCD47 / hSIRPα (hCD47 KI, female, 6 weeks old) were given 2.5 × 10 6 Human CD47-expressing mouse colon adenocarcinoma cells (MC38-hCD47, Biocytogen) were subcutaneously transplanted. Ten days after cancer cell transplantation, the size of the tumor tissue in each mouse was measured. Then, 20 mice were selected from the 25 mice and divided into four groups as follows to ensure equality (n=5 per group).

[0149] 1) Rat IgG administration group (control group) 2) Anti-PD-1 antibody administration group 3) Anti-CD47 antibody (3A5 antibody) administration group 4) Group receiving combination therapy with anti-PD-1 antibody and anti-CD47 antibody (3A5 antibody) Each experimental group received a 200 μg concentration of antibody intraperitoneally, administered four times at 5-day intervals. Cancer tissue size was measured regularly during antibody administration to assess cancer growth.

[0150] Similar to Example 10 described above, rat colon adenocarcinoma cells expressing human CD47 were transplanted into mice, and then the control antibody (Rat IgG), anti-PD-1 antibody, anti-CD47 antibody (Hu3A5(V10) antibody), and anti-PD-1 antibody + anti-CD47 antibody (Hu3A5(V10) antibody) were administered, respectively (Figure 7A).

[0151] As a result, we confirmed that tumor growth was suppressed in the group treated with Hu3A5(V10) antibody alone, and that the tumor growth inhibitory effect was most pronounced in the group treated with Hu3A5(V10) antibody and anti-PD-1 antibody in combination.

[0152] Furthermore, after the final antibody administration, the major organs (liver, lungs, and kidneys) of C57BL / 6-hCD47 / hSIRPα-lysed mice, 32 days after cancer cell transplantation and at the end of the experiment, were isolated and fixed in 10% formalin solution for one day. Each fixed tissue was embedded in paraffin wax, sectioned to a thickness of 5 μm, and mounted on a glass slide. Each slide was stained with H&E (hematoxylin and eosin) using an H&E staining kit (VECTOR laboratories, CAT #: H-3502). After staining, images of each tissue were acquired using a slide scanner (Vectra Polaris Imaging system, PerkinElmer).

[0153] As a result, as shown in Figure 8, no major tissue damage due to inflammation was observed even with antibody administration. [Industrial applicability] The humanized anti-CD47 antibody of the present invention not only blocks CD47-SIRPα binding, but also binds to cells that overexpress CD47, promoting macrophage activity by macrophages and suppressing the growth of tumors that express CD47. Therefore, it can be applied to the prevention or treatment of diseases or tumors in which the immune response is suppressed due to CD47 overexpression.

Claims

1. A humanized antibody or antigen-binding fragment thereof that specifically binds to CD47, comprising a heavy chain variable region indicated by the amino acid sequence of SEQ ID NO: 65 and a light chain variable region indicated by the amino acid sequence of SEQ ID NO:

66.

2. A polynucleotide encoding a humanized antibody or its antigen-binding fragment that specifically binds to CD47 as described in claim 1.

3. A vector comprising a polynucleotide encoding a humanized antibody or its antigen-binding fragment that specifically binds to CD47 as described in claim 1.

4. Recombinant cells that produce a humanized antibody or its antigen-binding fragment that specifically binds to CD47 transformed with the vector according to claim 3.

5. A pharmaceutical composition for the prevention or treatment of a disease mediated by cells overexpressing CD47, comprising a humanized antibody or its antigen-binding fragment that specifically binds to CD47 as described in claim 1.

6. Claim 5, a pharmaceutical composition for the prevention or treatment of a disease mediated by cells overexpressing CD47, characterized in that a humanized antibody or its antigen-binding fragment contained in the composition specifically binds to CD47, thereby preventing CD47 from interacting with signal-regulating protein α (SIRPα) or promoting macrophage-mediated phagocytosis of CD47-overexpressing cells.

7. Claim 5, a pharmaceutical composition for the prevention or treatment of a disease mediated by cells overexpressing CD47, further comprising an immune checkpoint inhibitor.

8. Claim 7, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, a pharmaceutical composition for the prevention or treatment of a disease mediated by cells overexpressing CD47.

9. A pharmaceutical composition for the prevention or treatment of a disease mediated by cells that overexpress CD47, characterized in that the disease mediated by cells that overexpress CD47 is a cancer or tumor that overexpresses CD47, according to claim 5.

10. A pharmaceutical composition for the prevention or treatment of a disease mediated by cells overexpressing CD47, characterized in that the cancer or tumor is selected from the group consisting of hematological cancer, ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastocyte-induced CNS tumor, monocytic leukemia, B-cell-induced leukemia, T-cell-induced leukemia, B-cell-induced lymphoma, T-cell-induced lymphoma, and mast cell-induced tumor.

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