Binding agent

Abstract

To provide antibody molecules and antigen-binding portions thereof that specifically bind to CD47 (differentiation cluster 47, also known as integrin-associated protein [IAP]).SOLUTION: In an embodiment of the invention, anti-CD47 antibody molecules and antigen-binding portions thereof specifically bind to human CD47 and cynomolgus monkey CD47. Medical uses of the anti-CD47 antibody molecules and antigen-binding portions of the invention are disclosed. The anti-CD47 antibody molecules and antigen-binding portions of the invention are modified and optimized binding molecules compared to the VxP037 murine / humanized anti-CD47 antibody described in WO 2014 / 093678 A2.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to CD47 (cluster of differentiation 47, integrin-associated protein [IAP]) The present invention relates to antibody molecules which specifically bind to the antibody molecule (also known as IgG1), and medical uses thereof. [Background technology]

[0002] CD47 (also known as integrin-associated protein [IAP]) is a key regulator of immune globulin expression. It is a transmembrane protein belonging to the globulin superfamily, and is involved in membrane integrins, thrombin thymus gland receptor 1 (TSP-1), and signal regulatory protein alpha (SIRPα) CD47 binds to several known partners, including IL-1, IL-2, and IL-1. CD47 is involved in the regulation of cell apoptosis, proliferation, and They are involved in various cellular processes such as cell adhesion, migration, and importantly, immune responses. CD47-SIRPα signaling plays an important role in the response to and angiogenesis. , a key molecular interaction that inhibits activation of phagocytosis by macrophages and other myeloid cells This is due to the fact that myeloid lineage-specific immune checkpoints are activated to promote tumor cell survival. It acts as a

[0003] Preclinical evidence indicates that blocking CD47-SIRPα signaling inhibits the phagocytic activity of macrophages. and inhibiting xenograft growth in multiple experimental models of hematological and solid malignancies. Macrophage activity is associated with tissue fibrosis and the formation of atherosclerotic plaques. It is also a recognized factor in the biology of inflammation-related tissue remodeling, such as osteoporosis. , the CD47-SIRPα signaling axis also has considerable therapeutic potential in non-cancerous diseases. Therefore, anti-CD47 mAbs may act as immunotherapeutic agents in cancer and other settings, and are currently It has the potential to amplify the effectiveness of established treatments.

[0004] The majority of currently approved antibody therapeutics are derived from immunized rodents. Many of these antibodies were developed through the "grafting" of mouse CDRs onto human v-gene framework sequences. These have undergone a process known as "humanization" (Nelson et al., 2010, Nat Rev Dru g Discov 9:767-774). This process is often imprecise and the results This leads to a decrease in the target binding affinity of the antibody. To restore the binding affinity of the original antibody, Mouse residues were introduced at key positions in the variable domain framework of the grafted v domain. (also known as a "reverse mutation").

[0005] Antibodies that have been humanized via CDR grafting and back-mutation contain entirely murine v domains. have been shown in clinical trials to induce a lower immune response rate compared to antibodies with However, the potential physical instability and immunogenicity still harbored by the grafted CDR loops remains. Because of the motif, antibodies humanized using this basic grafting method have yet to achieve significant clinical success. There is a risk of the emergence of novel anti-cancer drugs that target receptors on immune cells and whose pharmacological functions are mediated through antigen presentation. Antibodies such as CD47 inhibitors, which stimulate immune responses, can trigger anti-drug antibody responses. These anti-drug antibody responses in patients are associated with increased risk of developing May reduce half-life, efficacy, and safety. Animal testing of protein immunogenicity Because antibody engineering is often not predictive of human immune responses, therapeutic antibody engineering is often performed using purified proteins. Predicted human T cell epitope content, non-human germline amino acid content, and aggregation of The focus is on minimizing the impact.

[0006] Therefore, an ideal humanized antagonistic anti-CD47 antibody would be one that is based on a well-characterized human germline As many identical residues as possible to those found in both the framework and CDRs of the sequence are v domain. Townsend et al. (2015; PNAS 112: 15354-15359) CDRs from rabbit and mouse antibodies are grafted onto suitable human frameworks. , and then subject the antibodies to a human germline approach called "enhanced binary replacement." This approach has led to the generation of highly accurate antibody-antigen cocrystal structure data. In the absence of data, we demonstrated the fundamental plasticity of the original antibody paratope, but the C of a particular antibody Determine which residues in the DR loop can be converted in human germline sequences and in what combinations. Indeed it is still impossible to predict.

[0007] Therefore, it is preferable to maintain multiple functional properties of the molecule, and therefore the CDR germline Systems are complex and multifactorial, examples of which include: humans and test animal species (e.g. For example, the crab-eating monkey, also known as the macaque, or Macaca Target binding specificity, affinity, and v-domain binding to CD47 from both IL-1 and IL-2. Biophysical stability, and / or IgG expression. Antibody engineering studies have revealed that the major CDRs are Even a single residue mutation can dramatically affect all these desirable molecular properties. As shown.

[0008] WO 2014 / 093678A2 describes an antagonistic mouse model of VxP037. The authors describe the preparation of a humanized form of an anti-CD47 IgG molecule, VxP037. These humanized forms of VxP037 were produced using classical humanization techniques, namely Kaba by grafting defined murine CDRs onto human heavy and light chain framework sequences , some of the human framework residues are potential candidates for VxP037 mouse residues at the corresponding positions. For the above reasons, WO 2014 / 0936 Such a humanized form of VxP037 described in 78A2 is not ideal.

[0009] The present invention provides a number of optimized anti-CD47 antibodies and their medical uses. Summary of the Invention

[0010] In one embodiment of the invention, a method is provided comprising the steps of: Antibodies that specifically bind to mouse CD47 and / or to mouse CD47 or to an antigen-binding portion thereof. and an antibody molecule, wherein the antibody molecule, or an antigen-binding portion thereof, has a heavy chain variable region comprising: Including:

[0011] HCDR1 having the amino acids in the following order: GYT, or any amino acid ( For example, conservative substitutions of S, N, or R-FT, or TN, or NYYI. or a conservative substitution of IF, or any amino acid (e.g., V or G) (SEQ ID NO: 1); A conservative substitution of HCDR2:M or MGI having amino acids in the following order: or any amino acid (e.g., N, V, or D)-IN, or any amino acid (e.g., Y )-PV, or any amino acid (e.g., G or F)-D, or a conservative substitution of DG, is a conservative substitution of GDTN, or a conservative substitution of N(e.g., R)-Y, or a conservative substitution of YN or N(e.g., S)-PSFQG conservative substitution (SEQ ID NO: 2); and HCDR3:GGY, having the amino acids in the following order: For example, H, I, Q, or F)-T, or any amino acid (for example V or I)-M, or any any amino acid (e.g., T, R, P, A, or L)-D, or any amino acid (e.g., G) -R, or any amino acid (e.g., Q, N, Y, S, W, K, A, E, F, H, I, L, M, T, or V) (SEQ ID NO:3).

[0012] In an embodiment of the invention, the HCDR1 of the antibody molecule or antigen binding portion has the sequence GYTFTN YYVF (SEQ ID NO: 4) (V disclosed in WO 2014 / 093678A2) xP037 mouse / humanized antibody HCDR1) may be excluded, and / or the antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMDY (SEQ ID NO: 5) (International Publication No. 2014 VxP037 mouse / humanized antibody (HCDR3) disclosed in US Pat. No. 6,367,878, issued to May be excluded.

[0013] The antibody molecule or antigen binding portion may further comprise a light chain variable region having: LCDR1 having the amino acids in the following order: RSSQ, or QSL Conservative substitution, or conservative substitution of LL, or conservative substitution of LHSN, or any amino acid (e.g., Q, S, T, A, or G) or N(e.g., Q, S, T, or G)-G Conservative substitutions, or conservative substitutions of G (e.g., A)-Y, or any amino acid (e.g., N or is a conservative substitution of S)-T, or T (e.g., N)-YLH, or any amino acid (e.g., For example, D) (SEQ ID NO: 6); LCDR2:K having the following sequence of amino acids in the following order: or M)-V, or any amino acid (e.g. G)-SN, or any amino acid (e.g. Y)-RL, or any amino acid (e.g., F, A, or S)-S (SEQ ID NO: 7); and LCDR3:F having the amino acids in the following order: , M, S, T, or V)-QQ, or any amino acid (e.g., N, A, T, or S)- T, or any amino acid (e.g., L, M, or I)-H, or a conservative substitution of HT; any amino acid (e.g., V, I, A, or F)-P, or any amino acid (e.g., L)- R, or any amino acid (e.g., W)-T (SEQ ID NO:8).

[0014] In an embodiment of the invention, LCDR1 of the antibody molecule or antigen binding portion has the sequence RSSQSL VHSNGNTYLH (SEQ ID NO: 9) (disclosed in WO 2014 / 093678A2) and / or The LCDR2 of the antibody molecule or antigen-binding portion has the sequence KVSYRFS (SEQ ID NO: 10) (International VxP037 mouse / humanized antibody disclosed in Publication No. 2014 / 093678A2 LCDR2) and / or LCDR3 of the antibody molecule or antigen-binding portion may be: The sequence SQNTHVPRT (SEQ ID NO: 11) (WO 2014 / 093678 A2) The disclosed VxP037 mouse / humanized antibody LCDR3) may be omitted.

[0015] The above CDR sequences are defined using the "uniform" definitions presented in Table 1 and explained below. Instead, the CDR sequences of the present invention are based on structural biology and are consistent with the The shorter "AHo" definition (see Table 1) aims to unify the nomenclature of phosphoryl domains. (see

[0016] Using the shorter "AHo" CDR definition, in one embodiment the invention provides a method for identifying human CD4 7, and optionally specifically binds to cynomolgus monkey CD47, and / or mouse CD47. and an antibody molecule, or antigen-binding portion thereof, which is and a heavy chain variable region having:

[0017] HCDR1 having the amino acids in the following order: GSGYT, or any amino acid (e.g., S, N, or R)-FT, or conservative substitutions of TN, or NYY Conservative substitution (SEQ ID NO: 12); HCDR2:IN having the following sequence of amino acids in the following order: or any amino acid (e.g., G or F)-D, or a conservative substitution of DG. or a conservative substitution of GDTN, or a conservative substitution of N(e.g., R)-Y, or YN or a conservative substitution of N(e.g., S)-PSFQG (SEQ ID NO: 13). and HCDR3:GGY, having the amino acids in the following order: For example, H, I, Q, or F)-T, or any amino acid (for example V or I)-M, or any any amino acid (e.g., T, R, P, A, or L)-D, or any amino acid (e.g., G) (Sequence number 14).

[0018] Using the AHo definition, the HCDR1 of an antibody molecule or antigen binding portion has the sequence GSGYTF TNYY (SEQ ID NO: 15) (disclosed in WO 2014 / 093678A2) VxP037 mouse / humanized antibody HCDR1) may be excluded, and / or the antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMD (SEQ ID NO: 16) (International Publication No. WO 201 VxP037 mouse / humanized antibody HCDR3 disclosed in US Pat. No. 4 / 093678A2 may be excluded.

[0019] An antibody molecule or antigen-binding portion comprises CDRs defined using the AHo definitions as follows: and may further comprise a light chain variable region having: LCDR1 having the amino acids in the following order: SSQ or QSL conserved or a conservative substitution of LL, or a conservative substitution of LHSN, or any amino acid Storage of acid (e.g., Q, S, T, A, or G) or N (e.g., Q, S, T, or G)-G conservative substitutions of G (e.g. A)-Y, or any amino acid (e.g. N or S) -T, or a conservative substitution of T(e.g. N)-Y (SEQ ID NO: 17). LCDR2:K having the following sequence of amino acids in the following order: or M)-V, or any amino acid (e.g. G)-SN, or any amino acid (e.g. Y)-RL, or any amino acid (e.g., F, A, or S)-S (SEQ ID NO: 7); and LCDR3:Q having the amino acids in the following order: , A, T, or S)-T, or any amino acid (e.g., L, M, or I)-H, or H- Conservative substitutions of T, or any amino acid (e.g., V, I, A, or F)-P, or any amino acid (e.g. L)-R, or any amino acid (e.g. W) (SEQ ID NO: 18).

[0020] Using the AHo definition, the LCDR of an antibody molecule or antigen-binding portion in an embodiment of the present invention 1 has the sequence SSQSLVHSNGNTY (SEQ ID NO: 19) (WO 2014 / 093 678A2) and / or LCDR2 of the antibody molecule or antigen-binding portion may have the sequence KVSYRFS (sequence Column No. 10) (VxP037 disclosed in WO 2014 / 093678A2) The mouse / humanized antibody LCDR2) may be omitted, and / or the antibody molecule or antigen-binding portion The LCDR3 of this variant has the sequence NTHVPR (SEQ ID NO: 20) (WO 2014 / 0936 Even excluding the VxP037 mouse / humanized antibody LCDR3) disclosed in US Pat. No. 78A2, good.

[0021] According to the present invention, an antibody molecule as defined herein or an antigen binding thereof conjugated to a therapeutic agent Immunoconjugates comprising the moieties are also provided. In another aspect, the present invention relates to an antibody molecule or an antigen-binding portion thereof as defined herein. Nucleic acid molecules that encode the nucleic acid sequences are provided. Further provided is a vector comprising the nucleic acid molecule of the invention. Also provided is a host cell comprising a nucleic acid molecule or vector of the invention as defined herein.

[0022] In a further embodiment, the method of producing an anti-CD47 antibody and / or antigen-binding portion thereof comprises and wherein the antibody and / or antigen-binding portion thereof is subjected to the method of the present invention under conditions that result in expression and / or production of the antibody and / or antigen-binding portion thereof. and isolating the antibody and / or antigen-binding portion thereof from the host cell or culture. A method is provided comprising isolating the

[0023] In another aspect of the invention, an antibody molecule of the invention as defined herein or an antigen-binding a portion or an immunoconjugate of the invention as defined herein, or a pharmaceutical composition comprising the inventive vector as defined herein. .

[0024] Further provided is a method of enhancing an immune response in a subject, comprising administering to a subject a therapeutically effective amount of the compound of the present invention as defined herein. an antibody molecule or an antigen-binding portion thereof as defined herein, or an immunoconjugate of the invention as defined herein; means a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein or a method comprising administering an effective amount of the pharmaceutical composition of the invention as defined herein. Provided.

[0025] In a further aspect, there is provided a method of treating or preventing cancer in a subject, comprising administering to a subject ... as defined herein. or an antigen-binding portion thereof, Conjugates or nucleic acid molecules of the invention as defined herein or the invention as defined herein. or a pharmaceutical composition of the invention as defined herein. A method is provided that includes:

[0026] The present invention also relates to an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, The immunoconjugates of the invention as defined herein or the nucleic acid molecules of the invention as defined herein may be or a vector of the invention as defined herein, The present invention provides a pharmaceutical composition comprising:

[0027] In another aspect, the present invention provides a method for the treatment of cancer, comprising administering to a patient a therapeutic agent, the therapeutic agent being administered separately or in combination with a second therapeutic agent, e.g., an anticancer agent. The antibody molecule of the invention as defined herein, or Antigen-binding moieties, or immunoconjugates, or nucleic acid molecules, or vectors, or methods of treatment are provided. .

[0028] In a further aspect, a compound as defined herein in the manufacture of a medicament for the treatment of cancer. An antibody molecule of the invention or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein. The use of a pharmaceutical composition of the invention as defined herein is also provided.

[0029] The present invention also provides a method for treating or preventing ischemia-reperfusion injury, an autoimmune disease, or an inflammatory disease in a subject. A method of preventing, comprising administering to a subject an antibody molecule or antigen-binding portion thereof as defined herein, or An immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or or a pharmaceutical composition as defined herein. Provide the law.

[0030] In all aspects, the autoimmune or inflammatory disease is selected from the group consisting of arthritis, multiple sclerosis, psoriasis, From Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis, and ankylosing spondylitis It may be selected from the group consisting of:

[0031] In all embodiments, ischemia-reperfusion injury is a chronic condition that is characterized by a variety of conditions, including organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, and the like. , pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resection and reconstructive surgery, adnexal or It can occur with reattachment of other body parts, skin grafts or trauma.

[0032] It is also used in the treatment of ischemia-reperfusion injury, autoimmune diseases, or inflammatory diseases. An antibody molecule or an antigen-binding portion thereof as defined herein, or an immunoglobulin as defined herein. or a nucleic acid molecule as defined herein, or a vector as defined herein, Pharmaceutical compositions as defined herein are also provided.

[0033] Furthermore, the present invention is useful in the manufacture of a medicament for the treatment of ischemia-reperfusion injury, autoimmune disease, or inflammatory disease. an antibody molecule or an antigen-binding portion thereof as defined herein, or an immunoglobulin as defined herein An immunoconjugate, or a nucleic acid molecule as defined herein, or a vector as defined herein, The use of the pharmaceutical composition as defined herein is provided.

[0034] The present invention also relates to an antibody molecule as defined herein or an antigen-binding portion thereof, An immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, administering to a subject an effective amount of a vector or a pharmaceutical composition as defined herein. The present invention provides a method for treating or preventing a cardiovascular or fibrotic disease.

[0035] Also, an antibody molecule as defined herein for use in the treatment of a cardiovascular disease or a fibrotic disease. or an antigen-binding portion thereof, or an immunoconjugate as defined herein, or or a vector as defined herein, or a pharmaceutical composition as defined herein. will also be provided.

[0036] Furthermore, for the treatment of ischemia-reperfusion injury, autoimmune diseases, inflammatory diseases, or fibrotic diseases. The use of an antibody molecule or an antigen-binding portion thereof as defined herein, or An immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or The use of a vector or a pharmaceutical composition as defined herein is also provided.

[0037] In any aspect of the invention, the cardiovascular disease may be, for example, coronary heart disease or atherosclerotic heart disease. It may be arteriosclerosis.

[0038] In any embodiment of the present invention, the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, or the like. The disease may be selected from the group consisting of bronchitis, cystic fibrosis, bronchitis, and asthma.

[0039] The present invention also relates to human CD47, and optionally cynomolgus monkey CD47, and / or macaque CD47. The present invention provides a method for producing an antibody molecule, or an antigen-binding portion thereof, that specifically binds to mouse CD47. and the method comprises the steps of: (1) Grafting anti-CD47 CDRs of non-human origin onto a human v-domain framework producing a humanized anti-CD47 antibody molecule, or an antigen-binding portion thereof; (2) A humanized anti-CD47 antibody molecule or its antigen-binding portion comprising one or more mutations in the CDRs. generating a phage library of individual clones; (3) human CD47, and optionally cynomolgus monkey CD47, and / or mouse CD screening the phage library for binding to 47; (4) human CD47, and optionally cynomolgus monkey CD47, and / or mouse CD A step of selecting a clone having binding specificity to 47 from the screening step (3). Process; and (5) Extracting human CD47 and, optionally, crab CD47 from the clones selected from step (4). An antibody molecule that specifically binds to monkey CD47 and / or mouse CD47, or an antibody thereof Producing the original binding moiety.

[0040] This method is based on the clones selected in step (4), for example, Based on further exploratory mutagenesis at specific positions in the CDRs of the clones identified, additional Generate clones to enhance humanization and / or minimize human T-cell epitope content and / or improve the manufacturing properties of the antibody molecule or antigen-binding portion thereof produced in step (5). The method may include the further step of:

[0041] This method comprises the steps of: The immunogenicity of one or more v-domains is evaluated, e.g., by determining whether an appropriate Optionally, a further step of generating one or more additional mutations to reduce immunogenicity. Immunogenicity can be determined, for example, by in silico immunoassays as described herein. ) technology can be used to identify and evaluate the location of T cell epitopes. . [Brief description of the drawings]

[0042] [Figure 1] Direct binding ELISA of library-derived anti-CD47 scFv against human and mouse CD47-Fc proteins. Clones were obtained from three separate phage selection branches (A indicates branch A periprep ELISA, B indicates branch B periprep ELISA, and C indicates branch C periprep ELISA), where in each round phage populations were selected against biotinylated human, mouse, and / or cynomolgus CD47-Fc proteins. After each selection round, library-derived clones (black circles) were screened against both human and mouse CD47-Fc. The mean ± SD values ​​for each round are represented by grey bars. In each graph, the X-axis indicates the selection round ("R"), "H" indicates human, and "M" indicates mouse, and the Y-axis indicates the binding signal (OD450nm). [Figure 2A]Analysis of tolerance of CDR residues to mutations into germline. Plots of the frequency of retention of mouse amino acids in the CDRs of the ELISA-positive population of 854 unique scFv clones are shown for the VH (A) and VL (B) domains, respectively. Outside of HCDR3, only residues targeted for mutagenesis of human / mouse residues are plotted. In each plot, the CDR residues are shown on the X-axis and the Y-axis shows the percentage of retention of each mouse residue. CDR residues listed in brackets on the X-axis were identical to those found in the human germline used for grafting (IGKV2-28 and IGHV5-51). Residues in HCDR2 that are not in brackets but have a value set to 0 were mutated into the human germline during the grafting process. In both plots, the grey dashed line at 75% represents the cutoff for tolerance of substitution of mouse residues by human germline. [Figure 2B] Analysis of tolerance of CDR residues to mutations into germline. Plots of the frequency of retention of mouse amino acids in the CDRs of the ELISA-positive population of 854 unique scFv clones are shown for the VH (A) and VL (B) domains, respectively. Outside of HCDR3, only residues targeted for mutagenesis of human / mouse residues are plotted. In each plot, the CDR residues are shown on the X-axis and the Y-axis shows the percentage of retention of each mouse residue. CDR residues listed in brackets on the X-axis were identical to those found in the human germline used for grafting (IGKV2-28 and IGHV5-51). Residues in HCDR2 that are not in brackets but have a value set to 0 were mutated into the human germline during the grafting process. In both plots, the grey dashed line at 75% represents the cutoff for tolerance of substitution of mouse residues by human germline. [Figure 3A-D]Direct titration ELISA for IgG binding to human, mouse, and cynomolgus CD47-Fc proteins. Human IgG1 null form of chimeric anti-CD47 (mVH / mVL), library-derived clones were titrated (μg / ml) in a direct binding ELISA against human, mouse, and cynomolgus CD47-Fc proteins (A-H). mVH / mVL, library-derived clones, and designer clone MH showed binding activity to all three orthologs of CD47. Clone VH-A1 / VL-B1 binds to human and cynomolgus CD47, but not to mouse. Clone TTP lost nearly all binding function. The X-axis of each graph shows IgG concentration (μg / ml), and the Y-axis shows binding signal (OD450nm). [Figure 3E-H] Direct titration ELISA for IgG binding to human, mouse, and cynomolgus CD47-Fc proteins. Human IgG1 null form of chimeric anti-CD47 (mVH / mVL), library-derived clones were titrated (μg / ml) in a direct binding ELISA against human, mouse, and cynomolgus CD47-Fc proteins (A-H). mVH / mVL, library-derived clones, and designer clone MH showed binding activity to all three orthologs of CD47. Clone VH-A1 / VL-B1 binds to human and cynomolgus CD47, but not to mouse. Clone TTP lost nearly all binding function. The X-axis of each graph shows IgG concentration (μg / ml), and the Y-axis shows binding signal (OD450nm). [Figure 4]ELISA-based CD47-Fc-SIRPα competition assay. ELISA binding signals of human (A), cynomolgus (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of titrated competitive library-derived leads (A-D5, G-B6, D-H3, and VH-A1 / VL-B1 in IgG1 null form, isotype IgG1 as negative control, and mVH / mVL in IgG1 null form as positive control). All library-derived IgG and mVH / mVL showed a concentration-dependent decrease in binding of human, mouse, and cynomolgus CD47-Fc proteins, suggesting maintenance of a shared epitope. Notably, clone A-D5 showed significantly higher potency in neutralizing mouse CD47 compared to mVH / mVL, and VH-A1 / VL-B1 showed no ability to neutralize mouse CD47 activity. Neither the designer clones MH nor TTP showed a neutralization signal and for clarity are not plotted here. In each graph, the X-axis shows the antibody concentration (nM) and the Y-axis shows the binding signal (OD450nm). In the figure legends, "IC" refers to the isotype control. [Figure 5A-D]Binding specificity analysis of prioritized lead clones. For mVH / mVL in IgG1 (A) and IgG1 null (B) formats, and library-derived leads A-D5 (C), VH-A1 / VL-B1 (D), F-E7 (E), D-H3 (F), and G-B6 (G) in IgG1 null format, off-target homolog binding risk was investigated by direct ELISA of CD47-Fc orthologues and a panel of 14 human immunoglobulin superfamily proteins labeled on each X-axis ("B" refers to blank). Binding to human, cynomolgus, and mouse CD47-Fc (h / c / mCD47-Fc) was performed at an IgG concentration of 1 μg / ml. Binding to all other proteins was performed at an IgG concentration of 10 μg / ml. For each plot, the Y-axis indicates the binding signal (OD450nm). For almost all IgGs, binding was observed to hCD47-Fc, mCD37-Fc, and cCD47-Fc alone. No binding above background was observed with other human proteins. Notably, clone VH-A1 / VL-B1 again showed no reactivity to mouse CD47. [Figure 5E-G]Binding specificity analysis of prioritized lead clones. For mVH / mVL in IgG1 (A) and IgG1 null (B) formats, and library-derived leads A-D5 (C), VH-A1 / VL-B1 (D), F-E7 (E), D-H3 (F), and G-B6 (G) in IgG1 null format, off-target homolog binding risk was investigated by direct ELISA of CD47-Fc orthologues and a panel of 14 human immunoglobulin superfamily proteins labeled on each X-axis ("B" refers to blank). Binding to human, cynomolgus, and mouse CD47-Fc (h / c / mCD47-Fc) was performed at an IgG concentration of 1 μg / ml. Binding to all other proteins was performed at an IgG concentration of 10 μg / ml. For each plot, the Y-axis indicates the binding signal (OD450nm). For almost all IgGs, binding was observed to hCD47-Fc, mCD37-Fc, and cCD47-Fc alone. No binding above background was observed with other human proteins. Notably, clone VH-A1 / VL-B1 again showed no reactivity to mouse CD47. [Figure 6]Flow cytometry of binding to human and cynomolgus CD47+ CHO-K1 cells. Commercially available anti-CD47 antibody MS1991, human IgG1 ("I IgG1") and IgG4 ("I IgG4") isotype controls, and lead library derived IgG in both IgG1 null ("IgG1N") and IgG4 (S228P) forms were examined for specific binding to cynomolgus transfected CHO-K1 cells (A), human transfected CHO-K1 cells (B), and wild type (wt, i.e., untransfected) CHO-K1 cells (C). IgG was tested at concentrations ranging from 24 to 100,000 ng / ml. Concentration-dependent binding to both human and cynomolgus cell lines was observed for all CD47-specific antibodies, but not the isotype controls. For most antibodies, low levels of binding signals above background were observed for wild-type CHO-K1 cells, with a significantly stronger signal for mVH / mVL-derived IgG and a lower signal, especially for VH-A1 / VL-B1 IgG. In each graph, the X-axis shows each IgG tested and its concentration (ng / ml), and the Y-axis shows the mean fluorescence intensity (MFI). [Figure 7] Flow cytometry study of binding to human HL60 cells. Specific binding to HL60 cells was examined for IgG from the lead library, including the commercial anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls ("I IgG1" and "I IgG4", respectively), and both IgG1 null ("IgG1N") and IgG4 (S228P) forms. IgGs were tested at concentrations ranging from 24 to 100000ng / ml. Concentration-dependent binding was observed for all clones except the isotype control. In each graph, the X-axis shows each IgG tested and its concentration (ng / ml), and the Y-axis shows the mean fluorescence intensity (MFI). [Figure 8]Development risk ELISA. The assay showed that the IgG1 null forms of A-D5, G-B6, D-H3, VH-A1 / VL-B1, and mVH / mVL antibodies showed little or no binding to the negatively charged biomolecules insulin (A), double-stranded DNA (dsDNA) (B), and single-stranded DNA (ssDNA) (C). The X-axis of each graph shows the IgG concentration (μg / ml) and the Y-axis shows the binding signal (OD450nm). Strong off-target binding to these molecules observed with bococizumab and briakinumab analogues has been shown to be a high-risk indicator of poor clinical performance of therapeutic antibodies. [Figure 9] Direct titration ELISA of binding of designer IgG to human, mouse, and cynomolgus CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) in human IgG1 null form, designer A-D5 derived clones were titrated (μg / ml) in a direct binding ELISA against human (A), cynomolgus (B), and mouse (C) CD47-Fc proteins. In each graph, the X-axis shows IgG concentration (μg / ml) and the Y-axis shows binding signal (OD450nm). Most clones showed binding activity to all three orthologues of CD47, but clone A-D5.7 showed only weak binding to cynomolgus CD47 and clone A-D5.10 had lost almost all binding function to mouse CD47. In the figure legend, "IgG1NI" refers to the IgG1 null isotype. [Figure 10]ELISA-based CD47-Fc-SIRPα competition assay for designer IgG. The ELISA binding signals of human (A), cynomolgus (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of titrated competitive designer IgG in IgG1 null form and mVH / mVL in isotype IgG1 (represented as "IgG1NI") as a negative control and in IgG1 null form as a positive control. In each graph, the X-axis indicates the IgG concentration (nM) and the Y-axis indicates the binding signal (OD450nm). Notably, several A-D5-derived clones again showed a particularly marked increase in potency in neutralizing mouse CD47 compared to mVH / mVL. For their orthologues that showed weak ELISA binding signals, neither designer clones A-D5.7 nor A-D5.10 showed a neutralization signal and therefore are not plotted here for clarity. [Figure 11] Direct titration ELISA of A-D5.4-derived designer IgG binding to human, mouse, and cynomolgus CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) in human IgG1 null form, designer A-D5.4-derived clones were titrated (μg / ml) in a direct binding ELISA against human (A), cynomolgus (B), and (C) mouse CD47-Fc proteins. In each graph, the X-axis shows the IgG concentration (μg / ml) and the Y-axis shows the binding signal (OD450nm). All clones showed binding activity to all three orthologues of CD47. In the figure legend, "IgG1NI" refers to the IgG1 null isotype. [Figure 12]ELISA-based CD47-Fc-SIRPα competition assay for designer IgG. ELISA binding signals of human (A), cynomolgus (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of titrated competitive designer IgG in IgG1 null form, and isotype IgG1 (represented as "IgG1NI") as a negative control, and mVH / mVL in IgG1 null form as a positive control. In each graph, the X-axis shows the IgG concentration (nM) and the Y-axis shows the binding signal (OD450nm). Notably, several A-D5.4-derived clones again showed a marked increase in potency in neutralizing mouse CD47 compared to mVH / mVL. [Figure 13] Binding specificity analysis of designer clones A-D5.4 and A-D5.16. The off-target homolog binding risk of the IgG1 null forms A-D5.4 (A) and A-D5.16 (B) was investigated by direct ELISA against CD47-Fc orthologues and a panel of 14 human immunoglobulin superfamily proteins (labeled on the X-axis; "B" refers to blank). Binding to all proteins was performed at an IgG concentration of 10 μg / ml. In each plot, the Y-axis indicates the binding signal (OD450 nm). For both IgGs, binding to hCD47-Fc, mCD37-Fc, and cCD47-Fc alone was observed. No binding above background was observed with other human proteins. [Figure 14] Development risk ELISA for designer clones A-D5.4 and A-D5.16. The assay showed that the IgG1 null forms of A-D5.4 and A-D5.16 antibodies exhibited low binding (less than the negative control ustekinumab) to the negatively charged biomolecules insulin (A), double-stranded DNA (dsDNA) (B), and single-stranded DNA (ssDNA) (C). The X-axis of each graph shows the IgG concentration (μg / ml) and the Y-axis shows the binding signal (OD450nm). The strong off-target binding to these molecules observed with bococizumab and briakinumab analogues has been shown to be a high-risk indicator of poor clinical performance of therapeutic antibodies. [Figure 15]Flow cytometry of binding of library-derived and designer IgGs to CHO-K1 cells. Commercially available anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls (represented as "I IgG1" and "I IgG4", respectively), and lead IgGs A-D5, A-D5.4, and A-D5.16 in both IgG1 null and IgG4 forms were examined for specific binding on wild-type (i.e., non-transfected) CHO-K1 cells. IgGs were tested at concentrations ranging from 24 to 25,000 ng / ml. Concentration-dependent binding was observed for parental mVH / mVL antibodies in both IgG1 null and IgG4 forms, whereas weak or no binding was observed for isotype control, MS1991, and IgGs A-D5, A-D5.4, and A-D5.16 (both in IgG form). In each graph, the X-axis indicates the concentration of IgG (ng / ml) and the Y-axis indicates MFI. [Figure 16] Flow cytometry study of binding to human HL60 cells. Commercially available anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls (represented as "I IgG1" and "I IgG4", respectively), and lead IgG in both IgG1 null and IgG4 (S228P) forms were examined for specific binding to HL60 cells. IgG was tested at concentrations ranging from 24 to 100000 ng / ml. Concentration-dependent binding was observed for all clones except the isotype control. In each graph, the X-axis indicates the concentration of IgG (ng / ml) and the Y-axis indicates MFI. [Figure 17] T cell epitope peptide content in lead antibody v-domains. The v-domains of mVH / mVL, A-D5, A-D5.4, A-D5.16, and A-D5.16-DI antibodies were examined for the presence of germline (GE), high affinity foreign (HAF), low affinity foreign (LAF), and TCED+ T cell receptor epitopes. Both the VH and VL domains of mVH / mVL were found to contain multiple high-risk human T cell epitopes and several germline epitopes. High-risk epitope content was significantly reduced and germline epitope content was significantly improved in all lead clones. [Figure 18]Direct titration ELISA of A-D5.16 and A-D5.16-DI designer IgG binding to human, mouse, and cynomolgus CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) in human IgG1 null form, designer A-D5.16 and A-D5.16-DI clones were titrated in a direct binding ELISA against human (A), cynomolgus (B), and (C) mouse CD47-Fc proteins (μg / ml). All clones showed binding activity to all three orthologs of CD47. In each graph, the X-axis shows IgG concentration (μg / ml) and the Y-axis shows binding signal (OD450nm). [Figure 19] ELISA-based CD47-Fc-SIRPα competition assay for designer IgG. ELISA binding signals of human (A), cynomolgus (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of titrated competitive designer IgG in IgG1 null form and isotype IgG1 as a negative control, and mVH / mVL in IgG1 null form as a positive control. In each graph, the X-axis shows antibody concentration (nM) and the Y-axis shows binding signal (OD450nm). [Figure 20] Analysis of phagocytosis by flow cytometry. (A) Flow cytometric analysis of phagocytosis of CSFE-labeled HL60 cells by human CD14+ macrophages was performed for clones A-D5, A-D5.4, A-D5.16, and mVH / mVL in IgG4(S228P) form, and also for A-D5 in IgG1 null form (designated "IgG1 A-D5N") at multiple concentrations (indicated on the X-axis). The X-axis indicates antibody concentration (μg / ml) and the Y-axis indicates the % of cells that are CFSE+ and CD14+. (B) The analysis was then repeated with multiple human macrophage donors for A-D5 in IgG4 form and mVH / mVL at a standard concentration of 10 μg / ml. The X-axis indicates donor number, the Y-axis indicates the % of cells that are CFSE+ and CD14+, and "V" indicates vehicle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description of the Invention In a first aspect of the invention, a method is provided comprising the steps of: 7, and / or an antibody molecule or an antigen-binding portion thereof that specifically binds to mouse CD47 is provided. and wherein the antibody molecule or antigen-binding portion comprises a heavy chain variable region having:

[0044] HCDR1 having the amino acids in the following order: GYT, or any amino acid ( For example, conservative substitutions of S, N, or R-FT, or TN, or NYYI. or a conservative substitution of IF, or any amino acid (e.g., V or G) (SEQ ID NO: 1); A conservative substitution of HCDR2:M or MGI having amino acids in the following order: or any amino acid (e.g., N, V, or D)-IN, or any amino acid (e.g., Y )-PV, or any amino acid (e.g., G or F)-D, or a conservative substitution of DG, is a conservative substitution of GDTN, or a conservative substitution of N(e.g., R)-Y, or a conservative substitution of YN or N(e.g., S)-PSFQG conservative substitution (SEQ ID NO: 2); and HCDR3:GGY, having the amino acids in the following order: For example, H, I, Q, or F)-T, or any amino acid (for example V or I)-M, or any any amino acid (e.g., T, R, P, A, or L)-D, or any amino acid (e.g., G) -R, or any amino acid (e.g., Q, N, Y, S, W, K, A, E, F, H, I, L, M, T, or V) (SEQ ID NO:3).

[0045] In an embodiment of the invention, the HCDR1 of the antibody molecule or antigen binding portion has the sequence GYTFTN YYVF (SEQ ID NO: 4) (V disclosed in WO 2014 / 093678A2) xP037 mouse / humanized antibody HCDR1) may be excluded, and / or the antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMDY (SEQ ID NO: 5) (International Publication No. 2014 VxP037 mouse / humanized antibody (HCDR3) disclosed in US Pat. No. 6,367,878, issued to May be excluded.

[0046] In the present invention, the antibody molecule, or antigen-binding portion thereof, further comprises a light chain variable region having: May be included in: LCDR1 having the amino acids in the following order: RSSQ, or QSL Conservative substitution, or conservative substitution of LL, or conservative substitution of LHSN, or any amino acid (e.g., Q, S, T, A, or G) or N(e.g., Q, S, T, or G)-G Conservative substitutions, or conservative substitutions of G (e.g., A)-Y, or any amino acid (e.g., N or S)-T, or T(e.g., N)-YLH, or any amino acid (e.g., D) conservative substitution (SEQ ID NO:6); LCDR2:K having the following sequence of amino acids in the following order: or M)-V, or any amino acid (e.g. G)-SN, or any amino acid (e.g. Y)-RL, or any amino acid (F, A, or S)-S (SEQ ID NO: 7); and LCDR3:F having the amino acids in the following order: , M, S, T, or V)-QQ, or any amino acid (e.g., N, A, T, or S)- T, or any amino acid (e.g., L, M, or I)-H, or a conservative substitution of HT; any amino acid (e.g., V, I, A, or F)-P, or any amino acid (e.g., L)- R, or any amino acid (e.g., W)-T (SEQ ID NO:8).

[0047] In an embodiment of the invention, LCDR1 of the antibody molecule or antigen binding portion has the sequence RSSQSL VHSNGNTYLH (SEQ ID NO: 9) (disclosed in WO 2014 / 093678A2) and / or The LCDR2 of the antibody molecule or antigen-binding portion has the sequence KVSYRFS (SEQ ID NO: 10) (International VxP037 mouse / humanized antibody disclosed in Publication No. 2014 / 093678A2 LCDR2) and / or LCDR3 of the antibody molecule or antigen-binding portion may be: The sequence SQNTHVPRT (SEQ ID NO: 11) (WO 2014 / 093678 A2) The disclosed VxP037 mouse / humanized antibody LCDR3) may be omitted.

[0048] The above CDR sequences are defined using the "uniform" definitions provided in Table 1. Instead, the CDR sequences of the present invention are based on structural biology and are consistent with all immunoglobulin v domains. The shorter "AHo" definitions (see Table 1) aim to unify the main nomenclature. It can be defined using

[0049] Using the shorter "AHo" definition, in one embodiment the invention provides human CD47, and and optionally specifically binds to cynomolgus monkey CD47 and / or mouse CD47. An antibody molecule or antigen-binding portion thereof is provided, wherein the antibody molecule or antigen-binding portion comprises: The heavy chain variable region comprises:

[0050] HCDR1 having the amino acids in the following order: GSGYT, or any amino acid (e.g., S, N, or R)-FT, or conservative substitutions of TN, or NYY Conservative substitution, (SEQ ID NO: 12); HCDR2:IN having the following sequence of amino acids in the following order: or any amino acid (e.g., G or F)-D, or a conservative substitution of DG. or a conservative substitution of GDTN, or a conservative substitution of N(e.g., R)-Y, or YN or a conservative substitution of N(e.g., S)-PSFQG (SEQ ID NO: 13). and HCDR3:GGY, having the amino acids in the following order: For example, H, I, Q, or F)-T, or any amino acid (for example V or I)-M, or any any amino acid (e.g., T, R, P, A, or L)-D, or any amino acid (e.g., G) (Sequence number 14).

[0051] Using the AHo definition, the HCDR1 of an antibody molecule or antigen binding portion has the sequence GSGYTF TNYY (SEQ ID NO: 15) (disclosed in WO 2014 / 093678A2) VxP037 mouse / humanized antibody HCDR1) may be excluded, and / or the antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMD (SEQ ID NO: 16) (International Publication No. WO 201 VxP037 mouse / humanized antibody HCDR3 disclosed in US Pat. No. 4 / 093678A2 may be excluded.

[0052] The antibody molecule or antigen binding portion may further comprise a light chain variable region having: LCDR1 having the amino acids in the following order: SSQ or QSL conserved or a conservative substitution of LL, or a conservative substitution of LHSN, or any amino acid Storage of acid (e.g., Q, S, T, A, or G) or N (e.g., Q, S, T, or G)-G conservative substitutions of G (e.g. A)-Y, or any amino acid (e.g. N or S) -T, or a conservative substitution of T(e.g. N)-Y (SEQ ID NO: 17). LCDR2:K having the following sequence of amino acids in the following order: or M)-V, or any amino acid (e.g. G)-SN, or any amino acid (e.g. Y)-RL, or any amino acid (e.g., F, A, or S)-S (SEQ ID NO: 7); and LCDR3:Q having the amino acids in the following order: , A, T, or S)-T, or any amino acid (e.g., L, M, or I)-H, or H- Conservative substitutions of T, or any amino acid (e.g., V, I, A, or F)-P, or any amino acid (e.g. L)-R, or any amino acid (e.g. W) (SEQ ID NO: 18).

[0053] Using the AHo definition, the LCDR of an antibody molecule or antigen-binding portion in an embodiment of the present invention 1 has the sequence SSQSLVHSNGNTY (SEQ ID NO: 19) (WO 2014 / 093 678A2) and / or LCDR2 of the antibody molecule or antigen-binding portion may have the sequence KVSYRFS (sequence Column No. 10) (VxP037 disclosed in WO 2014 / 093678A2) The mouse / humanized antibody LCDR2) may be omitted, and / or the antibody molecule or antigen-binding portion The LCDR3 of this variant has the sequence NTHVPR (SEQ ID NO: 20) (WO 2014 / 0936 Even excluding the VxP037 mouse / humanized antibody LCDR3) disclosed in US Pat. No. 78A2, good.

[0054] As described in detail herein, the present inventors have Using the CDR sequences derived from the disclosed murine anti-CD47 antibody VxP037, a number of For the first time, we have succeeded in generating an optimized anti-CD47 antibody molecule. These antibody molecules bind to both human CD47 and cynomolgus monkey CD47 in part. The clones were selected to have binding specificity to mouse CD47 (in animal test species). (To facilitate research into the use of the antibody molecules described herein) , improved binding to the mouse orthologue of CD47, and improved murine CD47-SIRPα signaling improved neutralization potency, improved variable domain stability, high expression yields, and / or reduced immunogenicity. For example, we herein used the mouse anti-CD47 antibody VxP037 The precursor molecule of LCDR1 and LCDR2 has two major immunogenic risks, and the classical This is carried out using a novel humanization technique (as used in WO 2014 / 093678 A2) , which proves to be improved in the optimized antibody molecules described herein.

[0055] The antibody molecules or antigen-binding portions of the invention are selected from the group consisting of SEQ ID NOs: 4 (HCDR1), 123 (HCDR 2), 5(HCDR3), 9(LCDR1), 10(LCDR2), and 11(LCDR 3) The in silico immunogenicity may be improved compared to antibody molecules containing the CDR sequence of There is a gender.

[0056] The antibody molecules or antigen-binding portions of the invention are selected from the group consisting of SEQ ID NOs: 4 (HCDR1), 123 (HCDR 2), 5(HCDR3), 9(LCDR1), 10(LCDR2), 11(LCDR3) does not bind to hamster CD47, or For example, the antibody molecule or antigen-binding The moieties are SEQ ID NO: 4 (HCDR1), 123 (HCDR2), 5 (HCDR3), 9 (L Compared to antibodies containing the CDR sequences of CDR1), 10 (LCDR2), and 11 (LCDR3), and showed no binding to CHO cells or binding to CH As shown in FIG. 15, a representative antibody molecule of the present invention may exhibit reduced binding to O cells. It shows little or no cross-reactivity with CHO cells, but IgG1 null or IgG4 type The original mouse v domain of the cloned mVH / mVL showed a strong concentration-dependent increase in CHO cells. Drives sexual bonding.

[0057] A preferred optimized anti-CD47 antibody molecule of the present invention comprises the corresponding murine CDRs or other ( necessarily have the maximum number of human germline substitutions at amino acid positions (e.g., framework As described in detail in the Experimental Section below, we have demonstrated that anti-CD47 binding properties and A "fully humanized" antibody molecule is one that is "fully humanized" in terms of its specificity and / or other desirable characteristics. However, it was found that the RT-PCR was not "optimized" for the purpose.

[0058] The present invention relates to antibodies directed to the amino acid sequences of antibody molecules or antigen-binding portions thereof as defined herein. For example, the present invention encompasses functionally equivalent variable regions and modifications that largely differ in their properties. Antibody molecules that contain intact CDRs and variants with enhanced or decreased activity and / or affinity. and their corresponding antigen-binding portions. Antibodies with the desired binding affinity to a single residue can be obtained. The present invention also includes amino and / or carboxyl terminal fusions ranging in length from 1 to 500 ng / mL polypeptides containing 1 to 500 ng / mL amino acid residues. Insertions including the amino acid residues 1 and 2 are contemplated, as well as intrasequence insertions of single or multiple amino acid residues. Examples include antibody molecules with an N-terminal methionyl residue, or antibody molecules fused to an epitope tag. Other insertional variants of the antibody molecule include those that increase the half-life of the antibody in the blood circulation. Fusion of an enzyme or polypeptide that binds to the N-terminus or C-terminus of the antibody is also included.

[0059] The antibody molecules or antigen-binding portions of the present invention include glycosylated and non-glycosylated polypeptides. , as well as other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. The antibody molecules or antigen-binding portions of the present invention may include polypeptides having mutated Such translation can be achieved, for example, by adding, deleting, or substituting one or more amino acid residues. Post-translational modifications can be altered to create or remove glycosylation sites.

[0060] The antibody molecules or antigen-binding portions of the invention may be modified, e.g., by amino acid substitutions, to modify the Potential proteolytic sites can be removed.

[0061] In an antibody molecule or an antigen-binding portion thereof, HCDR1 has the amino acid sequence: GYT / S / N / RFT / NN / SYYI / VF / V / G (SEQ ID NO: 21) HCDR2 has the amino acid sequence: M / IGV / N / I / DIN / YPV / G / FN / DG / SDTN / R / KF / YN / SPSFQG(distribution HCDR3 may have the amino acid sequence: GGF / H / I / Q / YT / V / IM / T / R / P / A / LD / GY / Q / N / R / S / W / K / A / E / F / H / I / L / M / T / V (SEQ ID NO: 23). Alternatively, using the AHo definition, In an antibody molecule or an antigen-binding portion thereof, HCDR1 has the amino acid sequence: GSG- YT / S / N / RFT / NN / SYY (SEQ ID NO:24); HCD R2 is the amino acid sequence: IN / YPV / G / FN / DG / SDTN / R / KF / YN / SPSFQG (SEQ ID NO: 25); and HCD R3 has the amino acid sequence: GGF / H / I / Q / YT / V / IM / T / R / P / A / LD / G (SEQ ID NO:26).

[0062] For example, HCDR1 has the amino acid sequence: GYT / SFTNYYIF sequence number 27); HCDR2 may have the amino acid sequence: M / IGI / DINP - VN / DGDTN / RF / YN / SPSFQG (SEQ ID NO: 2 8); HCDR3 may have the amino acid sequence: GGF / YTM / PDY / R / K / I (SEQ ID NO: 29). Alternatively, using the AHo definition, HCDR1 can have It may have the amino acid sequence: GSGYT / SFTNYY (SEQ ID NO:30). HCDR2 has the amino acid sequence: INPVN / DGDTN / RF / Y -N / SPSFQG (SEQ ID NO:31); and HCDR3 can have an amino acid sequence similar to that of The polypeptide may have the amino acid sequence: GGF / YTM / PD (SEQ ID NO:32).

[0063] In an antibody molecule or an antigen-binding portion thereof, LCDR1 has the amino acid sequence: RSSQ / HSF / LL / VHSN / Q / AG / AY / N / SN / TYL -H / D (SEQ ID NO:33); LCDR2 can have the amino acid sequence: L / K / MV / G -SN / YRA / F / L / SS (SEQ ID NO:34); LCDR3 may have an amino acid sequence Acid sequence: F / L / M / S / T / VQQ / N / A / T / ST / L / M / IQ / H -T / V / I / A / FP / LR / WT (SEQ ID NO: 35). Using the Ho definition, in an antibody molecule or an antigen-binding portion thereof, LCDR1 has the amino acid sequence Column: SSQ / HSF / LL / VHSN / Q / AG / AY / N / S- LCDR2 can have the amino acid sequence: L / K / MV / GSN / YRA / F / L / SS (SEQ ID NO: 34); and LCD R3 is the amino acid sequence: Q / N / A / T / ST / L / M / IQ / HT / V / I / A / It may have FP / LR / W (sequence number 37).

[0064] For example, LCDR1 has the amino acid sequence: RSSQSLL / VHSN / Q / AGY / NN / TYLH / D (SEQ ID NO: 38); LCDR2 can have It has the amino acid sequence: L / KV / GSN / YRA / F / LS (SEQ ID NO: 39). and LCDR3 has the amino acid sequence: F / SQQ / N / AT / LQ / H -T / VPRT (SEQ ID NO: 40). Alternatively, using the AHo definition, LCDR1 has the amino acid sequence: SSQSLL / VHSN / Q / AGY LCDR2 may have the amino acid sequence: L / KV / GSN / YRA / F / LS (SEQ ID NO: 39); and LCDR 3 has the amino acid sequence: Q / N / AT / LQ / HT / VPR (SEQ ID NO:42). possible.

[0065] In certain embodiments of the invention, antibody sequences are defined using unified CDR definitions. The polypeptide or antigen-binding portion may comprise:

[0066] (a) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGDI NPVNGDTNYSPSFQG (SEQ ID NO: 44) (HCDR2), GGYTPDY (SEQ ID NO: Row number 45) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KGSNRFS (SEQ ID NO: 47) (LCDR2), and SQNLHVPRT (SEQ ID NO: 48) (LCDR3) [Clone D-H3]; or

[0067] (b) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Sequence number 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5]; or

[0068] (c) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), IGDI NPVNGDTNFSPSFQG (SEQ ID NO: 55) (HCDR2), GGYTMDK (SEQ ID NO: Sequence number 56) (HCDR3), RSSQSLVHSNGYTYLH (SEQ ID NO: 57) (L CDR1), KGSYRAS (SEQ ID NO:58) (LCDR2), and SQNTQTPRT (SEQ ID NO:59) (LCDR3) [clone G-B6]; or

[0069] (d) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVNGDTNYNPSFQG (SEQ ID NO: 60) (HCDR2), GGYTMGK (SEQ ID NO: Row number 61) (HCDR3), RSSQSLVHSNGNTYLD (SEQ ID NO: 62) (L CDR1), KGSYRFS (SEQ ID NO: 63) (LCDR2), and SQATHTPRT (SEQ ID NO: 64) (LCDR3) [clone F-E7]; or

[0070] (e) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGFTMDY (SEQ ID NO: Row number 66) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KGSNRAS (SEQ ID NO: 67) (LCDR2), and SQNTHTPRT (SEQ ID NO: 68) (LCDR3) [clone VH-A1 / VL-B1]; or

[0071] (f) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), IGII NPVDGDTRYSPSFQG (sequence number 69) (HCDR2), GGYTMDI (sequence number Row number 70) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), LGSNRFS (SEQ ID NO: 71) (LCDR2), and SQNTQTPRT (SEQ ID NO:59) (LCDR3) [Clone MH]; or

[0072] (g) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (sequence number 65) (HCDR2), GGYTMDI (sequence number Row number 70) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), LGSNRAS (SEQ ID NO: 72) (LCDR2), and SQATQTPRT (SEQ ID NO: 73) (LCDR3) [Clone TTP]; or

[0073] (h) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Sequence number 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.1]; or

[0074] (i) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYNPSFQG (SEQ ID NO: 74) (HCDR2), GGYTMDR (SEQ ID NO: Sequence number 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.2]; or

[0075] (j) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Sequence number 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.3]; or

[0076] (k) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.4]; or

[0077] (l) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.5]; or

[0078] (m) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2), and FQNTQTPRT (SEQ ID NO: 76) (LCDR3) [clone A-D5.6]; or

[0079] (n) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), LGSNRLS (SEQ ID NO: 77) (LCDR2), and FQNTQTPRT (SEQ ID NO:76) (LCDR3) [clone A-D5.7]; or

[0080] (o) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSQGYTYLH (SEQ ID NO: 78) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.8]; or

[0081] (p) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Sequence number 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQQTHTPRT (SEQ ID NO:79) (LCDR3) [clone A-D5.9]; or

[0082] (q) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSQGYTYLH (SEQ ID NO: 78) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQQTHTPRT (SEQ ID NO:79) (LCDR3) [clone A-D5.10]; or

[0083] (r) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.11]; or

[0084] (s) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYNPSFQG (SEQ ID NO: 74) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.12]; or

[0085] (t) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.13]; or

[0086] (u) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSSGYNYLH (SEQ ID NO: 80) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.14]; or

[0087] (v) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSGGYNYLH (SEQ ID NO: 81) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.15]; or

[0088] (w) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSAGYNYLH (SEQ ID NO: 82) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.16]; or

[0089] (x) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSTGYNYLH (SEQ ID NO: 83) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.17]; or

[0090] (y) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSNAYNYLH (SEQ ID NO: 84) (L CDR1), KVSNRLS (SEQ ID NO:53) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.18]; or

[0091] (z) amino acid sequence GYSFTNYYIF (SEQ ID NO: 43) (HCDR1), MGII NPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: Row number 51) (HCDR3), RSSQSLLHSAGYNYLH (SEQ ID NO: 82) (L CDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2), and FQNTHTPRT (SEQ ID NO:54) (LCDR3) [clone A-D5.16-DI]; or

[0092] (z.1) amino acid sequence GYTFTNYYIF (SEQ ID NO: 49) (HCDR1), MG IINPVDGDTNYNPSFQG (SEQ ID NO:50) (HCDR2), GGYTMDR (SEQ ID NO: 51) (HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2), and FQNTHTP RT (SEQ ID NO:54) (LCDR3) [clone A-D5-DI].

[0093] In the above specific embodiment of the invention, the CDRs are instead defined using the AHo CDR definitions. Thus, an antibody molecule or antigen-binding portion can be defined as comprising:

[0094] (a) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV NGDTNYSPSFQG (SEQ ID NO: 87) (HCDR2), GGYTPD (SEQ ID NO: 8 8) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), K GSNRFS (SEQ ID NO: 47) (LCDR2), and NLHVPR (SEQ ID NO: 90) (L CDR3) [clone D-H3]; or

[0095] (b) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), K VSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (L CDR3) [clone A-D5]; or

[0096] (c) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV NGDTNFSPSFQG (SEQ ID NO: 94) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLVHSNGYTY (SEQ ID NO: 95) (LCDR1), K GSYRAS (SEQ ID NO: 58) (LCDR2), and NTQTPR (SEQ ID NO: 96) (L CDR3) [clone G-B6]; or

[0097] (d) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV NGDTNYNPSFQG (SEQ ID NO: 97) (HCDR2), GGYTMG (SEQ ID NO: 9 8) (HCDR3), SSQSLVHSNGNTY (SEQ ID NO: 19) (LCDR1), K GSYRFS (SEQ ID NO: 63) (LCDR2), and ATHTPR (SEQ ID NO: 99) (L CDR3) [clone F-E7]; or

[0098] (e) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGFTMD (SEQ ID NO: 101) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1) , KGSNRAS (SEQ ID NO: 67) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone VH-A1 / VL-B1]; or

[0099] (f) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRFS (SEQ ID NO: 71) (LCDR2), and NTQTPR (SEQ ID NO: 96) ( LCDR3) [clone MH]; or

[0100] (g) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRAS (SEQ ID NO: 72) (LCDR2), and ATQTPR (SEQ ID NO: 102) (LCDR3) [clone TTP]; or

[0101] (h) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), K VSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (L CDR3) [clone A-D5.1]; or

[0102] (i) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) ( LCDR3) [clone A-D5.2]; or

[0103] (j) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) ( LCDR3) [clone A-D5.3]; or

[0104] (k) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), K VSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (L CDR3) [clone A-D5.4]; or

[0105] (l) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), K GSNRLS (SEQ ID NO: 75) (LCDR2), and NTHTPR (SEQ ID NO: 93) (L CDR3) [clone A-D5.5]; or

[0106] (m) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), K GSNRLS (SEQ ID NO: 75) (LCDR2), and NTQTPR (SEQ ID NO: 96) (L CDR3) [clone A-D5.6]; or

[0107] (n) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), L GSNRLS (SEQ ID NO: 77) (LCDR2), and NTQTPR (SEQ ID NO: 96) (L CDR3) [clone A-D5.7]; or

[0108] (o) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) ( LCDR3) [clone A-D5.8]; or

[0109] (p) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), K VSNRLS (SEQ ID NO: 53) (LCDR2), and QTHTPR (SEQ ID NO: 105) ( LCDR3) [clone A-D5.9]; or

[0110] (q) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and QTHTPR (SEQ ID NO: 105) (LCDR3) [clone A-D5.10]; or

[0111] (r) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 1 6) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), K VSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (L CDR3) [clone A-D5.11]; or

[0112] (s) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) ( LCDR3) [clone A-D5.12]; or

[0113] (t) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) ( LCDR3) [clone A-D5.13]; or

[0114] (u) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSSGYNY (SEQ ID NO: 106) (LCDR1) , KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.14]; or

[0115] (v) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSGGYNY (SEQ ID NO: 107) (LCDR1) , KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.15]; or

[0116] (w) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1) , KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.16]; or

[0117] (x) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSTGYNY (SEQ ID NO: 109) (LCDR1) , KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.17]; or

[0118] (y) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNAYNY (SEQ ID NO: 110) (LCDR1) , KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.18]; or

[0119] (z) amino acid sequence GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPV DGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1) , KVSNRFS (SEQ ID NO: 85) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3) [clone A-D5.16-DI]; or

[0120] (z.1) amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), IN PVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: No. 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1) , KVSNRFS (SEQ ID NO: 85) (LCDR2), and NTHTPR (SEQ ID NO: 93) LCDR3) [clone A-D5-DI].

[0121] Antibody molecules or antigen-binding portions (as defined using the AHo definition) of the present invention include: The amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) or GSGYSFTNYY (SEQ ID NO: HCDR1 with no. 86); Amino acid sequence INPVDGDTNYNPSFQG (SEQ ID NO: 91) or INPVDGD HCDR2 having TRYSPSFQG (SEQ ID NO: 100); and HCDR3 having the amino acid sequence GGYTMD (SEQ ID NO: 16), Optionally further comprising: Amino acid sequence SSQSLLHSNGYNY (SEQ ID NO: 89) or SSQSLLHSNG L having YTY (SEQ ID NO: 92) or SSQSLLHSAGYNY (SEQ ID NO: 108) CDR1; The amino acid sequence KVSNRLS (SEQ ID NO: 53) or KVSNRFS (SEQ ID NO: 85) LCDR2 having; and LCDR3 having the amino acid sequence NTHTPR (SEQ ID NO:93). .

[0122] The antibody molecules or antigen-binding portions described above may alternatively be expressed as equivalent unified CD4 antibodies as disclosed herein. It can be defined using the R definition.

[0123] In a particular embodiment of the invention, the antibody molecule or antigen-binding portion is Loan A-D5, Clone A-D5.4, or Clone A-D5.16, or Clone A -D5.16-DI, or clone A-D5-DI, or the CDR sequences of each of these clones It may contain the six CDR sequences in any suitable combination of strings.

[0124] For example, in an antibody molecule or antigen-binding portion defined using the unified CDR definitions, HC DR1 has the amino acid sequence: GYT / SFTNYYIF (SEQ ID NO:27) HCDR2 may have the amino acid sequence: MGIINPVDGDT -N / RYN / SPSFQG (SEQ ID NO: 111); HCDR3 can have the amino acid sequence: GGYTMDR (SEQ ID NO:51); LCDR1 is the amino acid sequence: RSSQSLLHSN / AGYN / TY- LH (SEQ ID NO: 112); LCDR2 may have the amino acid sequence: KVSNR and LCDR3 can have the amino acid sequence: F- QNTHTPRT (SEQ ID NO:54).

[0125] Alternatively, in an antibody molecule or antigen-binding portion defined using the AHo definition, HC DR1 has the amino acid sequence: GSGYT / SFTNYY (SEQ ID NO:30) HCDR2 may have the amino acid sequence: INPVDGDTN / RY -N / SPSFQG (SEQ ID NO: 114); HCDR3 may have the amino acid sequence LCDR1 may have the amino acid sequence: SSQSLLHSN / AGYN / TY (SEQ ID NO: 115) LCDR2 has the amino acid sequence: KVSNRL / FS (SEQ ID NO: 113 ) and LCDR3 can have the amino acid sequence: NTHTPR (SEQ ID NO: 93).

[0126] Antibody molecules or antigen-binding portions as defined herein may, for example, contain glycosylation sites (N-linked or O-linked), deamination sites, phosphorylation sites, or isomerization / fragmentation sites. It may contain one or more substitutions, deletions, and / or insertions that remove post-translational modification (PTM) sites.

[0127] More than 350 types of PTMs are known. The major forms of PTMs include phosphorylation, glycosylation, and Sylation (N- and O-linked), sumoylation, palmitoylation, acetylation, sulfation, myristoylation These include ylation, prenylation, and methylation (of K and R residues). Statistical methods for identifying putative amino acid sites that contribute to a nucleotide sequence are known in the art (Zhou et al., 2016, Nature Protocols 1: 1318-1321), e.g. substitutions, deletions, and / or insertions. and then optionally removing such sites by (a) binding activity and / or (b) Testing (experimental and / or theoretical) of the disappearance of the PTM is contemplated.

[0128] For example, VxP037 mouse LCDR1 (as defined herein, i.e., amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9)) is a nucleotide sequence that is substituted at residues 10 (N) and / or 12 (N). (N) has been identified as a putative deamination site. For example, conservative substitutions (e.g. , S, A, Q, or D, etc.) in the equivalent position of the LCDR1 of the present invention. Deletion of either site is envisaged (e.g. clone A-D5.8). Clones A-D5 and other clones in Tables 3 and 4, or clones A-D5.11 to A-D5.12 in Table 5 -D5.18).

[0129] Similarly, VxP037 mouse LCDR3 (as defined herein, i.e., amino acid sequence The string SQNTHVPRT (SEQ ID NO:11) has a putative deamination site at residue 3 (N). For example, conservative or non-conservative substitutions (e.g., A, S, H, D, T, Deletion of this site at the equivalent position of LCDR3 of the present invention by any of the following amino acids: K, G, E, Q, or R Removal of the clones is envisioned (eg, as in clone F-E7 and other clones in Tables 3 and 4).

[0130] Similarly, VxP037 mouse HCDR3 (as defined herein, i.e., amino acid sequence The string GGYTMDY (SEQ ID NO:5) was identified as having a putative oxidation site at residue 5 (M). For example, conservative or non-conservative substitutions (e.g., P, A, T, S, L, F, W, V, Removal of this site in the equivalent position of the HCDR3 of the present invention by a nucleotide sequence of I, Y or R is conceivable. (e.g., clone D-H3 and other clones in Tables 3 and 4).

[0131] The antibody molecule, or antigen-binding portion thereof, can be human, humanized, or chimeric. An antibody molecule, or an antigen-binding portion thereof, comprises one or more human variable domains into which the CDRs are inserted. The framework scaffold may include: The antibody molecule or antigen-binding portion thereof is an IGHV5 antibody having a corresponding HCDR sequence inserted therein. -51 human germline scaffold. The antibody molecule or antigen-binding portion thereof may comprise an IGKV2 antibody having a corresponding LCDR sequence inserted therein. -28 human germline scaffold.

[0132] An antibody molecule, or an antigen-binding portion thereof, may comprise immunologically inactive constant regions. The antibody molecule or its antigen-binding portion may be a Fab fragment, a F(ab)2 fragment, a Fv fragment, a tetramer, Antibodies, tetravalent antibodies, multispecific antibodies (e.g., bivalent antibodies), single domain antibodies (e.g., shark Antibody [V NAR antibody] or a fragment thereof, or camel antibody [V H H antibody or its fragment), The antibody molecule and its method of construction and use can be, for example, For example, see Holliger & Hudson (2005, Nature Biotechnol. 23(9): 1126-1136). There are.

[0133] In another aspect of the invention, an antibody of the invention as defined herein conjugated to a therapeutic agent. Immunoconjugates comprising the molecule or an antigen-binding portion thereof are provided.

[0134] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, anti-angiogenic agents, and the like. agents, antiproliferative agents, proapoptotic agents, cytostatic and cytolytic enzymes (e.g. RNA Further therapeutic agents include immunomodulatory agents, antiangiogenic agents, antiproliferative agents, or anticancer agents. These drug descriptions include therapeutic nucleic acids, such as genes encoding apoptosis-promoting agents. are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above terms. Cut.

[0135] Examples of therapeutic agents suitable for use in immunoconjugates include taxanes, maytansine, CC-106 5, and duocarmycins, calicheamicins and other enediynes, and auristachios. Other examples include antifolates, vinca alkaloids, and anthracyclines. These include plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioactive Isotopes and photosensitizers can also be used in immunoconjugates. In addition, two types of immunoconjugates, such as liposomes and polymers, can be used. The following carriers can be used as cytotoxic agents to form conjugates. Suitable cytotoxins include These include agents that inhibit or prevent the function and / or cause the destruction of cells. Potential cytotoxins include antibiotics, inhibitors of tubulin polymerization, and enzymes that bind to and destroy DNA. and alkylating agents for protein synthesis or essential cellular proteins (protein kinases, phosphatases, etc.). These drugs include those that disrupt the function of certain enzymes (such as catalyses, topoisomerases, enzymes, and cyclins). .

[0136] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, , idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, Rubicin, Nogalamycin, Menogaril, Pitarubicin, Valrubicin, Cytarabine, Gelatin Mucitabine, trifluridine, ancitabine, enocitabine, azacytidine, doxifur Lysine, pentostatin, broxuridine, capecitabine, cladribine, decitabine , floxuridine, fludarabine, gougerotin, puromycin, tegafur, Azofurin, Adriamycin, Cisplatin, Carboplatin, Cyclophosphamide, Dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, Mechlorethamine, prednisone, procarbazine, methotrexate, fluorouracil , etoposide, taxol, taxol analogues, cisplatin, carboplatin, etc. latin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epinephrine, Rubicin, actinomycin, autramycin, azaserine, bleomycin, tamoxifen Sifen, Idarubicin, Dolastatin / Auristatin, Hemiasterin, Esperama These include isin, and maytansinoids.

[0137] Suitable immunomodulatory agents include antihormonal agents that block hormone action on tumors; Suppresses cytokine production, downregulates self-antigen expression, or masks MHC antigens These include immunosuppressants.

[0138] Nucleic acid molecules encoding the antibody molecules of the invention as defined herein or antigen-binding portions thereof are also provided. Provided. Further provided is a vector comprising a nucleic acid molecule of the invention as defined herein. Also provided is a host cell comprising a nucleic acid molecule or vector of the invention as defined herein.

[0139] In a further embodiment, the expression and / or production of the antibody and / or antigen-binding portion thereof is and culturing the host cells of the present invention under conditions that favor the production of antibodies and / or their antigens from the host cells or culture. Methods for producing anti-CD47 antibodies and / or antigen-binding portions thereof, including isolating the original binding portions. A method is provided.

[0140] In another aspect of the invention, an antibody molecule of the invention as defined herein or an antigen-binding a portion, a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein A pharmaceutical composition comprising the ter is provided.

[0141] An antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or a nucleic acid molecule of the invention as defined herein or An effective amount of a vector of the invention as defined herein or a pharmaceutical composition of the invention as defined herein Further provided is a method of enhancing an immune response in a subject comprising administering

[0142] In a further embodiment, an antibody molecule or an antigen-binding portion thereof of the invention as defined herein. or an immunoconjugate of the invention as defined herein, or a nucleoside of the invention as defined herein. an acid molecule or a vector of the invention as defined herein or a compound of the invention as defined herein A method for treating or preventing cancer in a subject is provided, comprising administering an effective amount of a pharmaceutical composition of the formula: will be done.

[0143] Cancers include, for example, pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, Ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, Selected from the group consisting of salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and cancer of the blood tissue. It can be done.

[0144] The present invention also relates to an antibody molecule of the invention as defined herein for use in the treatment of cancer. or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, Also provided is a pharmaceutical composition of the invention defined as

[0145] In another aspect, the present invention provides an antibody molecule, or an antigen-binding portion thereof, or an immunoglobulin for use. The immunoconjugate, or nucleic acid molecule, or vector may be administered in combination with a second therapeutic agent, e.g., an anti-cancer agent. The therapeutic agents of the invention as defined herein for separate, sequential or simultaneous use in combination with The present invention provides a method of treatment for

[0146] In a further aspect, a compound as defined herein in the manufacture of a medicament for the treatment of cancer. The antibody molecule of the invention or an antigen-binding portion thereof, or the immunoglobulin of the invention as defined herein or a nucleic acid molecule of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein. There is provided the use of a vector or a pharmaceutical composition of the invention as defined herein.

[0147] The present invention also relates to an antibody molecule or an antigen-binding portion thereof as defined herein, or or a nucleic acid molecule as defined herein, The method comprises administering to a subject an effective amount of a vector or a pharmaceutical composition as defined herein. Methods for treating or preventing ischemia-reperfusion injury, autoimmune disease, or inflammatory disease in a subject to provide.

[0148] In all embodiments, ischemia-reperfusion injury is a chronic condition that is characterized by a variety of conditions, including organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, and the like. , pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resection and reconstructive surgery, adnexal or This can occur with reattachment of other body parts, skin grafts, or trauma.

[0149] Autoimmune or inflammatory diseases include arthritis, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, The present invention relates to a method for treating a pulmonary artery disease, the method comprising administering to a patient a therapeutically effective amount of the present invention ... It is possible.

[0150] The present invention also provides a method for treating ischemia-reperfusion injury, autoimmune disease, or inflammatory disease. An antibody molecule or antigen-binding portion thereof as defined herein, or an immunobinding or a nucleic acid molecule as defined herein, or a vector as defined herein, Pharmaceutical compositions as defined herein are also provided.

[0151] Furthermore, the present invention is useful in the manufacture of a medicament for the treatment of ischemia-reperfusion injury, autoimmune disease, or inflammatory disease. an antibody molecule or an antigen-binding portion thereof as defined herein, or an immunoglobulin as defined herein An immunoconjugate, or a nucleic acid molecule as defined herein, or a vector as defined herein, The use of the pharmaceutical composition as defined herein is provided.

[0152] The present invention also relates to an antibody molecule as defined herein or an antigen-binding portion thereof, An immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, administering to a subject an effective amount of a vector or a pharmaceutical composition as defined herein. The present invention provides a method for treating or preventing a cardiovascular or fibrotic disease.

[0153] Also, an antibody as defined herein for use in the treatment of a cardiovascular disease or a fibrotic disease. A molecule or an antigen-binding portion thereof, or an immunoconjugate as defined herein, or or a vector as defined herein, or a pharmaceutical composition as defined herein. Compositions are also provided.

[0154] Further, the use of a compound as defined herein in the manufacture of a medicament for the treatment of a cardiovascular disease or a fibrotic disease. or an immunoconjugate as defined herein, or A nucleic acid molecule as defined herein, or a vector as defined herein, or Use of the pharmaceutical composition is provided.

[0155] In any aspect of the invention, the cardiovascular disease may be, for example, coronary heart disease or atherosclerotic heart disease. It may be arteriosclerosis.

[0156] In any embodiment of the present invention, the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, or the like. The disease may be selected from the group consisting of bronchitis, asthma, cystic fibrosis, and bronchitis.

[0157] The pharmaceutical composition of the present invention may include a pharma- ceutically acceptable excipient. The vehicle does not induce secondary reactions and, for example, facilitates administration of the anti-CD47 antibody molecule over its lifetime. and / or availability in the body, or increased solubility in solution. The compound or combination of compounds that go into the composition may be used. Vehicles are known and can be adapted by the person skilled in the art as a function of the mode of administration of the anti-CD47 antibody molecule. It will be.

[0158] In some embodiments, the anti-CD47 antibody molecule is provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules may be reconstituted in sterile water and administered to an individual. It may be mixed with saline prior to administration.

[0159] The anti-CD47 antibody molecule is typically administered in the form of a pharmaceutical composition, which contains at least one other component in addition to the antibody molecule. That is, the pharmaceutical composition may comprise, in addition to the anti-CD47 antibody molecule, a pharmaceutical The composition may include any suitable excipients, carriers, buffer substances, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the anti-CD47 antibody molecule. The exact nature of the or other substance will depend on the route of administration, which may include, but is not limited to, the administration of bovine serum albumin (BSA), as discussed below. The administration may be by way of a flow, infusion, injection, or other suitable route.

[0160] For parenteral administration, e.g., by injection, e.g., subcutaneous or intravenous, the anti-CD47 antibody molecule can be administered The pharmaceutical composition is a parenteral formulation that is pyrogen-free and has suitable pH, isotonicity, and stability. The pharmaceutical composition may be in the form of a physiologically acceptable aqueous solution. Prepare appropriate solutions using isotonic vehicles such as Gel Injection, Lactated Ringer's Injection, etc. It is quite possible to add preservatives, stabilizers, buffers, antioxidants, and and / or other additives may be used, such as buffers, e.g., phosphates, citrates, etc. Acid salts, other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (chlorides, etc.) Octadecyldimethylbenzylammonium, Hexamethonium chloride, Benzalkonium chloride Benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl phenyl Parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexyl 3'-pentanol; and m-cresol; low molecular weight polypeptides; proteins Proteins, e.g., serum albumin, gelatin, immunoglobulins; hydrophilic polymers, e.g., poly Vinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine , arginine, or lysine; monosaccharides, including glucose, mannose, or dextrin; Sugars and other carbohydrates; chelating agents such as EDTA; sugars, e.g. mannitol, trehalose, etc. Halo, sorbitol; salt-forming counterions, e.g. sodium; metal complexes, e.g. Zn -protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLU RONICS (registered trademark), or polyethylene glycol (PEG), etc.

[0161] Pharmaceutical compositions comprising anti-CD47 antibody molecules may be used alone or in combination with other treatments. They can be administered simultaneously or sequentially depending on the condition being treated.

[0162] The anti-CD47 antibody molecules described herein can be used for prophylactic or preventative treatment (e.g., treatment of to reduce the risk of developing symptoms, delay their onset, or reduce their severity after onset treatment of the human or animal body, including prior to the onset of symptoms in an individual, to reduce the severity of The therapeutic method may be used by administering an anti-CD47 antibody molecule to an individual in need thereof. The method may include:

[0163] Administration is typically in a "therapeutically effective amount," which is sufficient to show benefit to a patient. The beneficial benefit may be at least an amelioration of at least one symptom. The rate and time course of administration will depend on the nature and severity of the subject being treated, the particular mammal being treated, and the individual The clinical condition of the patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the schedule of administration, and The prescription of treatment, e.g., dosage, etc., will depend on other factors known to the medical practitioner. It is within the responsibility of the medical practitioner and other physicians to determine the severity of the symptoms and / or the progression of the disease being treated. Appropriate doses of antibody molecules are known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antib ody,Immunoconjugates and Radiopharmaceuticals 4: 915-922). The specific dosage is Depending on the type of medication being administered, the Physician's The therapeutically effective amount or appropriate amount of an antibody molecule may be appropriately determined. The appropriate dose can be determined by comparing its in vitro and in vivo activity in animal models. Methods are known for extrapolating effective doses from mice and other test animals to humans. The exact dose will depend on many factors, including whether the antibody is intended for prophylaxis or treatment, the location of the area to be treated, and the dosage. The size and location, the exact nature of the antibody (whole antibody, fragment, etc.), and the detectable It depends on the nature of the label or other molecule.

[0164] Typical antibody doses range from 100 μg to 1 g for systemic administration and 100 μg to 1 g for local administration. Range: 1 μg to 1 mg. An initial higher loading dose is followed by one or more lower doses. Typically, the antibody is a whole antibody, e.g., an IgG1 or IgG4 isotype. This is a single treatment dose for adult patients, adjusted proportionally for children and infants. Other antibody forms may be administered in proportion to their molecular weight. Treatment may be administered daily, twice weekly, or at the discretion of the physician. The treatment schedule for an individual may be repeated at weekly or monthly intervals. The dosage will depend on the pharmacokinetic and pharmacodynamic properties of the compound, the route of administration, and the nature of the condition being treated.

[0165] The treatment may be periodic, with the period between administrations being about 2 weeks or more, for example about 3 weeks or more, For example, the treatment may be once every 4 weeks or more, once every month or more, once every 5 weeks or more, or once every 6 weeks or more. Treatment may be every 2-4 weeks or every 4-8 weeks. Treatment may be administered before and / or after surgery. and / or may be administered or applied directly to the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

[0166] In some embodiments, the anti-CD47 antibody molecules described herein are administered by subcutaneous injection. Subcutaneous injections can be administered, for example, using an autoinjector for long-term prophylaxis / treatment. It can be administered.

[0167] In some preferred embodiments, the therapeutic effect of the anti-CD47 antibody molecule is dose-dependent. For example, the therapeutic efficacy of a single administration of an anti-CD47 antibody molecule may last for several half-lives. The results are as follows: 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or The symptoms may last for six or more months.

[0168] The present invention also relates to human CD47, and optionally cynomolgus monkey CD47, and / or macaque CD47. The present invention provides a method for producing an antibody molecule, or an antigen-binding portion thereof, that specifically binds to mouse CD47. and the method comprises the steps of: (1) Grafting anti-CD47 CDRs of non-human origin onto a human v-domain framework producing a humanized anti-CD47 antibody molecule, or an antigen-binding portion thereof; (2) A humanized anti-CD47 antibody molecule or its antigen-binding portion comprising one or more mutations in the CDRs. generating a phage library of individual clones; (3) human CD47, and optionally cynomolgus monkey CD47, and / or mouse CD screening the phage library for binding to 47; (4) human CD47, and optionally cynomolgus monkey CD47, and / or mouse CD A step of selecting a clone having binding specificity to 47 from the screening step (3). Process; and (5) Extracting human CD47 and, optionally, crab CD47 from the clones selected from step (4). An antibody molecule that specifically binds to monkey CD47 and / or mouse CD47, or an antibody thereof Producing the original binding moiety.

[0169] This method is based on the clones selected in step (4), for example, Based on further exploratory mutagenesis at specific positions in the CDRs of the clones identified, additional Generate clones to enhance humanization and minimize human T cell epitope content, and / or or improving the manufacturing properties of the antibody molecule or antigen-binding portion thereof produced in step (5). It may further include:

[0170] This method comprises the steps of: The immunogenicity of one or more v-domains is evaluated, e.g., by determining whether an appropriate Optionally, a further step of generating one or more additional mutations to reduce immunogenicity. Immunogenicity can be assessed, for example, using the in silico techniques described herein. This can be assessed by localizing T cell epitopes.

[0171] Refinement that can be applied to the above method is described in Example 1 below.

[0172] As used herein, the term "CD47" refers to a compound that possesses at least one of the biological activities of CD47. As used herein, the term "IAP" refers to integrin-associated proteins (IAPs) and variants thereof that retain the IAP domain. When used, CD47 is expressed in all mammalian cells, including humans, rats, mice, and chickens. The term "CD47" includes variants of human CD47, such as IgE, IL-1, and IgE-1. The term is used to include all species, species, and species homologs. The antibodies of the present invention may be used in non-human species. Cross-reacts with CD47 from human leukocytes, especially CD47 from cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the antibody is fully specific for human CD47. may exhibit no non-human cross-reactivity.

[0173] As used herein, the terms "antagonist" or "antibody" used in the context of the antibodies of the invention are used interchangeably. Anti-CD47 antagonist antibodies (interchangeably referred to as "anti-CD47 antibodies") are antibodies that bind to CD4 7 and the biological activity of CD47 mediated by CD47 signaling and / or downstream pathways. CD47 signaling, such as receptor binding and / or induction of cellular responses to CD47, is involved in the induction of Block, antagonize, inhibit, or reduce (substantially) the biological activity of CD47, including downstream pathways mediated by CD47. For the purposes of the present invention, the term "anti-CD47 antibody" includes antibodies that can The term "agonist antibody" refers to an antibody that inhibits CD47 itself and the biological activity of CD47 (including but not limited to). (ability to enhance activation of phagocytosis by cells of the myeloid lineage, but not to inhibit the proliferation of the endothelial cells) or activity or biological activity Any term, heading, or other term or effect that substantially nullifies, reduces, or neutralizes to any significant degree and functional status and characteristics.

[0174] CD47 binds with higher affinity, avidity, and more readily and efficiently than other receptors. and / or for a longer period of time, they “specifically interact” with CD47, "preferentially bind," "bind," or "interact."

[0175] An "antibody molecule" refers to at least one antigen-recognizing polypeptide located in the variable region of an immunoglobulin molecule. Target specificity for carbohydrates, polynucleotides, lipids, polypeptides, etc. via the site As used herein, the term "antibody molecule" refers to an immunoglobulin molecule capable of binding to a , native polyclonal or monoclonal antibodies, as well as any antigen-binding fragments thereof. (e.g., "antigen-binding portion") or single chains thereof, fusion proteins including antibodies, and, particularly limited Although not limited to these, scFv, single domain antibodies (e.g. shark antibodies [V NAR Antibody]Also and its fragments, and camelid antibodies [V H H antibody or its fragment), maxibody, minibody , intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv and other modified configurations of immunoglobulin molecules that contain antigen recognition sites, including

[0176] An "antibody molecule" refers to any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof). The antibody does not have to be of a particular class, including antibodies of any class. Depending on the amino acid sequence, immunoglobulins can be assigned to different classes. There are five major classes of globulins: IgA, IgD, IgE, IgG, and IgM. Some of these are further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, They may be classified as IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins The location is well known.

[0177] As used herein, the term "antigen-binding portion" of an antibody molecule refers to an antibody that specifically binds to CD47. It refers to one or more fragments of an intact antibody that retain the ability to bind antigen. can be carried out by fragments of an intact antibody. The term "antigen-binding portion" of an antibody molecule Examples of binding fragments included in - ;Fab' - F(ab')2; VH and CH1 domain an Fd fragment consisting of the VL and VH domains of a single arm of an antibody; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; Includes single domain antibody (dAb) fragments and isolated complementarity determining regions (CDRs) .

[0178] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Immunoglobulin heavy chain Although the boundaries of the Fc region of a human IgG heavy chain can vary, the Fc region of a human IgG heavy chain typically consists of the Cys The stretch from amino acid residue 226 or Pro230 to its carboxyl terminus The numbering of residues in the Fc region is based on the EU index numbering of Kabat. The Fc region of a globulin generally comprises two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in a dimeric or monomeric form.

[0179] A "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are and are connected by three complementarity determining regions (CDRs), also known as hypervariable regions. It consists of four framework regions (FR) and contributes to the formation of the antigen-binding site of an antibody. For example, when selecting FRs flanking the CDRs in humanizing or optimizing an antibody, the same standard FRs from an antibody containing the CDR sequences of the class are preferred.

[0180] The definition of CDRs used in this application is based on immunoglobulin repertoire analysis and isolated Based on a combination of structural analyses of antibodies and their cocrystals with antigens, It combines domains used in many different and often conflicting schemes created by (Swindells et al., 2016, abYsis: Integrated Antibody Sequencing and Structure-Management,Analysis, and Prediction. J Mol Biol. [PMID: 27561707; Ep The definition of CDR used here ("Harmonized The "definition" incorporates the lessons of all such previous insights and defines a novel class of target-binding complementarity mediating Contains all the relevant loop positions needed to find the complete possible residue context. .

[0181] Table 1 shows the CDRs defined herein compared to known alternative systems for defining the same CDRs. The amino acid sequences of the VxP037 mouse anti-CD47 antibody CDRs are shown in the "unified" scheme. show.

[0182] As used herein, the term "conservative substitution" refers to replacing an amino acid with a non-functional amino acid. A conservative substitution refers to a substitution of an amino acid with another amino acid that does not significantly or adversely change the amino acid sequence. A suitable example is a sequence that substitutes one amino acid with a value >= 0 in the BLOSUM62 substitution matrix: The first step is to replace the amino acid with another amino acid (Henikoff & Henikoff, 1992, PNAS 89: 10915-10 (see 919).

[0183] ARNDCQEGHILKMFPSTWYV A 4 -1 -2 -2 0-1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 R -1 5 0 -2 -3 1 0-2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3 N -2 0 6 1 -3 0 0 0 1-3 -3 0 -2 -3 -2 1 0 -4 -2 -3 D -2 -2 1 6 -3 0 2-1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 C 0 -3 -3 -3 9-3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 Q -1 1 0 0 -3 5 2-2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2 E -1 0 0 2 -4 2 5-2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2 G 0 -2 0 -1 -3-2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 H -2 0 1 -1 -3 0 0-2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3 I -1 -3 -3 -3-1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 L -1 -2 -3 -4-1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1 K -1 2 0 -1 -3 1 1-2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 M -1 -1 -2 -3-1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1 F -2 -3 -3 -3-2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1 P -1 -2 -2 -1-3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2 S 1 -1 1 0 -1 0 0 0-1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2 T 0 -1 0 -1 -1-1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0 W -3 -3 -4 -4-2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3 Y -2 -2 -2 -3-2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 V 0 -3 -3 -3 -1-2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4.

[0184] The term "monoclonal antibody" (Mab) refers to any antibody, e.g., eukaryotic, prokaryotic, or fungal. Antibodies or antigen-binding portions thereof derived from a single copy or clone, including a phage clone. Preferably, the monoclonal antibodies of the present invention are homogeneous. The chromosomes are present in a uniform or substantially uniform population.

[0185] "Humanized" antibody molecules are chimeric immunoglobulin molecules that contain minimal sequence derived from non-human immunoglobulin. Immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F( ab')2, or other antigen-binding subsequence of an antibody, Humanized antibodies refer to forms of the polypeptide or antigen-binding portion thereof in which the CDR residues of the receptor are not modified in the desired manner. CDRs of non-human species such as mouse, rat, or rabbit with specificity, affinity, and capacity It may be a human immunoglobulin (recipient antibody) in which the residues of stomach.

[0186] "Human antibody or fully human antibody" refers to a transgenic mouse carrying a human antibody gene. It refers to an antibody molecule or antigen-binding portion thereof derived from mouse or human cells.

[0187] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another. The term refers to an antibody molecule or antigen-binding portion thereof derived from a species, e.g., a mouse antibody having variable region sequences and the constant region sequences are derived from a human antibody.

[0188] "Antibody-drug conjugates" and "immunoconjugates" refer to compounds that bind to CD47 and are capable of inhibiting cytotoxic and cytotoxic effects. Antibody molecules or their antigen binding sites, including antibody derivatives conjugated to antiproliferative and / or therapeutic agents. Refers to the joint part.

[0189] The antibody molecules of the present invention, or antigen-binding portions thereof, can be produced by techniques known in the art, e.g. recombinant techniques. The method may be carried out using techniques such as phage display techniques, synthetic techniques, or other techniques readily available in the art. It can also be produced using a combination of other known techniques.

[0190] The term "epitope" refers to a region of an antibody molecule that is present in one or more of the antigen-binding regions of the antibody molecule. or the portion of a molecule that can be recognized and bound by an antigen-binding portion thereof. A pitope consists of a defined region of primary, secondary, or tertiary protein structure and is the target of an antibody. The secondary structural unit or structural domain of the target recognized by the original binding region or its antigen-binding portion. Similarly, epitopes include defined sequences of molecules such as amino acids or sugar side chains. Consists of chemically active surface groups with specific three-dimensional structural characteristics and specific charge properties As used herein, the term "antigenic epitope" is understood in the art to mean Any method known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography, may be used. The antibody molecule has specificity as determined by structural analysis or related structural determination methods (e.g., NMR). is defined as a portion of a polypeptide that can specifically bind to a polypeptide.

[0191] The term "binding affinity" or "KD" refers to the dissociation rate of a particular antigen-antibody interaction. The KD is the dissociation rate (off rate (k off ) and the association rate or "on Rate (k on )" That is, KD is the ratio of k off / k on is equal to molar concentration (M) The smaller the KD, the stronger the binding affinity. Therefore, a KD of 1 μM D is 1n K of M D The KD values ​​of antibodies are well established in the art. One method for determining the KD value of an antibody is to measure the surface Surface plasmon resonance (SPR), typically a biosensor system such as a Biacore® system The first step is to use a stem.

[0192] The term "potency" is a measure of biological activity and is expressed as IC 50 or a CD as described herein. Antibodies against the antigen CD47 that inhibit 50% of the activity as measured by the CD47 activity assay or It can be specified as the effective concentration of the antibody drug conjugate.

[0193] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to a compound that is capable of producing a desired therapeutic result. An effective amount refers to the amount necessary (at a dosage and for a period of time and by a means of administration) to achieve the desired therapeutic effect in a subject. At least the minimum amount of an active agent required to provide a therapeutic benefit, but less than a toxic amount .

[0194] "Inhibit" or "neutralize" as used herein in reference to a biological activity of an antibody molecule of the present invention. The term "antibody" refers to, for example, but not limited to, the production of antibody molecules against CD47. The antibody substantially antagonizes or inhibits the progression or severity of the disease, including the biological activity or binding interaction. "Destructive" means the ability to prevent, inhibit, slow down, destroy, eliminate, stop, reduce, or reverse.

[0195] A "host cell" refers to a cell that is a recipient of a vector for integration of a polynucleotide insert. The term "host cell" includes an individual cell or cell culture that may be a recipient or has been a recipient. A cell contains the progeny of a single host cell, and the progeny may vary due to natural, accidental, or deliberate mutations. Therefore, they may not be completely identical to the original parent cell (in terms of morphology or genomic DNA complement). Host cells can be transfected in vivo with a polynucleotide of the invention. The present invention includes cells that have been cultured.

[0196] As used herein, a "vector" refers to a vector that conveys one or more genes or sequences of interest into a host cell. Vectors refer to constructs capable of delivering and preferably expressing a gene sequence. Examples of vectors include, but are not limited to, including, but not limited to, viral vectors, naked DNA or RNA expression vectors, Plasmid, cosmid, or phage vector, DNA associated with cationic condensation agent or RNA expression vectors, liposome-encapsulated DNA or RNA expression vectors, and promoters These include certain eukaryotic cells such as inducer cells.

[0197] As used herein, the term "treat" refers to, unless otherwise specified, Reversal of the disorder or condition to which the term applies, or of one or more symptoms of such disorder or condition As used herein, the term "prostate cancer" refers to the prevention, amelioration, inhibition of progression, slowing of progression, delaying of onset, or prevention. The term "treatment" as used herein, unless otherwise specified, refers to treatment as defined above. The term "treat" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, references herein to "treatment" include curative, palliative and This includes preventive and prophylactic treatments.

[0198] Whenever an embodiment is described herein with the phrase "comprising," it is understood that the phrase "consists of" is also intended to be used interchangeably. and / or similar embodiments described by the term "consisting essentially of" are also provided. do.

[0199] Aspects or embodiments of the invention relate to Markush groups or other groups in the alternative. When described in a group, the invention includes the entire group and not just the entire group listed as a whole. This includes each member of the main group and all possible subgroups of the main group, but does not include any subgroup of the main group. The present invention also includes the main group in which one or more of the sub-members are absent. The present application envisages the explicit exclusion of any one or more of the group members of the invention being claimed.

[0200] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present invention. The meaning of the term "incompatible" is the same as that commonly understood by a person skilled in the art to which the invention pertains. In all cases, the present specification, including definitions, will control. "comprises" or "comprising", etc. A variation of is meant to include the stated integer or group of integers but not any other integer or group of integers. It is understood that no exclusion of groups is meant. Numerous terms shall include the plural and plural terms shall include the singular. Examples following the words "eg" or "for example" are not exhaustive or exclusive. It is not intended to be.

[0201] The practice of the present invention may involve, unless otherwise indicated, techniques such as molecular biology (including recombinant techniques), microbiology, , using conventional techniques of cell biology, biochemistry, and immunology, which are within the skill of the art. do. EXAMPLES

[0202] Specific non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings.

[0203] Example 1 Generation and characterization of optimized anti-CD47 therapeutic antibodies Introduction In this example, we successfully generated a panel of competitive and optimized anti-CD47 antibodies. These anti-CD47 antibodies are well expressed, biophysically stable, highly soluble, and suitable for It has the greatest identity to the human germline.

[0204] Materials and Methods IgG cloning, transient expression and purification The DNA sequence encoding the antibody v domain is isolated from another plasmid via restriction-ligation cloning. The antibodies were cloned into separate IgG heavy and light chain expression cassettes in a plasmid vector. Two forms of human IgG were expressed: IgG4 with the hinge-stabilizing S228P mutation IgG4 with and lower hinge mutations that minimize Fcγ receptor-driven effector functions IgG1 Null-IgG1 with L234A / L235A / G237A. Manufacturer's profile Transient incubation with endotoxin-free IgG-expressing plasmid preparations according to protocol. After transfection, IgG was expressed in HEK-293expi cells. IgG was purified using a one-step protocol: 1, pre-equilibrated with PBS (pH 7.4). The conditioned medium was added (undiluted) to a 5 ml ProA Sepharose column. The column was then loaded with 5 column volumes. After washing with a volume of PBS (pH 7.4), the protein was diluted with 100 mM glycine (pH 2.7 ) and dialyzed against PBS (pH 7.4) using a dialysis membrane with a 30 kDa cutoff. .

[0205] IgG titration binding ELISA To coat the GreinerBio-One High bind ELISA plates, use target proteins. Proteins were diluted to 1 μg / ml in carbonate buffer and added at 100 μl per well overnight at 4°C. The coated plate was washed three times with PBS (pH 7.4) and then diluted with PBS (380 After blocking with 1% BSA in PBS (µl / well) for 1 h at room temperature, the plates were incubated in PBS-Tween 20 ( The plates were washed three times with PBST. Then, CD47 antibody (100 μl / well; diluted in PBST) was added. The plate was then washed three times with PBST and incubated at room temperature for 1 hour. Goat anti-human kappa chain-HRP was added (100 μl / well) for 1 hour at room temperature. Wash the plates three times with PBST and twice with PBS, then add 100 μl of TMB per well. The reaction was stopped by adding 100 μl of 2M H2SO4 / well, and the plates were The OD was read at 450 nm using a thermometer reader.

[0206] Anti-CD47 antibodies were tested for polyreactivity by ELISA. Antigens and non-target antigens were plated on a 96-well Nunc maxisorp plate at 100 ng / well in carbonate buffer. Plates were then coated with PBS overnight at 4°C. The plates were then washed three times with PBS and diluted with 100 mM NaCl in PBS. After blocking with 100% BSA, the sections were washed three times with PBS-Tween 20. A series of dilutions was applied, the plates were washed three times with PBS-Tween 20, and then goat anti-human kappa Secondary antibody, ribonuclease-HRP (1:4,000), was applied. The wells were then washed with PBS-Tween 2 Wash 3 times with 0 and 2 times with PBS and add 100 μl of TMB peroxidase substrate per well. The reaction was stopped by adding 100 μl of 2M H2SO4, and the absorbance was adjusted to 45 IgG binding analysis by ELISA on negatively charged biomolecular surfaces was performed as follows: This was performed as previously described (see Mouquet et al., 2010, Nature 467: 591-595). .

[0207] CD47 library generation and selection The CD47 scFv repertoire was assembled by bulk oligo synthesis and PCR. The amplified scFv repertoire was then cloned into a phagemid vector by restriction-ligation. The phage repertoire was essentially as previously described in detail. Rescue was performed as previously described (Finlay et al., 2011, Methods Mol Biol 681: 383 -401). Streptavidin magnetic microbeads were loaded with CD47-Fc protein (human or cytoplasmic Coat the beads with 5% skim milk protein (Isal), wash the beads three times with PBS, and Phage selection was performed by resuspending the cells in PBS (pH 7.4) (MPBS) containing These beads were coated with 200 nM of target protein in round 1 of selection. In subsequent rounds, the antibodies were coated at 100, 50, and 10 nM.

[0208] CD47-SIRPα binding competition assay A competitive ELISA assay was performed to determine the optimal binding site for blocking the binding interaction between CD47 and SIRPα. Greiner Bio-One High bind ELISA was established to examine the potency of the purified leads. To coat the plates, add 10 μg / ml human S in carbonate coating buffer. IRPα-Fc was added at 100 μl per well overnight at 4°C. Wash the plates three times with PBS (pH 7.4) and add 1% BSA in PBS (380 μl / well). After blocking with PBST (PBSCl) for 1 hour at room temperature, the cells were washed three times with PBST. Next, biotinylated human, mouse, or cynomolgus IgG were incubated with or without the addition of competitor IgG. CD47-Fc was added at 0.2 μg / ml in PBS at 100 μl / well for 60 min at room temperature. The plate was then washed three times with PBST and incubated with streptavidin-HRP at room temperature. The plates were then washed three times with PBST and twice with PBS. After washing twice, 100 μl of TMB was added per well. The reaction was stopped by adding O4 / well and the OD measured at 450 nm on a plate reader. I read it.

[0209] Antibody v-domain T cell epitope content: an in silico analysis In silico techniques based on identifying the location of T cell epitopes in therapeutic antibodies and proteins ( The potential immunogenicity of antibody v-domains was assessed using iTope (registered trademark). We have used the ELISA kit (registered trademark) to identify peptides that bind promiscuous high affinity to human MHC class II. The VL and VH sequences of the primary lead were analyzed. Promiscuous high affinity MHC class II binding The peptides are intended to identify T cell epitopes, which are high-risk indicators of clinical immunogenicity of drug proteins. iTope® software is a peptide amine. The acid side chains and specific sites within the open-ended binding groove of 34 human MHC class II alleles Favourable interactions with the binding pocket (specific pocket positions: p1, p4, p6, p7, and p9) These alleles are those that are most commonly found in a particular ethnic group. Represents the most common HLA-DR alleles found worldwide, without weighting due to Twenty alleles contain an "open" p1 configuration and 14 alleles contain a p1 region at position 8 It contains a "closed" conformation in which glycine at position 3 is replaced by valine. Residue positions are determined by 8 amino acid overlapping 9-mer peptides spanning the test protein sequence. This process is accomplished by the in silico generation of It distinguishes between peptides that bind and those that do not bind with high accuracy.

[0210] Furthermore, in vitro human T cell epitope mapping analysis of other protein sequences has demonstrated For matches with previously identified T cell epitopes, TECD® (T Cell Epitope Sequences were analyzed using the TECD Database® search. Large-scale (>10,000 peptides) database of peptides derived from protein and antibody sequences It is used to search any test sequence against the database.

[0211] Cancer cell phagocytosis analysis Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation. CD14+ PBMCs were isolated by magnetic cell separation using CD14 microbeads. In parallel, green CFSE (carboxyfluorescein diacetate, succinimidyl esterase) was A total of 1.25 × 10 HL-60 cells were labeled using a cellular tracer dye. 6 Labeled HL-60 cells were incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. The cells were preincubated in a well plate in the presence of anti-CD47 antibody. After that, 5×10 5 CD14 positive cells were added to each well and incubated for another 1 h under the same culture conditions. The cells were then incubated for 2 h. The cells were then harvested by vigorous pipetting and resuspended in ice-cold 4% paraformaldehyde. Fix with aldehyde for 10 min and incubate with Fc receptor binding inhibitor monoclonal antibody for 10 min. Following the blocking step, cells were stained with Alexa Fluor 647 (AF647). Incubate with conjugated anti-human CD14 antibody for 30 min at room temperature and then resuspend in 4% paraformaldehyde. The tissue was then fixed in aldehyde for an additional 5 min.

[0212] Cells were analyzed for side and forward scatter properties along with CFSE and AF647 fluorescence intensity data. Analyze with a BD Fortessa flow cytometer to record at least 1 x 10 4 A Data was captured until an F647 positive event was recorded. After data was acquired, Analyses were performed using FlowJo software (version 10.4.2). Cell debris was gated out by scatter properties (SSC area vs. FSC area). Single cells were also , gated by SSC area vs. SSC height, then by FSC area vs. FSC height. From the remaining single cell population, CFSE and CD14 double positive cells were isolated from vehicle-treated controls. Gating was performed using quadrant gates positioned based on the population of CD14 positive cells in the experiment. GraphPadPrism software (version 7.0a) was used to measure CD14-positive The percentage of CFSE positive cells from the sex population was calculated and plotted.

[0213] Results and Discussion CDR grafting into suitable human germline v genes Using CDR grafting, we generated the antagonistic murine anti-CD47 IgG VP037 (mVH / mV L; see WO 2014 / 093678 and Table 2) were first synthesized from human germline The immunoglobulin v-domain framework sequence scaffold was engineered to provide optimal drug-like properties. To focus our engineering efforts on a final lead therapeutic IgG compound with , known to have good solubility and are highly frequent in the expressed human antibody repertoire. The "preferred" germline scaffolds IGHV5-51 and IGKV2-28 used in We chose to graft the CDRs of the parent antibody.

[0214] These scaffolds and the grafted CDR definitions are outlined in Table 2. Heavy chain of mouse anti-CD47 antibody The light and light chain sequences are also shown in Table 2. This process of CDR grafting is well known, but specific A set of human v-domain sequences are provided as acceptor frameworks suitable for non-human CDR grafting. There are still problems predicting whether it will work. Using the wrong framework This can result in loss of target binding function, protein stability issues, or ultimately impaired expression of IgG. Therefore, the IGHV5-51 / IGKV2-28 graft is This was carried forward as a template for mutagenesis and selection of improved clones.

[0215] Library generation and screening The CDR-grafted IGHV5-51 / IGKV2-28 v-domain sequence is VL-VH s The mutagenesis library was then synthesized by bulk oligo synthesis and assembly in a cFv format. The cassettes were generated. The final scFv library was then transferred to a phage display vector. Ligated and transformed into E. coli via electroporation to obtain 1.3 x 10 9 1 independent claw The quality of the library build was verified by sequencing 96 clones. The sequencing data consisted of either mouse or human germline residues at each dispersed position. The results showed that the coding positions were effectively sampled at a frequency of about 50%. The clones were rescued using helper phage M13 and isolated into three separate branches, A, B, C, D, E ... Selected with biotinylated human, mouse, and cynomolgus CD47-Fc proteins in B and C was carried out.

[0216] Post-selection screening (Figure 1) and DNA sequencing confirmed the human content within the CDRs. Significantly increased abundance of 854 unique human and mouse CD47-binding scFv clones Although the presence of these genes was revealed, the framework sequences remained completely germline. Among 854 clones, germline mutations were observed in all CDRs (Table 3). The lead clone was assayed using ELISA for binding to both human and mouse CD47-Fc. The sequences were ranked based on the level of CDR germline to signal (Figure 1). The v-domains of the top four ranked clones and the two most humanized observed heavy chains and The fifth clone ("VH-A1 / VL-B1"), which combined the V and light chain v domains, These were then subcloned into an IgG expression vector for further studies as follows (Table 4).

[0217] All CDRs of lead clones derived directly from library selection were germline Although mutations were observed, sequence analysis may allow further design of clones to maximize humanization. Thus, 854 binding signals for human and mouse proteins remained. Using sequence-specific hits, the CDRs of this functionally characterized population were identified in mice The amino acid retention frequency was analyzed. The positional amino acid retention frequency was V H and V L Found in domain The RFs were expressed as the percentage of residues that were identified by a series of combinatorial analyses (Figure 2A and B). In the context of natorial design, the target-binding paratope may not be essential and may be a germline was considered a position that was likely to be open to

[0218] Designs containing only mouse residues with RF>75% were designated "MH" (MH = maximally humanized). Another design combines the five most humanized CDRs observed in the population, which we named . A number of clones ("TTP" = Theoretical Possible Total) were also produced. The clones were generated by gene synthesis (the four library clones above and the positive pair (along with control mVH / mVL and negative control isotype-matched non-CD47 reactive v-domain) The human expression vectors were cloned for production as IgG1 null and IgG4(S228P). All IgGs were cloned by transient transfection of HEK-293 cells. It was easily expressed and purified from

[0219] Lead IgG specificity and potency characteristics The purified IgG was tested against human, mouse, and cynomolgus monkeys in a direct titer ELISA format. We then tested the binding of the clone to CD47-Fc (Figure 3). Lone MH, A-D5, and D-H3 bind to all three orthologs of CD47 Two clones (G-B6 and F-E7) retained the affinity to mouse CD 47, and one clone (VH-A1 / VL-B1) showed reduced binding to both human and mouse The antibody maintained equivalent binding to both human and mouse CD47 but lost cross-reactivity with mouse CD47. However, one clone (TTP) showed almost complete loss of binding function.

[0220] In the CD47-SIRPα blocking assay (Figure 4), A-D5, G-B6, F-E7, and and D-H3 all inhibited SIRPα- showed concentration-dependent inhibition of human, mouse, and cynomolgus CD47 interactions with Fc In particular, VH-A1 / VL-B1 IgG1 potently inhibited human and cynomolgus CD47. Interestingly, clone MH showed inhibition of E Although LISA showed binding to all three orthologues of CD47, Clone TTP was also negative in all inhibition assays. It was.

[0221] Ensure that the lead clones have not lost target specificity during the mutation and reselection process. To confirm this, 14 purified human proteins from the immunoglobulin superfamily were The lead and control IgG1 clones were tested for binding to the panel (Figure 5). All IgGs showed binding signals to CD47-Fc at 1 μg / ml (human O D450nm>2.0, cynomolgus monkey>2.0, mouse>1.25), and other proteins There was no detectable binding to IgG (OD450nm < 0.1). One notable exception was , VH-A1 / V1-B1 clone, which again targets human and cynomolgus CD47 The results showed strong binding to mouse CD47 but no signal for mouse CD47.

[0222] Flow cytometric analysis of binding specificity of lead IgG at cell membranes Concentration-dependent binding of antibodies against CD47 to the cell surface was examined by flow cytometry. CHO-K1 cells were transfected with human, mouse, or cynomolgus CD47. Stably transfected with full-length cDNA. Anti-CD47 IgG mVH / m VL, VH-A1 / VL-B1, A-D5, G-B6, F-E7, and D-H3, and A Isotype control IgG1 was administered in both IgG1 null and IgG4(S228P) form. Together with the commercially available mouse anti-human CD47 monoclonal MS1991, Human, cynomolgus monkey, or wild-type control ("wt"; The binding to CHO-K1 (transfected or untransfected) was tested. All IgGs except for the control showed concentration-dependent binding to human and cynomolgus monkey CD47+ cells. The maximum MFI in each case is shown, which is the maximum MFI for non-transfected CHO-K1. The signal was more than 10-fold higher than that observed for binding (Figure 6). Measurable binding to T cells was observed for all clones except VH-A1 / VL-B1. However, mVH / mVL IgG was only observed at high antibody concentrations. The strongest reaction was observed at a low concentration of 0.01 mg / ml, with a signal 10-fold higher than the "no antibody" negative control. This background binding was due to the fact that the original mouse antibody VxP037 reacts with mouse CD4 47, as well as cross-reactivity to hamsters. Antibodies for clinical use are usually produced in CHO cell cultures, so Minimization of this cross-reactivity to mustard CD47 is favorable for therapeutic proteins. The high IgG binding affinity for the hamster CD47 protein is desirable in the production process. No co-purified CD47 host cell protein (which was prepared to avoid immunogenicity in patients) This can lead to a significant increase in the content of

[0223] To examine the binding of the lead IgG to CD47+ human cancer cells, HL60 cells (human acute myeloid leukemia virus type 2 (AML)) were cultured in vitro. Myeloid leukemia-derived IgG1 was also used in the flow cytometry analysis described above. All antibodies except for the control IgG showed strong concentration-dependent binding to HL60 cells (Figure 7). ).

[0224] Lead IgG analysis by "developmental" ELISA assay The art has reported the use of IgG4-directed immunosorbent assays intended for therapeutic use on a number of indicated biological substrates. This conjugate has low bioavailability and short in vivo half-life, which increases the risk of poor performance in patients. Three such biological substrates are known to be markers of insulin resistance. Therefore, these three substrates were used to perform ELISA. Coating rates and investigating binding of an IgG1 null version of an optimized lead antibody The binding signals of these human IgG-based antibodies were found to be polyreactive and of low performance. Known and progressing in clinical trials (bococizumab and briakinumab, human IgG1 analogs) The results were compared to a "positive control" human IgG antibody that had been stopped. In particular, the IgG1 ustekinumab analog reacts with the same therapeutic target as brikinumab, but is more IgG1 ustekinumab, which has a long pK and has been successfully approved as a therapeutic product In the ELISA analysis shown in Figure 8, the positive control antibody inhibited all three substrates. The expected strong reactivity against the IgG antibody was observed, whereas the negative control showed low reactivity. Finally, all IgG1 null lead proteins tested were negative for all three substrates. The binding of the optimized clones A-D5, G-B6 was less than that of the control. , D-H3, and VH-A1 / VL-B1 maintain highly specific target-driven binding It emphasizes:

[0225] Analysis of designer IgG based on lead clone A-D5 As described above, clone A-D5 showed high affinity for human, cynomolgus monkey, and mouse CD47. Highly specific binding, low off-target binding potential, improved neutralization of mouse CD47, CHO Reduced background binding to cells and multiple human germline mutations in the CDRs However, the A-D5 sequence, as a clone derived from the library, was Many non-germ cell studies have been found to be potentially unnecessary, suggesting that the data found in A and 2B may be of little use. The cytoplasmic (mouse-derived) residues were retained. A-D5 sequence and all other library-derived The clones also showed high expression at the apex of the long and flexible IGKV2-28 germline template CDR-L1 loop. Therefore, we identified "N" in CDR-L1, which is at high risk of deamination. The human germline sequences of the A-D5 variable domains were simultaneously optimized to retain the "G" motif. In an attempt to increase the size of the deamination site and minimize the content of high-risk deamination motifs in the protein, A series of designer clones were created. The experiment was carried out in two phases. The A-D5.1 to A-D5.10 clones contained the CDR sequences outlined in Table 4. The clone was expressed and purified in an IgG1 null form and expressed the E For target binding by LISA (Fig. 9A, B, C) and for all orthologs Neutralization of the CD47-SIRPαB interaction (Figure 10A, B, C) was examined. At this stage, human germline and these improvements may be associated with a modest loss of efficacy. Furthermore, target binding ELISA and CD47-SIRPα interaction were Despite the associated reduction in potency of both neutralization of The first mutation we attempted to remove (via a conservative N to Q substitution) was successful (Figure 10A , B, C).

[0226] In the second phase, a series of further mutations were made to the best performing variant in the first phase, A-D5.4. Nine variants were designed (Table 5). These nine variants were designed to confers the “NG” deamination risk motif. The "N" in the nucleotide sequence may be replaced with conservative or non-conservative mutations such as S, G, A, or T. or a further substitution of CDR-H2 of AD.4 with or without replacing the "G" residue with A. These clones were again expressed and purified in the IgG1 null form, and all Target binding by ELISA for all CD47 orthologues (Figure 11A, B, C) , and for neutralization of the CD47-SIRPα interaction for all orthologs (Fig. 1 2A, B, C) were examined. At this stage, there was no loss of potency compared to clone A-D5. We found that the human germline content of CDR-H2 could be increased by an additional two residues. In addition, a mutation that seeks to remove the “NG” motif in CDR-L1 via an N substitution The study was successful and clone A-D5.16 was identified, which contains a nonconservative N to A mutation. and CD47-SIRP target binding ELISA against both A-D5 and mVL / mVH. The maximally humanized CD4+ antibody was shown to have only a slight (approximately 3-fold) decrease in either A or neutralization potency. The CDR-L1 sequence of AD.16 was RSSQSLLHS A GYNYL H " (SEQ ID NO: 82) (and clones A-D5.14, A-D5.15, A-D5.17 , and A-D5.28) contain only two non-germline residues (underlined) and are the largest human An optimal balance between germline content and maximum stability properties was achieved.

[0227] Next, A-D5 derivatives A-D5.4 and A-D5.16 were designed to induce the mutation and reselection process. To confirm that there was no loss of target specificity during the study, I was analyzed for maintenance of binding specificity. We also tested the gG1 null form; 14 purified human Both clones were tested for binding to a panel of target proteins (Figure 13). IgG showed binding signals to CD47-Fc at 10 μg / ml (human, cynomolgus monkey). All of the human and mouse strains were >2.0 OD450nm, with no detectable effect against other proteins. There was no significant binding (OD450nm<0.1). The "development" ELISA shown in Figure 14 In our analysis, the positive control antibody showed the expected strong reactivity with all three substrates; The negative controls showed low reactivity. Importantly, A-D5.4 and A-D5.16 Ig Both G1 null lead proteins showed results below the binding of the negative controls to all three substrates. This finding was supported by the optimized clones A-D5, A-D5.4, and These results highlight the maintenance of highly specific target-driven binding in A-D5.16 and A-D5.16.

[0228] Finally, the A-D5 derived mutants, A-D5.4 and A-D5.16, were transfected with wild-type CHO Binding to HL60 (Figure 15) and HL60 (Figure 16) cells was measured by flow cytometry. These analyses revealed that clonal mVH / mVL of IgG1 null or IgG4 form The original mouse v domain of drives strong concentration-dependent binding to CHO cells, whereas Clones A-D5, A-D5.4, and A-D5.16 bind both IgG forms We confirmed that the signal was mediated little or not at all (Figure 15). The mVH / mVL, A-D5, A-D5.4, and A-D5.16, are all derived from human CD4 The antibody showed strong binding to 47+ HL60 cells (Figure 16), indicating that human CD47 was bound to the cell membrane. This was retained in our optimized clones, but the reactivity to hamster CD47 was altered. It showed that it was improved.

[0229] Antibody v-domain T cell epitope analysis In silico techniques based on identifying the location of T cell epitopes in therapeutic antibodies and proteins ( Abzena, Ltd.) to assess the immunogenicity of both the mVH / mVL and lead antibody v-domains. Analysis of the v-domain sequences was performed for each of the 34 MHC class II allotypes. Overlapping 9-mer peptides (each overlapping the last peptide by 8 residues) were tested against Each 9-mer was analyzed for potential "matches" and interactions with MHC class II molecules. Peptide scores were calculated by the software on a scale of 0 to 1. It produced a high average binding score (>0.55 using the iTope® scoring function). The peptides with >50% (i.e., 17 of 34 alleles) M HC class II-binding peptides have high binding affinity (score > 0.6); The peptides are "high affinity" peptides that are considered at high risk of containing CD4+ T cell epitopes. Low affinity MHC class II binding peptides were defined as: Binds to a large number of alleles (>50%) with a binding score above 0.55 (but the majority (number not exceeding 0.6). Further analysis of the sequences was carried out using TCED®. This sequence was used to perform a BLAST search on TCED®, a product of Abzena Ltd. Irrelevant T-cell responses stimulated in previous in vitro T-cell epitope mapping studies identified high sequence homology between peptides (T cell epitopes) from various proteins / antibodies .

[0230] The peptides were divided into four classes: high affinity foreign ("HAF" - highly immunogenic risk); Low Affinity ("LAF" - low immunogenicity risk), TECD+ (previously TCED (registered trademark) (epitopes identified in the "genomic target" database), and germline epitopes ("GE"-M Human germline peptide sequences with high HC class II binding affinity. Germline epitopes The 9-mer peptide has been validated in previous studies using a broad range of germline peptides. As such, these peptides are unlikely to be immunogenic due to T cell tolerance (i.e., (Recognized by the host as “self”). Importantly, such germline v-domain endogenous The pitope (further supported by similar sequences in human antibody constant regions) is a key regulator of antigen presentation. They also compete for MHC class II occupancy at the cell membrane, achieving the "activation threshold" required for T cell stimulation. Therefore, a high GE content reduces the risk of exogenous peptide presentation sufficient to achieve , a valuable quality in the clinical development of antibody therapeutics.

[0231] As shown in Figure 17, the major lead v-domains are more highly fragmented than the mVH / mVL domains. The v-domain engineering process performed here showed significant beneficial changes in epitope content. The process maintains anti-CD47 potency without the need for murine residues in the framework. We succeeded in selecting antibodies that could bind to both the heavy and light chains of mVH / mVL (Table 2). Multiple HAF and LAF epitopes were found in the framework of the domains, all of which The GE epitope was absent from the library-derived and designer leads (Figure 17). The amount of β-glucose was also significantly increased (3 to 14 or more in all leads), especially in the lead c-type. In the VH region of the lone read, GE content increased from 0 to 9 in all reads, and TCED+ epitopes The number of repeats decreased from 3 to 2 for all leads (Table 8). The epitope was also eliminated by a germline mutation found in the CDR of the lead clone For example, the TCED+ peptide found in LCDR-1 of mVH / mVL, “LVH SNGNTY" (SEQ ID NO: 116) (and thus any previously humanized by CDR grafting) The VxP037 form of the mutated V>L at position 2 and V>L at position 3 in most lead clones. The LC sequence of the mVH / mVL sequence was similarly excluded by N>Y at position 7 (Tables 4 and 5). DR2 is composed of three foreign peptides spanning the VL framework 2-LCDR2-framework 3. These included two HAF peptides ("LLIYKV SYR" (SEQ ID NO: 117) and "YRFSGVPDR" (SEQ ID NO: 118)) and one The LAF peptide ('LIYKVSYRF' (SEQ ID NO: 119)) was included. Inserting 2 into the human germline framework IGKV2-28 does not completely address this issue. The sequence "LLIYKVSYRFSGVPDR" (sequence No. 120) maintains 100% identity in the IGKV2-28 grafted sequence (Table 2). Clones A-D5, A-D5.4, and A-D5.16 contained one H Although AF and two LAF peptides were still found, the HAF sequence “YRFSGVPDR” ( SEQ ID NO: 118) was deleted at position 1 by the mutation Y>N. However, surprisingly , which have been identified within the functional binding population during library screening (Table 3), and The LCDR2 sequence KVSNRFS (SEQ ID NO: 4) contained a single human germline mutation Y>N. No. 85) was found to completely improve all predicted foreign epitopes in this region. (HAF, LAF, and TECD+ peptides were not predicted), but two additional GE sequences were found. The lead clone A-D5 and all of its designer derivatives were also generated (Figure 18). Conductors (Tables 4 and 5) are TECD+ spanning the VL framework 3 and LCDR3 regions. It was also observed that the HAF peptide "VGVYYCFQN" (SEQ ID NO: 121) was retained. Mutations from V to A, T, or F at position 1 of this peptide all corresponded to the predicted epitope. It was found that this disrupts the peptide's loop structure and eliminates the immunogenicity risk of this peptide.

[0232] Based on the above findings, A-D5.16 was used to form the clone “A-D5.16-DI”. It was possible to design maximally deimmunized light chain sequences paired with VH sequences (Table 4). A-D5.16-DI is the LCDR sequence "RSSQSLLHSAGYNYLH" (sequence No. 82), LCDR2 sequence "KVSNRFS" (SEQ ID NO: 85), and framework 2-LCDR3 sequence " AGVYYC FQNTHTPRT" (sequence number 122) (frame The clone contained the IgG1 null form (the 2 residues are underlined). are readily expressed, comparable to mVH / mVL IgG (which is reduced in mice, FIG. 18). It was found to retain target binding affinity for human, cynomolgus monkey, and mouse CD47. (Figure 18A-C). A-D5.16-DI also showed improved results compared to A-D5.16. showed effective CD47-SIRPα inhibition and expressed mVH / It was found to show similar inhibition to mVL and was slightly reduced in mice (Figure 19). These findings support the LCDR sequence "RSSQSLLHSNGYTYLH" (SEQ ID NO:52). (or SSQSLLHSNGYTY using the AHo definition, SEQ ID NO: 92) LCDR 2 sequence "KVSNRFS" (SEQ ID NO: 85), and framework 2-LCDR3 sequence " AGVYYC FQNTHTPRT" (SEQ ID NO: 12s2) (framework 2 residues underlined) The resulting deimmunized clone A-D5.4-DI has a light chain design that includes the following: did.

[0233] Cancer cell phagocytosis Inhibition of CD47 in driving phagocytosis of HL60 human cancer cells by human primary macrophages A study was conducted to examine the relative efficacy of the IgG4 ( S228P) forms, mVH / mVL, A-D5, A-D5.4, and A-D5.16 All of these induced significant phagocytosis at all concentrations tested. 5 showed no significant potency at 20 μg / ml and inhibited Fc gamma receptor 1 to induce phagocytosis. Unexpectedly, IgG4 A-D5, A-D5.4, and A-D5. D5.16 was significantly more potent at both 1 and 10 μg / ml when compared to mVH / mVL. This phenomenon was then examined in human macrophages from four separate donors. Therefore, IgG4 A-D5 and mVH / mVL were examined (Figure 20B). This confirms the higher potency shown in FIG. 20A, with A-D5 being the most potent in all donors. The results were significantly more potent at 100-fold increase in β-lactamase activity (Figure 20B).

[0234] Although the present invention has been described with reference to preferred or exemplary embodiments, it is understood by those skilled in the art that the spirit and scope of the present invention may be readily understood by those skilled in the art. and that various modifications and changes thereto can be effected without departing from the scope thereof; and will recognize that such modifications are expressly contemplated herein. LIMITATIONS ON CERTAIN EMBODIMENTS DISCLOSED AND CLAIMS APPENDED HEREIN is not intended, and no limitation should be inferred.

[0235] All documents cited herein are incorporated by reference in their entirety. .

[0236] [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

Table 3-5

Table 4-1

Table 4-2

Table 5

Claims

1. An antibody molecule, or antigen-binding portion thereof, that specifically binds to human CD47 and cynomolgus CD47, the antibody molecule or antigen-binding portion comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), and a light chain variable region (VL) comprising light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3); the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are (a) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTTPR (SEQ ID NO: 93) (LCDR3), respectively; or (b) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTTPR (SEQ ID NO: 93) (LCDR3), respectively; or (c) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2), and NTHTTPR (SEQ ID NO: 93) (LCDR3), respectively; or (d) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2), and NTQTPR (SEQ ID NO: 96) (LCDR3), respectively; or (e) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRLS (SEQ ID NO: 77) (LCDR2), and NTQTPR (SEQ ID NO: 96) (LCDR3), respectively; or (f) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3), respectively; or (g) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and QTHTPR (SEQ ID NO: 105) (LCDR3), respectively; or (h) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2), and QTHTPR (SEQ ID NO: 105) (LCDR3), respectively; or (i) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2), and NTHTTPR (SEQ ID NO: 93) (LCDR3), respectively; or (j) an antibody molecule or antigen-binding portion comprising the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2), and NTHTPR (SEQ ID NO: 93) (LCDR3), respectively.

2. The antibody molecule or antigen-binding portion of claim 1, comprising one or more substitutions, deletions, or insertions that remove post-translational modification sites, such as glycosylation sites, deamination sites, phosphorylation sites, or isomerization / fragmentation sites.

3. The antibody molecule or antigen-binding portion of claim 1 or 2, wherein the antibody molecule or antigen-binding portion is humanized or chimeric.

4. An antibody molecule or antigen-binding portion according to any one of claims 1 to 3, comprising one or more human variable domain framework scaffolds into which the CDRs are inserted.

5. 5. The antibody molecule or antigen-binding portion of any one of claims 1 to 4, comprising an IGHV5-51 human germline scaffold into which the corresponding HCDR sequences have been inserted.

6. 6. The antibody molecule or antigen-binding portion of any one of claims 1 to 5, comprising an IGKV2-28 human germline scaffold into which the corresponding LCDR sequences have been inserted.

7. 7. The antibody molecule or antigen-binding portion of any one of claims 1 to 6, wherein the VH comprises: (a) amino acid residues 1 to 23 of SEQ ID NO:141; (b) amino acid residues 34-50 of SEQ ID NO:141; (c) amino acid residues 67-98 of SEQ ID NO:141; (d) amino acid residues 105-116 of SEQ ID NO: 141; or (e) any combination of these, an antibody molecule or antigen-binding portion comprising a sequence having at least 90% sequence identity to.

8. 8. The antibody molecule or antigen-binding portion of claim 7, wherein the VH is: (a) amino acid residues 1 to 23 of SEQ ID NO:141; (b) amino acid residues 34-50 of SEQ ID NO:141; (c) amino acid residues 67-98 of SEQ ID NO:141; (d) amino acid residues 105-116 of SEQ ID NO: 141; or (e) any combination thereof.

9. 9. The antibody molecule or antigen-binding portion of any one of claims 1 to 8, wherein the VL comprises: (a) amino acid residues 1 to 24 of SEQ ID NO:143; (b) amino acid residues 38-54 of SEQ ID NO:143; (c) amino acid residues 62-95 of SEQ ID NO:143; (d) amino acid residues 102-112 of SEQ ID NO: 143; or (e) any combination of these, an antibody molecule or antigen-binding portion comprising a sequence having at least 90% sequence identity to.

10. 10. The antibody molecule or antigen-binding portion of claim 9, wherein the VL comprises: (a) amino acid residues 1 to 24 of SEQ ID NO:143; (b) amino acid residues 38-54 of SEQ ID NO:143; (c) amino acid residues 62-95 of SEQ ID NO:143; (d) amino acid residues 102-112 of SEQ ID NO: 143; or (e) any combination thereof.

11. The antibody molecule or antigen-binding portion of any one of claims 1 to 10, wherein the VH comprises a sequence having at least 85% sequence identity to SEQ ID NO:

141.

12. 12. The antibody molecule or antigen-binding portion of any one of claims 1 to 11, wherein the VL comprises a sequence having at least 85% sequence identity to SEQ ID NO:

143.

13. 13. An antibody molecule or antigen-binding portion according to any one of claims 1 to 12, comprising an immunologically inactive constant region.

14. The antibody molecule or antigen-binding portion may be a Fab fragment, F(ab) ２ The antibody molecule or antigen-binding portion of any one of claims 1 to 13, which is a fragment, Fv fragment, a tetrameric antibody, a tetravalent antibody, a multispecific antibody (e.g. a bivalent antibody), a monoclonal antibody, or a fusion protein.

15. 15. An immunoconjugate comprising an antibody molecule, or an antigen-binding portion thereof, as defined in any one of claims 1 to 14, conjugated to a therapeutic agent.

16. A nucleic acid molecule encoding an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising a nucleic acid molecule as defined in claim 16 or a vector as defined in claim 17.

19. 20. A method for producing an anti-CD47 antibody or antigen-binding portion thereof, comprising culturing a host cell as defined in claim 18 under conditions resulting in the expression or production of said antibody or antigen-binding portion thereof, and isolating said antibody or antigen-binding portion thereof from the host cell or culture.

20. A pharmaceutical composition comprising an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17.

21. A composition for enhancing an immune response in a subject, comprising an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20.

22. 20. Use of an antibody molecule or an antigen-binding portion thereof as defined in any of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20, in the manufacture of a medicament for the treatment of cancer.

23. A composition for the treatment of cancer, comprising an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14, or comprising an immunoconjugate as defined in claim 15, or comprising a nucleic acid molecule as defined in claim 16, or comprising a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20.

24. 24. The composition of claim 23, wherein the cancer is selected from the group consisting of pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and blood tissue cancer.

25. The composition of claim 21 , wherein the antibody, antigen-binding portion thereof, immunoconjugate, nucleic acid molecule, vector, or pharmaceutical composition is for use in combination with a second therapeutic agent, such as an anti-cancer agent, either separately, sequentially, or simultaneously.

26. Use of an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20, in the manufacture of a medicament for the treatment of ischemia-reperfusion injury, an autoimmune disease, or an inflammatory disease.

27. Use of an antibody molecule or an antigen-binding portion thereof as defined in any of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20, in the manufacture of a medicament for the treatment of a cardiovascular or fibrotic disease.

28. 28. The use according to claim 27, wherein the fibrotic disease is selected from the group consisting of myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis and asthma.

29. 28. The use according to claim 27, wherein the cardiovascular disease is coronary heart disease or atherosclerosis.

30. A composition for the treatment of ischemia-reperfusion injury, an autoimmune disease, or an inflammatory disease, comprising an antibody molecule or an antigen-binding portion thereof as defined in any one of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20.

31. A composition for the treatment of a cardiovascular or fibrotic disease, comprising an antibody molecule or an antigen-binding portion thereof as defined in any of claims 1 to 14, or an immunoconjugate as defined in claim 15, or a nucleic acid molecule as defined in claim 16, or a vector as defined in claim 17, or a pharmaceutical composition as defined in claim 20.

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