Binder

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 the use of CD47 (cluster of differentiation 47, integrin-associated protein [IAP]) The present invention relates to antibody molecules that specifically bind to IgG1 (also known as IgG1a), and their medical uses. [Background technology]

[0002] CD47 (also known as integrin-associated protein [IAP]) is a It is a transmembrane protein belonging to the globulin superfamily, and is involved in membrane integrins, thrombins, and transspondin-1 (TSP-1), and signal regulatory protein alpha (SIRPα) CD47 binds to several known partners, including α- and β-glucanase (GlcNAc), which mediates cell apoptosis, proliferation, and They are involved in various cellular processes such as cell proliferation, 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 activation of myeloid lineage-specific immune checkpoints to promote tumor cell survival. It functions as a

[0003] Preclinical evidence suggests that blocking CD47-SIRPα signaling inhibits the phagocytic activity of macrophages. It has been shown to enhance the efficacy of IFN-γ-glucan and inhibit the growth of xenografts in many 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 inflammatory bowel disease. , the CD47-SIRPα signaling axis also has considerable therapeutic potential in non-cancerous diseases. Therefore, anti-CD47 mAbs act as immunotherapeutic agents in cancer and other settings, and 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. have undergone a process known as "humanization" (Nelson et al., 2010, Nat Rev Dru g Discov 9:767-774). This process is often inaccurate and results in 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 humanized via CDR grafting and backmutation contain entirely murine v domains. have been shown in clinical trials to induce a lower rate of immune response 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 see significant clinical application. There is a risk of the emergence of new drugs that target receptors on immune cells and whose pharmacological function is via antigen presentation. Antibodies such as CD47 inhibitors, which stimulate immune responses, can induce anti-drug antibody responses. These anti-drug antibody responses in patients are associated with increased risk of developing drugs in clinical use. May reduce half-life, efficacy, and safety. Animal testing of protein immunogenicity Because antibodies often do not predict the human immune response, therapeutic antibody engineering relies on 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, the ideal humanized antagonistic anti-CD47 antibody would be a well-characterized human germline antibody. As many identical residues as possible to those found in both the framework and CDRs of the sequence are identified. 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 co-crystal structure data. In the absence of data, we demonstrated the fundamental plasticity of the original antibody paratope, but not the C of a particular antibody. Determine which residues in the DR loop can be converted in what combinations in the human germline. Indeed, it is still impossible to predict.

[0007] Therefore, it is preferable to maintain the CDR germline because multiple functional properties of the molecule must be maintained. The system is a complex, multifactorial problem, and in this example includes: humans and test animal species (e.g. For example, the crab-eating macaque, also known as the long-tailed macaque, or Macaca Target binding specificity, affinity, and V domain activity for CD47 from both α fascicularis and α fascicularis Biophysical stability and / or IgG expression. Antibody engineering studies have revealed that the single CDRs of the major Even a single residue mutation can dramatically affect all these desirable molecular properties. is shown.

[0008] WO 2014 / 093678A2 describes an antagonistic mouse model called "VxP037" describes the preparation of a humanized form of an anti-CD47 IgG molecule, VxP037. These humanized forms of VxP037 were synthesized using classical humanization techniques, i.e., Kaba by grafting defined murine CDRs onto human heavy and light chain framework sequences. , some of the human framework residues are hidden in the VxP037 mouse residues at the corresponding positions. For the above reasons, WO 2014 / 0936 Such a humanized form of VxP037 as 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 mouse CD47 or antigen-binding portions thereof and an antibody molecule, wherein the antibody molecule, or antigen-binding portion thereof, has a heavy chain variable region comprising: Including:

[0011] HCDR1 having 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. Conservative substitutions 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 DG conservative substitutions, or is a conservative substitution in GDTN, or a conservative substitution in N(e.g., R)-Y, or a conservative substitution in YN conservative substitution, or N(e.g., S)-PSFQG conservative substitution (SEQ ID NO: 2); and HCDR3:GGY, having 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, 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) 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. 6,273,787. May be excluded.

[0013] The antibody molecule or antigen-binding portion may further comprise a light chain variable region having: LCDR1:RSSQ or QSL having amino acids in the following order: 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 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 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; 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)-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 murine / 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 / 093678A2) The disclosed VxP037 murine / 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 all immunoglobulins. The shorter "AHo" definition (see Table 1) aims to unify the nomenclature of phosphorylation domains. (see

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

[0017] HCDR1 having the amino acids in the following order: GSGYT, or any amino acid 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: Conservative substitutions of any amino acid (e.g., G or F) with D, or 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 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) (SEQ ID NO: 14).

[0018] Using the AHo definition, 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 may exclude HCDR1) and / or antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMD (SEQ ID NO: 16) (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:SSQ or QSL, which has the amino acids in the following order: 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, or 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 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 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 amino acid (e.g., L)-R, or any amino acid (e.g., W) (SEQ ID NO: 18).

[0020] Using the definition of AHo, 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 may exclude LCDR2) and / or the antibody molecule or antigen-binding portion The LCDR3 of this gene is represented by 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 moiety are also provided. In another aspect, the present invention provides an antibody molecule, or antigen-binding portion thereof, as defined herein. Nucleic acid molecules that encode the nucleic acid are provided. Further provided are vectors comprising the nucleic acid molecules 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 produced under conditions that result in the expression and / or production of the antibody. and culturing the host cells and extracting the antibody and / or antigen-binding portion thereof from the host cells or culture. isolating the compound.

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

[0024] Further provided is a method of enhancing an immune response in a subject, comprising administering to a subject a compound of the invention as defined herein. an antibody molecule or antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, is 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 present 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 a method of treating or preventing cancer, as defined herein. an antibody molecule of the invention or an antigen-binding portion thereof, or an immunoglobulin of the invention as defined herein Conjugates or nucleic acid molecules of the invention as defined herein or the invention as defined herein. or a pharmaceutical composition of the present invention as defined herein. A method is provided that includes:

[0026] The present invention also provides 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 used in combination with a vector of the invention as defined herein, 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 treating a cancer patient, comprising administering a compound to a patient in combination with a second therapeutic agent, such as an anti-cancer agent, separately or in conjunction with the compound. an antibody molecule of the invention as defined herein, or a combination thereof, for subsequent or simultaneous use 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, autoimmune disease, or inflammatory disease in a subject. A method of preventing the infection of a patient, comprising administering to said patient 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 a nucleic acid molecule as defined herein a method comprising administering an effective amount of a vector 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 aspects, ischemia-reperfusion injury is a serious condition that can occur in organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, and other conditions. , pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resection and reconstruction, 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 as defined herein or an antigen-binding portion thereof, or an immunoglobulin as defined herein or a nucleic acid molecule as defined herein, or a vector as defined herein, or Pharmaceutical compositions as defined herein are also provided.

[0033] Furthermore, the present invention is useful in the manufacture of medicaments for the treatment of ischemia-reperfusion injury, autoimmune diseases, or inflammatory diseases. an antibody molecule or 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, or 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 cardiovascular or fibrotic diseases.

[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 an immunoconjugate as defined herein. a nucleic acid molecule comprising the nucleic acid molecule, or a vector as defined herein, or a pharmaceutical composition as defined herein is also provided.

[0036] Furthermore, for the treatment of ischemia-reperfusion injury, autoimmune diseases, inflammatory diseases, or fibrotic diseases. 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 a nucleic acid molecule as defined herein Use of the vector or 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 may be 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 provides a method for the production of 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. The method includes the following steps: (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 containing one or more mutations in the CDRs; generating a phage library of 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 clones having binding specificity to 47 from the screening step (3). 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 Clones are generated 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 assessed, e.g., by determining the presence of a desired gene in the CDR and framework regions. Optionally, generating one or more additional mutations to reduce immunogenicity. Immunogenicity can be measured, for example, by in silico immunoassays as described herein. ) technology can be used to identify and evaluate the location of T cell epitopes. . [Brief explanation of the drawings]

[0042] [Figure 1] Direct binding ELISA of library-derived anti-CD47 scFv against human and mouse CD47-Fc protein. 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 protein. 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 gray 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 CDR residue tolerance to germline mutations. Plots of the frequency of mouse amino acid retention in the CDRs of the ELISA-positive population of 854 unique scFv clones are shown for the VH (A) and VL (B) domains, respectively. Except for within HCDR3, only residues targeted for human / mouse mutagenesis are plotted. In each plot, CDR residues are shown on the X-axis, and the Y-axis indicates 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 within HCDR2 that are not in brackets but are set to a value of 0 were mutated to the human germline during the grafting process. In both plots, the gray dashed line at 75% represents the cutoff for tolerance of mouse residue substitution with human germline. [Figure 2B] Analysis of CDR residue tolerance to germline mutations. Plots of the frequency of mouse amino acid retention in the CDRs of the ELISA-positive population of 854 unique scFv clones are shown for the VH (A) and VL (B) domains, respectively. Except for within HCDR3, only residues targeted for human / mouse mutagenesis are plotted. In each plot, CDR residues are shown on the X-axis, and the Y-axis indicates 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 within HCDR2 that are not in brackets but are set to a value of 0 were mutated to the human germline during the grafting process. In both plots, the gray dashed line at 75% represents the cutoff for tolerance of mouse residue substitution with human germline. [Figure 3A-D]Direct titration ELISA for IgG binding to human, mouse, and cynomolgus monkey CD47-Fc proteins. Human IgG1 null chimeric anti-CD47 (mVH / mVL) and library-derived clones were titrated (μg / ml) against human, mouse, and cynomolgus monkey CD47-Fc proteins (A-H) by direct binding ELISA. mVH / mVL, library-derived clones, and designer clone MH exhibited binding activity to all three CD47 orthologs. Clone VH-A1 / VL-B1 binds to human and cynomolgus monkey CD47 but not to mouse. Clone TTP lost almost all binding function. The x-axis of each graph represents IgG concentration (μg / ml), and the y-axis represents binding signal (OD450nm). [Figure 3E-H] Direct titration ELISA for IgG binding to human, mouse, and cynomolgus monkey CD47-Fc proteins. Human IgG1 null chimeric anti-CD47 (mVH / mVL) and library-derived clones were titrated (μg / ml) against human, mouse, and cynomolgus monkey CD47-Fc proteins (A-H) by direct binding ELISA. mVH / mVL, library-derived clones, and designer clone MH exhibited binding activity to all three CD47 orthologs. Clone VH-A1 / VL-B1 binds to human and cynomolgus monkey CD47 but not to mouse. Clone TTP lost almost all binding function. The x-axis of each graph represents IgG concentration (μg / ml), and the y-axis represents binding signal (OD450nm). [Figure 4]ELISA-based CD47-Fc-SIRPα competition assay. ELISA binding signals of human (A), cynomolgus monkey (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of titrated competitive library leads (IgG1 null forms A-D5, G-B6, D-H3, and VH-A1 / VL-B1; isotype IgG1 as a negative control; and IgG1 null forms mVH / mVL as a positive control). All library-derived IgGs and mVH / mVL showed a concentration-dependent decrease in binding of human, mouse, and cynomolgus monkey CD47-Fc proteins, suggesting the maintenance of a shared epitope. Notably, clone A-D5 showed significantly higher potency in neutralizing mouse CD47 compared to mVH / mVL, while VH-A1 / VL-B1 showed no ability to neutralize mouse CD47 activity. Neither designer clone MH nor TTP showed a neutralization signal and, for clarity, are not plotted here. In each graph, the X-axis represents antibody concentration (nM) and the Y-axis represents binding signal (OD450nm). In the figure legend, "IC" refers to the isotype control. [Figure 5A-D]Binding specificity analysis of prioritized lead clones. For the IgG1 (A) and IgG1-null (B) mVH / mVL forms, and the IgG1-null library-derived leads A-D5 (C), VH-A1 / VL-B1 (D), F-E7 (E), D-H3 (F), and G-B6 (G), off-target homolog binding risk was examined by direct ELISA of CD47-Fc orthologs and a panel of 14 human immunoglobulin superfamily proteins labeled on each X-axis ("B" indicates blank). Binding to human, cynomolgus monkey, 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). Nearly all IgGs were observed to bind 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 the IgG1 (A) and IgG1-null (B) mVH / mVL forms, and the IgG1-null library-derived leads A-D5 (C), VH-A1 / VL-B1 (D), F-E7 (E), D-H3 (F), and G-B6 (G), off-target homolog binding risk was examined by direct ELISA of CD47-Fc orthologs and a panel of 14 human immunoglobulin superfamily proteins labeled on each X-axis ("B" indicates blank). Binding to human, cynomolgus monkey, 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). Nearly all IgGs were observed to bind 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 monkey CD47+ CHO-K1 cells. The commercially available anti-CD47 antibody MS1991, human IgG1 ("I IgG1") and IgG4 ("I IgG4") isotype controls, and both IgG1 null ("IgG1N") and IgG4 (S228P) forms of lead library-derived IgG were tested for specific binding to cynomolgus monkey-transfected CHO-K1 cells (A), human-transfected CHO-K1 cells (B), and wild-type (wt, i.e., untransfected) CHO-K1 cells (C). IgGs were tested at concentrations ranging from 24 to 100,000 ng / ml. Concentration-dependent binding to both human and cynomolgus monkey cell lines was observed for all CD47-specific antibodies, but not for the isotype controls. For most antibodies, low levels of binding signals above background were observed against wild-type CHO-K1 cells, with significantly stronger signals for mVH / mVL-derived IgGs and particularly low signals 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 mean fluorescence intensity (MFI). [Figure 7] Flow cytometry study of binding to human HL60 cells. Specific binding to HL60 cells was examined for the commercially available anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls ("I IgG1" and "I IgG4", respectively), and IgG1 null ("IgG1N") and IgG4 (S228P) forms of IgGs from the lead library. IgGs were tested at concentrations ranging from 24 to 100,000 ng / 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 mean fluorescence intensity (MFI). [Figure 8]Development risk ELISA. This assay demonstrated that the IgG1 null forms of A-D5, G-B6, D-H3, VH-A1 / VL-B1, and mVH / mVL antibodies exhibited little to 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 represents IgG concentration (μg / ml), and the y-axis represents the binding signal (OD450nm). Strong off-target binding to these molecules, as observed with bococizumab and briakinumab analogs, has been shown to be a high-risk indicator of poor clinical performance of therapeutic antibodies. [Figure 9] Direct titration ELISA for binding of designer IgG to human, mouse, and cynomolgus monkey CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) clones derived from designer A-D5, a human IgG1 null form, were titrated (μg / ml) in a direct binding ELISA against human (A), cynomolgus monkey (B), and mouse (C) CD47-Fc proteins. In each graph, the X-axis represents IgG concentration (μg / ml), and the Y-axis represents binding signal (OD450nm). Most clones exhibited binding activity to all three CD47 orthologs, but clone A-D5.7 exhibited only weak binding to cynomolgus monkey CD47, and clone A-D5.10 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. ELISA binding signals of human (A), cynomolgus monkey (B), and mouse (C) CD47-Fc proteins to plate-bound human SIRPα were examined in the presence of a titrated competitive designer IgG containing an IgG1 null form, an isotype IgG1 (designated "IgG1NI") as a negative control, and an IgG1 null form of mVH / mVL as a positive control. In each graph, the X-axis represents the IgG concentration (nM), and the Y-axis represents the binding signal (OD450nm). Notably, several A-D5-derived clones again demonstrated particularly pronounced increases in potency in neutralizing mouse CD47 compared to mVH / mVL. In contrast, the orthologues that showed weak ELISA binding signals, designer clones A-D5.7 and A-D5.10, did not exhibit neutralization signals and are therefore not plotted here for clarity. [Figure 11] Direct titration ELISA of A-D5.4-derived designer IgG binding to human, mouse, and cynomolgus monkey CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) human IgG1 null forms, designer A-D5.4-derived clones, were titrated in a direct binding ELISA against human (A), cynomolgus monkey (B), and mouse (C) CD47-Fc proteins (μg / ml). In each graph, the X-axis represents IgG concentration (μg / ml), and the Y-axis represents binding signal (OD450nm). All clones showed binding activity to all three orthologs 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 monkey (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, isotype IgG1 (designated "IgG1NI") as a negative control, and IgG1 null form mVH / mVL as a positive control. In each graph, the X-axis represents IgG concentration (nM), and the Y-axis represents binding signal (OD450nm). Notably, several A-D5.4-derived clones again showed significantly increased 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 risk of off-target homolog binding 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" indicates blank). Binding to all proteins was performed at an IgG concentration of 10 μg / ml. For each plot, the Y-axis indicates the binding signal (OD450 nm). Binding to hCD47-Fc, mCD37-Fc, and cCD47-Fc alone was observed for both IgGs. 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. This assay demonstrated that the IgG1-null forms of the 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 represents IgG concentration (μg / ml), and the y-axis represents the binding signal (OD450nm). The strong off-target binding to these molecules observed with bococizumab and briakinumab analogs has been shown to be a high-risk indicator of poor clinical performance for therapeutic antibodies. [Figure 15]Flow cytometry analysis of library-derived and designer IgG binding to CHO-K1 cells. The commercially available anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls (designated "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 tested for specific binding on wild-type (i.e., untransfected) CHO-K1 cells. IgGs were tested at concentrations ranging from 24 to 25,000 ng / ml. Concentration-dependent binding was observed for the parental mVH / mVL antibodies in both IgG1 null and IgG4 forms, whereas weak or no binding was observed for the 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 IgG concentration (ng / ml), and the Y-axis indicates MFI. [Figure 16] Flow cytometry study of binding to human HL60 cells. The commercially available anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls (represented as "I IgG1" and "I IgG4," respectively), and the lead IgG in both IgG1 null and IgG4 (S228P) forms were tested for specific binding to HL60 cells. IgGs were tested at concentrations ranging from 24 to 100,000 ng / ml. Concentration-dependent binding was observed for all clones except the isotype control. In each graph, the X-axis represents the IgG concentration (ng / ml), and the Y-axis represents 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. All lead clones showed a significant reduction in high-risk epitope content and a significant improvement in germline epitope content. [Figure 18]Direct titration ELISA of A-D5.16 and A-D5.16-DI designer IgG binding to human, mouse, and cynomolgus monkey CD47-Fc proteins. Chimeric anti-CD47 (mVH / mVL) human IgG1 null forms, designer A-D5.16 and A-D5.16-DI clones, were titrated (μg / ml) in a direct binding ELISA against human (A), cynomolgus monkey (B), and mouse (C) CD47-Fc proteins. All clones showed binding activity to all three orthologs of CD47. In each graph, the X-axis represents IgG concentration (μg / ml), and the Y-axis represents the binding signal (OD450nm). [Figure 19] ELISA-based CD47-Fc-SIRPα competition assay for designer IgG. ELISA binding signals of human (A), cynomolgus monkey (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, isotype IgG1 as a negative control, and mVH / mVL in IgG1 null form as a positive control. In each graph, the X-axis represents antibody concentration (nM), and the Y-axis represents binding signal (OD450nm). [Figure 20] Analysis of phagocytosis by flow cytometry. (A) Flow cytometry 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 the IgG1 null form of A-D5 (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 the IgG4 form of A-D5 and mVH / mVL at a standard concentration of 10 μg / ml. The X-axis indicates the donor number, and the Y-axis indicates the % of cells that are CFSE+ and CD14+, with "V" indicating vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0043] (Detailed Description of the Invention) In a first aspect of the invention, a method is provided for the preparation of a human CD47 antibody, and optionally a cynomolgus monkey CD4 7, and / or an antibody molecule, or an antigen-binding portion thereof, that specifically binds to mouse CD47. provided herein, wherein the antibody molecule or antigen-binding portion comprises a heavy chain variable region having:

[0044] HCDR1 having 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. Conservative substitutions 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 DG conservative substitutions, or is a conservative substitution in GDTN, or a conservative substitution in N(e.g., R)-Y, or a conservative substitution in YN conservative substitution, or N(e.g., S)-PSFQG conservative substitution (SEQ ID NO: 2); and HCDR3:GGY, having 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, 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) 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. 6,273,787. 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:RSSQ or QSL having amino acids in the following order: 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 conservative substitutions, or any amino acid (e.g., D) conservative substitution (SEQ ID NO: 6); LCDR2:K having 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 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; 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)-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 murine / 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 / 093678A2) The disclosed VxP037 murine / 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 in the present invention are based on structural biology and are identical to those of all immunoglobulin v domains. The shorter "AHo" definition (see Table 1) aims 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: comprising a heavy chain variable region having:

[0050] HCDR1 having the amino acids in the following order: GSGYT, or any amino acid 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: Conservative substitutions of any amino acid (e.g., G or F) with D, or 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 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) (SEQ ID NO: 14).

[0051] Using the AHo definition, 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 may exclude HCDR1) and / or antibody molecule or The HCDR3 of the antigen-binding portion has the sequence GGYTMD (SEQ ID NO: 16) (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:SSQ or QSL, which has the amino acids in the following order: 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, or 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 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 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 amino acid (e.g., L)-R, or any amino acid (e.g., W) (SEQ ID NO: 18).

[0053] Using the definition of AHo, 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 may exclude LCDR2) and / or the antibody molecule or antigen-binding portion The LCDR3 of this gene is represented by 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 developed a method for producing a compound according to the present invention as disclosed in WO 2014 / 093678A2. The CDR sequences from the disclosed murine anti-CD47 antibody VxP037 were used to generate 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). Further refinement of the optimized antibody molecules described herein will , improved binding to the mouse orthologue of CD47, and murine CD47-SIRPα signaling improved neutralization potency, improved variable domain stability, high expression yield, and / or immunogenicity of the antibody. For example, we herein report a mouse anti-CD47 antibody, VxP037. The precursor molecule of LCDR1 and LCDR2 has two major immunogenic risks, and This is carried out using a humanization technique (as used in WO 2014 / 093678A2) , which proves to be improved in the optimized antibody molecules described herein.

[0055] The antibody molecules or antigen-binding portions of the invention may comprise 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 sequences of There is a gender.

[0056] The antibody molecules or antigen-binding portions of the invention may comprise SEQ ID NOs: 4 (HCDR1), 123 (HCDR 2), 5(HCDR3), 9(LCDR1), 10(LCDR2), 11(LCDR3) or does not bind to hamster CD47 compared to antibody molecules containing the CDR sequences of For example, an antibody molecule or antigen-binding The portions are SEQ ID NOs: 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 CHO cells as measured by flow cytometry. As shown in Figure 15, representative antibody molecules of the present invention may exhibit reduced binding to 0 cells. shows little or no cross-reactivity with CHO cells, but The original mouse v domain of the cloned mVH / mVL showed a strong concentration-dependent response to CHO cells. Drives sexual bonding.

[0057] A preferred optimized anti-CD47 antibody molecule of the present invention may comprise the corresponding murine CDRs or other ( necessarily have the maximum number of human germline substitutions at amino acid positions (e.g., framework) As detailed in the experimental section below, we have investigated the anti-CD47 binding properties and A "maximally humanized" antibody molecule is one that is "maximally humanized" in terms of its specificity and / or other desirable characteristics. We found that the system is not "optimized for" the

[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 thereof that are largely dependent on their properties. Antibody molecules containing intact CDRs and variants with enhanced or decreased activity and / or affinity. and its corresponding antigen-binding portion. Antibodies with the desired binding affinity to one or more of the nucleotides can be obtained. The present invention also includes amino and / or carboxyl terminal fusions ranging in length from 1 to 1000 nucleotides, up to polypeptides containing 1 to 2000 nucleotides. Insertions containing the amino acid sequence of the amino acid sequence 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 bloodstream. 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 may be prepared using 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 be mutated to include polypeptides having an oxidized form. Such translation can be achieved, for example, by adding, removing, 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, for example by amino acid substitution, to modify the Potential proteolytic sites can be removed.

[0061] In an antibody molecule or 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, and 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 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 amino acid sequence may be 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 LCDR2 may have the amino acid sequence: L / K / MV / G -SN / YRA / F / L / SS (SEQ ID NO: 34); LCDR3 may have 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 antigen-binding portion thereof, LCDR1 has the amino acid sequence Column: SSQ / HSF / LL / VHSN / Q / AG / AY / N / S- LCDR2 may 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 / FP / LR / W (SEQ ID NO: 37).

[0064] For example, LCDR1 has the amino acid sequence: RSSQSLL / VHSN / Q LCDR2 may have the sequence: / AGY / NN / TYLH / D (SEQ ID NO: 38); 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 molecules 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: Sequence 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: 51) 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: 56) 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: 61) Sequence 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: Sequence 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 (SEQ ID NO: 69) (HCDR2), GGYTMDI (SEQ ID NO: Sequence 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 (SEQ ID NO: 65) (HCDR2), GGYTMDI (SEQ ID NO: Sequence 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: 51) 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: 75) 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: 66) 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: 51) Sequence 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: 51) Sequence 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: 51) Sequence 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: 51) Sequence 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: 51) Sequence 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: 51) 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: 51) Sequence 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: 51) Sequence 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: 75) Sequence 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: 66) Sequence 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: 66) Sequence 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: 66) Sequence 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: 66) Sequence 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: 66) Sequence 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: 66) Sequence 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: 66) Sequence 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. can be used to define an antibody molecule or antigen-binding portion to include:

[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 number 86); Amino acid sequence INPVDGDTNYNPSFQG (SEQ ID NO: 91) or INPVDGD HCDR2 having TRYSPSFQG (SEQ ID NO: 100); and an 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 with 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 CD4s as disclosed herein. It can be defined using the R definition.

[0123] In certain embodiments of the invention, the antibody molecule or antigen-binding portion is Clone 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 six CDR sequences in any suitable combination.

[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 may 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 may have the amino acid sequence: NTHTPR (SEQ ID NO: 93).

[0126] Antibody molecules or antigen-binding portions as defined herein may contain, for example, glycosylation sites (N-linked or O-linkage), 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 types 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 are known in the art (Zhou et al., 2016, Nature Protocols 1: 1318-1321), e.g., substitutions, deletions, and / or insertions. and then optionally determining (a) binding activity and / or (b) Testing (experimental and / or theoretical) of the loss of PTMs is contemplated.

[0128] For example, VxP037 mouse LCDR1 (as defined herein, i.e., amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9)) at residues 10 (N) and / or 12 (N) has been identified as a putative deamination site. For example, conservative substitutions (e.g. , S, A, Q, or D, etc.) in the equivalent positions 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 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 in the equivalent position of LCDR3 of the present invention by any of the following: Removal is envisioned (for example, 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 sequence 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 (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 also be included. 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 be 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 inert 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 its fragment, or camel antibody [V H H antibody or its fragment), monoclonal antibody The antibody molecule and its methods of construction and use are described in, 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 antigen-binding portions thereof are provided.

[0134] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulators, 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 anticoagulants. 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 auristatins Other examples include antifolates, vinca alkaloids, and anthracyclines. These include plant toxins, other bioactive proteins, enzymes (i.e., ADEPTs), radioactive Isotopes and photosensitizers can also be used in immunoconjugates. In addition, liposomes and polymers can be used as immunoconjugates. 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 destruction of cells. Potential cytotoxins include antibiotics, inhibitors of tubulin polymerization, and enzymes that bind to and destroy DNA. alkylating agents, and enzymes for protein synthesis or essential cellular proteins (protein kinases, phosphatase inhibitors, These drugs disrupt the function of certain enzymes (e.g., ATPases, 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, azacitidine, doxiflur 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. cyclosporin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epinephrine Rubicin, actinomycin, autramycin, azaserine, bleomycin, tamoxacin Xifen, idarubicin, dolastatin / auristatin, hemiasterin, esperama These include isin, and maytansinoids.

[0137] Suitable immunomodulatory agents include antihormonal agents that block hormone action on tumors; suppress cytokine production, downregulate self-antigen expression, or mask 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 antibodies and / or antigen-binding portions thereof is and culturing the host cells of the present invention under conditions suitable for the production of antibodies and / or their antigens from the host cells or culture. and isolating the original binding portion. A method is provided.

[0140] In another aspect of the invention, an antibody molecule of the invention as defined herein or an antigen binding thereof 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 compound is provided.

[0141] An antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or 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 aspect, an antibody molecule or 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 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, cancer of the brain or central nervous system, cancer of the peripheral nervous system, 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 or a compound thereof 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, or Also provided is a pharmaceutical composition of the invention defined as

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

[0146] 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 immunoglobulin of the invention as defined herein 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 antigen-binding portion thereof as defined herein, or a an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a a method for treating a subject, comprising administering an effective amount of a vector comprising the compound of formula (I) 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 aspects, ischemia-reperfusion injury is a serious condition that can occur in organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, and other conditions. , pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resection and reconstruction, adnexal or It 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, disease, lupus, Graves' disease, Hashimoto's thyroiditis, and ankylosing spondylitis. It is possible.

[0150] Also disclosed is 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 molecule as defined herein or a nucleic acid molecule as defined herein, or a vector as defined herein, or a nucleic acid molecule as defined herein Pharmaceutical compositions as defined herein are also provided.

[0151] Furthermore, the present invention is useful in the manufacture of medicaments for the treatment of ischemia-reperfusion injury, autoimmune diseases, or inflammatory diseases. an antibody molecule or 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, or 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 cardiovascular or fibrotic diseases.

[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 a nucleic acid molecule as defined herein, 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 cardiovascular or fibrotic diseases. an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, or a vector as defined herein, or a 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 may be 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 compositions of the present invention may comprise a pharmaceutically acceptable excipient. The vehicle does not induce secondary reactions and facilitates administration of, for example, anti-CD47 antibody molecules over their 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 pharmaceutically acceptable. Vehicles are known and can be adapted by those 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 in 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 compound 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 commercially acceptable excipients, carriers, buffers, 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, bovine serum albumin (BSA), as discussed below. This 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 pyrogen-free, parenteral formulation having suitable pH, isotonicity, and stability. The formulation may be in the form of a physiologically acceptable aqueous solution. Prepare an appropriate solution using an isotonic vehicle such as Gel Injection or Lactated Ringer's Injection It is quite possible to add preservatives, stabilizers, buffers, antioxidants, and and / or other additives may be used, such as buffers, e.g., phosphates, citric acid, Acid salts, other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (chlorides Octadecyldimethylbenzylammonium, hexamethonium chloride, benzalkonium chloride Benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl phenol Parabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexyl 3'-pentanol; 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, and histidine , arginine, or lysine; monosaccharides, including glucose, mannose, or dextrin; sugars and other carbohydrates; chelating agents such as EDTA; sugars, e.g., mannitol, treacle, 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®, or polyethylene glycol (PEG), etc.

[0161] Pharmaceutical compositions containing 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 may be used for prophylactic or preventative treatment (e.g., in an individual). to reduce the risk of developing symptoms, delay their onset, or reduce the severity of symptoms after they have developed treatment of the human or animal body, including treatment prior to the onset of symptoms in an individual to reduce the severity of the condition The therapeutic method may be used in a therapeutic method comprising administering an anti-CD47 antibody molecule to an individual in need thereof. This 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 improvement in 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 patient's clinical condition, 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 under treatment. 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 These may be indicated in the specification or by a physician's prescription depending on the type of medication being administered. The therapeutically effective amount or appropriate amount of the antibody molecule may be appropriately used. 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 prophylactic or therapeutic, the location of the area to be treated, and the type of antibody to be administered. the size and location, the exact nature of the antibody (whole antibody, fragment, etc.), and the detectable molecules attached to the antibody. 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 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 and should be 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 regimen will depend on the pharmacokinetic and pharmacodynamic properties of the compound, the route of administration, and the nature of the condition being treated.

[0165] Treatment may be periodic, with the period between doses being about 2 weeks or more, for example about 3 weeks or more, It may be once every four weeks or more, once every about one month or more, once every five weeks or more, or once every six weeks or more. Treatment may be every 2-4 weeks or every 4-8 weeks. Treatment may be given before and / or after surgery. and / or can 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 auto-injector 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 in individuals. The symptoms may last for several months or longer.

[0168] The present invention also provides a method for the production of 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. The method includes the following steps: (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 containing one or more mutations in the CDRs; generating a phage library of 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 clones having binding specificity to 47 from the screening step (3). 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 assessed, e.g., by determining the presence of a desired gene in the CDR and framework regions. Optionally, generating one or more additional mutations to reduce immunogenicity. Immunogenicity can be assessed, for example, using in silico techniques described herein. This can be assessed by identifying the location of T cell epitopes.

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

[0172] As used herein, the term "CD47" refers to a molecule 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 region. When used, CD47 is expressed in all mammals, including humans, rats, mice, and chickens. The term "CD47" includes variants of human CD47, including the native sequence CD47 of various species. The term "antibody" is used to include isoforms and species homologs. The antibodies of the present invention may be used in non-human species. Cross-reacts with CD47 derived from human leukocytes, especially CD47 derived from cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the antibody is fully specific for human CD47. It is possible that the antibody may not exhibit 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. Anti-CD47 antagonist antibodies refer to antibodies that can inhibit the CD47 signaling pathway and / or downstream pathways. CD47 signaling, such as receptor binding and / or induction of cellular responses to CD47, Block, antagonize, inhibit, or reduce (e.g., suppress) 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 capable of inhibiting CD47 expression (including significantly reducing CD47 expression). "Antigonist antibodies" include antibodies that inhibit CD47 itself and the biological activity of CD47 (including but not limited to). (the ability to enhance the activation of phagocytosis by cells of the myeloid lineage, but not the ability to enhance the activity or biological activity of 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 readily than other receptors. and / or binds to CD47 for a longer period of time, "preferentially bind," "bind," or "interact."

[0175] An "antibody molecule" is an immunoglobulin molecule that contains at least one antigen-recognizing antibody located in the variable region of the immunoglobulin molecule. Target specificity for carbohydrates, polynucleotides, lipids, polypeptides, etc. via sites As used herein, the term "antibody molecule" refers to an immunoglobulin molecule capable of binding to a target antigen. , native polyclonal or monoclonal antibodies as well as any antigen-binding fragments thereof. (e.g., "antigen-binding portion") or single chains thereof, fusion proteins comprising antibodies, and, particularly limited Although not intended to be used in this context, scFv, single domain antibodies (e.g. shark antibodies [V NAR Antibody]Also fragments thereof, 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 containing antigen recognition sites including:

[0176] An "antibody molecule" is any class of antibody molecule, such as IgG, IgA, or IgM (or subclasses thereof). The antibody does not have to be of a particular 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 IgA1. The heavy chain constant regions corresponding to the different classes of α, α- ... The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are called 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. Refers to one or more fragments of an intact antibody that retain the ability to bind. Antigen-binding function of an antibody molecule can be performed 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 domains Fd fragments consisting of the VL and VH domains of a single arm of an antibody; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; Single domain antibody (dAb) fragments and isolated complementarity determining regions (CDRs) are included. .

[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 might vary, the Fc region of a human IgG heavy chain typically consists of the Cys The stretch from the amino acid residue at position 226 or Pro230 to its carboxyl terminus The numbering of residues in the Fc region is that of the EU index 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 is known in the art, the variable regions of heavy and light chains are and connected by three complementarity-determining regions (CDRs), also known as hypervariable regions It consists of four framework regions (FRs) 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 antibodies containing 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 analysis of antibodies and their co-crystallization with antigens, It combines domains used in many different and often contradictory schemes that have been created (Swindell 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" or "harmonized") is given in the review by [see the review by] [ub 22 August 2016]. The "definition" incorporates the lessons of all such previous insights and defines a novel target-binding complementarity mediating Contains all the relevant loop positions needed to find the complete residue context for the .

[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-conservative one that retains functional activity. 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. A suitable example is to substitute one amino acid with a value >= 0 in the BLOSUM62 substitution matrix below. 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, whether eukaryotic, prokaryotic, or fungal. Antibodies or antigen-binding portions thereof derived from a single copy or clone, including phage clones. Preferably, the monoclonal antibodies of the present invention are homogeneous. The nucleotides 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 antibodies or antigen-binding portions 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 that have specificity, affinity, and potency It may be a human immunoglobulin (recipient antibody) in which the residues of the 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. It refers to an antibody molecule or antigen-binding portion thereof derived from a species, e.g., a mouse antibody whose variable region sequences are derived from a mouse antibody. 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 agents, cytotoxicity, and immunological damage. 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. techniques, 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 in one or more of its antigen-binding regions. 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 antigen-binding region or its antigen-binding portion. Similarly, epitopes can be 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 The term "antigenic epitope" as used herein means a molecule that is known in the art. Any known method, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography. 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. 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 the 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 used in biosensor systems such as Biacore® systems. The key 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 an antibody against the antigen CD47 that inhibits 50% of the activity measured in 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 dose that achieves the desired therapeutic result. An effective amount refers to the amount necessary (at the dosage, duration, and means of administration) to achieve the desired effect. At least the minimum amount of an active agent required to provide a therapeutic benefit, but less than a toxic amount .

[0194] As used herein, "inhibit" or "neutralize" refers 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, inhibits, or inhibits the progression or severity of the disease, including the biological activity or binding interaction. "Destruction" means the ability to prevent, suppress, slow down, destroy, eliminate, stop, reduce, or reverse any activity that is harmful to humans or the environment.

[0195] A "host cell" is 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 host 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 necessarily 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 cells are included.

[0196] As used herein, a "vector" refers to a vector that carries one or more genes or sequences of interest in a host cell. A vector refers to a construct capable of delivering and preferably expressing a gene. Examples of vectors include, among others, including, but not limited to, viral vectors, naked DNA or RNA expression vectors, Plasmid, cosmid, or phage vector, DNA associated with cationic condensing 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, unless otherwise specified, the term "treating" refers to such 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 "prevent" refers to the alleviation, inhibition of progression, slowing of progression, delaying of onset, or prevention of a disease. The term "treatment", as used herein, unless otherwise specified, refers to therapeutic treatment as defined above. The term "treating" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, references herein to "treatment" include references to curative, palliative, and This includes preventative and prophylactic treatment.

[0198] Whenever an embodiment is described herein with the phrase "comprising," it should be understood that the phrase "consisting of" is also used. and / or analogous 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 place of them. When described in a group, the invention applies to the group and not just the entire group listed as a whole. It includes each member of the main group and all possible subgroups of the main group, but does not include the group. The present invention also encompasses the main group in which one or more of the members is absent. The invention contemplates the explicit exclusion of any one or more of the group members.

[0200] Unless otherwise defined, all technical and scientific terms used herein are defined by the present invention. The meaning of the term "conflict" is the same as that commonly understood by a person skilled in the art. In all cases, the present specification, including definitions, will control. The word "comprises" or "comprising" Variations of "a" or "b" are intended to mean the inclusion of the stated integer or group of integers, but not of any other integer or group of integers. It is understood that this does not mean excluding groups. 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 techniques such as molecular biology (including recombinant techniques), microbiology, and the like, unless otherwise indicated. , using conventional techniques of cell biology, biochemistry, and immunology, which are within the skill of the art. do. [Example]

[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 antagonistic and optimized anti-CD47 antibodies. These anti-CD47 antibodies are well expressed, biophysically stable, highly soluble, and suitable for It has the greatest identity with the human germline.

[0204] Materials and Methods IgG cloning, transient expression, and purification The DNA sequence encoding the antibody v domain is then cloned into another promoter via restriction-ligation cloning. The antibodies were cloned into separate IgG heavy and light chain expression cassettes in a plasmid vector. , expressed in two forms of human IgG: IgG4 with the hinge-stabilizing S228P mutation Lower hinge mutations that minimize IgG4 and Fcγ receptor-driven effector functions IgG1 Null-IgG1 with L234A / L235A / G237A. Manufacturer's profile Transient transfection with endotoxin-free IgG expression plasmid preparations according to protocol. After transfection, IgG was expressed in HEK-293expi cells. was purified using a one-step protocol: 1 pre-equilibrated with PBS (pH 7.4) Conditioned medium was added (undiluted) to a 5 ml ProA Sepharose column. After washing with 100 mL 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 titer measurement binding ELISA To coat the GreinerBio-One High bind ELISA plate, use target proteins. Protein was 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 with PBS (380 After blocking with 1% BSA 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 on a thermometer reader.

[0206] Anti-CD47 antibodies were tested for polyreactivity by ELISA. and non-target antigens were plated onto a 96-well Nunc maxisorp plate at 100 ng / well in carbonate buffer. The plates were then coated with PBS overnight at 4°C. The plates were then washed three times with PBS and diluted with 1 mL of PBS. After blocking with 100% BSA, the cells were washed three times with PBS-Tween 20. Next, a diluted solution of the primary antibody was added. A series of dilutions was applied, and the plate was washed three times with PBS-Tween 20. Then, goat anti-human kappa antibody was added. Secondary antibody, ribosomal HRP (1:4,000), was applied. The wells were then resuspended in PBS-Tween 2. Wash three times with PBS and twice with PBS. Add 100 μl of TMB peroxidase substrate per well. The reaction was stopped by adding 100 μl of 2 M H2SO4, and the absorbance was adjusted to 45 The ELISA analysis of IgG binding 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 large-scale 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 cynomolgus monkey). Coat the beads with PBS (Isal), wash the beads three times with PBS, and then add 5% skim milk protein. Phage selection was performed by resuspending the cells in PBS (pH 7.4) (MPBS) containing 1000 kJ / ml. 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 identify the optimal blocking factor for 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 (PBST) for 1 hour at room temperature, the cells were washed three times with PBST. Next, biotinylated human, mouse, or cynomolgus IgG were added 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 streptavidin-HRP was added at room temperature. The plate was then washed three times with PBST and twice with PBS. After washing twice, 100 μl of TMB was added per well. 100 μl of 2M H2S The reaction was stopped by adding 04 / well and the OD measured at 450 nm on a plate reader. I read.

[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 NIH trademark to identify peptides that bind promiscuously to human MHC class II with high affinity. The VL and VH sequences of the primary leads were analyzed. The peptides are intended to identify T cell epitopes, which are high-risk indicators of clinical immunogenicity of drug proteins. The iTope® software is believed to correlate with the presence of peptides. The amino acid side chains and specific regions within the open-ended binding groove of 34 human MHC class II alleles Favorable interaction with the binding pocket (specific pocket positions: p1, p4, p6, p7, and p9) These alleles are those 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 at position 8 It contains a "closed" conformation in which glycine at position 3 is replaced by valine. Residue positions are determined by 9-mer peptides with eight amino acid overlaps spanning the test protein sequence. This process is achieved by the in silico generation of proteins that bind to MHC class II molecules. It discriminates with high accuracy between peptides that bind and those that do not.

[0210] Furthermore, in vitro human T cell epitope mapping analysis of other protein sequences has shown For matches to previously identified T cell epitopes, TECD® (T Cell Epitope Sequences were analyzed using a 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) A total of 1.25 × 10 HL-60 cells were labeled using a tert-butyl ether (Tet-B) cell tracer dye. 6 Labeled HL-60 cells were incubated for 1 hour at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours. The cells were pre-incubated in the presence of anti-CD47 antibody in a well plate. After that, 5 x 10 5 CD14-positive cells were added to each well and further incubated under the same culture conditions for 1 hour. The cells were then collected by vigorous pipetting and resuspended in ice-cold 4% paraformaldehyde. Fixation with aldehyde for 10 min and immunoblotting 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 minutes at room temperature and then resuspend in 4% paraformaldehyde. The cells were then fixed in aldehyde for an additional 5 minutes.

[0212] Cells were analyzed for side and forward scatter properties along with CFSE and AF647 fluorescence intensity data. Record at least 1 x 10 cells and analyze with a BD Fortessa flow cytometer. 4 A Data was captured until an F647 positive event was recorded. Analysis was performed using FlowJo software (version 10.4.2). Cell debris was gated out by scatter characteristics (SSC area vs. FSC area). Single cells were also , gated by SSC area vs. SSC height, and then by FSC area vs. FSC height. From the remaining single cell population, CFSE and CD14 double positive cells were isolated from the 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 cells. 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 CDR grafting was used to generate the antagonistic murine anti-CD47 IgG VP037 (mVH / mV L; see WO 2014 / 093678 and Table 2) were first isolated from human germline Optimal drug-like properties were obtained by incorporating immunoglobulin v domain framework sequence scaffolds into cytosolic systems. To focus our engineering efforts on a final lead therapeutic IgG compound with , known to have good solubility and are frequently expressed in the 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 murine 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 is prepared as acceptor frameworks suitable for non-human CDR grafting. There are still problems predicting whether it will work. This can lead to loss of target binding function, protein stability issues, or ultimately impaired IgG expression. Therefore, the IGHV5-51 / IGKV2-28 graft is It 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 sequences are VL-VH s Mutagenesis library by bulk oligo synthesis and assembly coupled to 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 produce 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. showed that the coding positions were effectively sampled at a frequency of approximately 50%. The branches were rescued using helper phage M13 and isolated into three separate branches: A, B, C, D, E, E, F ... B, C: Selected with biotinylated human, mouse, and cynomolgus monkey CD47-Fc proteins was carried out.

[0216] Post-selection screening (Figure 1) and DNA sequencing confirmed the human content within the CDRs. Significantly increased presence of 854 unique human and mouse CD47-binding scFv clones The presence of these genes was revealed, but the framework sequences remained completely germline. Among the 854 clones, germline mutations were observed in all CDRs (Table 3). The lead clone was assayed by ELISA for binding to both human and mouse CD47-Fc. The sequences were ranked based on the level of CDR germline relative to the signal (Figure 1). The v-domains of the top four ranked clones and the two most humanized heavy chains and The fifth clone ("VH-A1 / VL-B1"), which combined the VH and light chain v domains, It was 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 that maximize humanization. Therefore, 854 binding signals for human and mouse proteins remained. Using sequence-specific hits, we identified CDRs in this functionally characterized population of mice. The amino acid retention frequency was analyzed. The positional amino acid retention frequency was V H and V L Found in the domain The RF values ​​were expressed as the percentage of residues that were identified (Fig. 2A and B). Mouse residues with RF < 75% were identified as residues that ... 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 was developed that combined the five most humanized CDRs observed in the population. Necrolone ("TTP" = Theoretical Possible Total) was also produced. The clones were generated by gene synthesis (the four library-derived clones mentioned above and the positive pair). (along with a reference mVH / mVL and a negative control isotype-matched non-CD47 reactive v-domain) , cloned into a human expression vector 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 the

[0219] Specificity and potency characteristics of lead IgG The purified IgG described above was tested in a direct titration ELISA format against human, mouse, and cynomolgus monkeys. The binding of CD47-Fc to the clone was tested (Figure 3). Lone MH, A-D5, and D-H3 bind to all three orthologs of CD47 Two clones (G-B6 and F-E7) retained their affinity to mouse CD4. 47, and one clone (VH-A1 / VL-B1) showed reduced binding to both human and mouse It 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 Both D-H3 and D-H3 inhibited SIRPα- showed concentration-dependent inhibition of human, mouse, and cynomolgus CD47 interactions with Fc In particular, VH-A1 / VL-B1 IgG1 potently inhibits 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 clone has 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 the IgG (OD450nm<0.1). One notable exception was , VH-A1 / V1-B1 clone, which again targets human and cynomolgus CD47 showed strong binding to mouse CD47 but no signal for mouse CD47.

[0222] Flow cytometric analysis of the binding specificity of lead IgG at cell membranes Concentration-dependent binding of antibodies against CD47 to the cell surface was determined by flow cytometry. CHO-K1 cells were transfected with human, mouse, or cynomolgus monkey 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) forms. Together with commercially available mouse anti-human CD47 monoclonal antibody MS1991, 100,000-24 Human, cynomolgus monkey, or wild-type control ("wt", i.e., thyroid) were tested at concentrations ranging from 1000 to 1000 ng / ml. The antibodies were tested for binding to CHO-K1 (transfected or untransfected). All IgGs except the control showed concentration-dependent binding to human and cynomolgus monkey CD47+ cells. The maximum MFI in each case is shown. The signal was more than 10-fold higher than that observed for CHO-K1 w 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 low concentrations of 0.05 µg / ml, with a signal more than 10-fold higher than the "no antibody" negative control. This background binding was due to the fact that the original murine antibody VxP037 reacted with mouse CD4 47, as well as cross-reactivity to hamsters. Antibodies for clinical use are usually produced in CHO cell culture, so Minimizing this cross-reactivity to mutant CD47 is desirable 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 certain substances (which must be removed from the formulation).

[0223] To examine the binding of lead IgG to CD47+ human cancer cells, HL60 cells (human IgG) were cultured. Myeloid leukemia-derived IgG4-associated leukemia (MMLL) 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 demonstrated the ability to bind IgG intended for therapeutic use to several key biological substrates. This conjugate has low bioavailability and a short in vivo half-life, which increases the risk of poor performance in patients. Three such biological substrates are known to be indicators of insulin resistance. Therefore, these three substrates were used to perform ELISA experiments. The binding of an IgG1 null version of the optimized lead antibody was investigated using a 100% IgG1-coated platelet. The binding signals of these human IgG-based antibodies were found to be polyreactive and poorly performing. Known and progressing in clinical trials (bococizumab and briakinumab, human IgG1 analogs) The results were compared with a "positive control" human IgG antibody that had been stopped. In this study, the IgG1 ustekinumab analogue reacts with the same therapeutic target as brikinumab but is more effective. 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 was observed against the IgG1 antibody, whereas the negative control showed low reactivity. Finally, all IgG1 null lead proteins tested were negative for all three substrates. The optimized clones A-D5, G-B6 showed binding below 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, as a clone derived from the library, the A-D5 sequence was Many non-germ cell types were potentially unnecessary, as evidenced by the data found in A and 2B. 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 variable domains of A-D5 were simultaneously optimized. In an attempt to increase the size of the protein and minimize the content of high-risk deamination motifs in the protein, A series of designer clones were created. This experiment was carried out in two phases. Clones A-D5.1 to A-D5.10 contained the CDR sequences outlined in Table 4. The clones were expressed and purified in an IgG1 null form, and all CD47 orthologues were expressed in an IgG1 null form. for target binding by LISA (Fig. 9A, B, C) and for all orthologs Neutralization of the CD47-SIRPαB interaction (Fig. 10A, B, C) was investigated. At this stage, these improvements in the human germline 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 the 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 performed on the best-performing mutant from the first phase, A-D5.4. These nine mutants were designed to mimic the "NG" deamination risk motif. The "N" in the fragment may be replaced with conservative and non-conservative mutations such as S, G, A, and T, or not. or a further modification of CDR-H2 of AD.4 with or without replacing the "G" residue with A. These clones were again expressed and purified in an IgG1 null form, and all Target binding by ELISA for all CD47 orthologs (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 two additional residues. Furthermore, mutations that attempt to remove the "NG" motif in CDR-L1 via N substitutions The study was successful and clone A-D5.16 was identified, which contains a non-conservative N to A mutation. and CD47-SIRP target binding ELISA against both A-D5 and mVL / mVH. The maximal humanized CD4+ antibody yielded 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 characteristics was achieved.

[0227] Next, A-D5 derivatives A-D5.4 and A-D5.16 were developed by the mutation and reselection process. To ensure that there was no loss of target specificity during the assay, I was analyzed to maintain binding specificity. 14 purified human immunoglobulins from the immunoglobulin superfamily were also tested; 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, and no detectable activity against other proteins. There was no significant binding (OD450nm<0.1). "Development" ELISA shown in Figure 14 In our analysis, the positive control antibody showed the expected strong reactivity with all three substrates; The negative control showed low reactivity. Importantly, A-D5.4 and A-D5.16 Ig Both G1 null lead proteins showed binding results below those of the negative controls for all three substrates. This finding was supported by the optimized clones A-D5, A-D5.4, and This highlights 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 the clonal mVH / mVL were either IgG1 null or IgG4 forms. 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 little or no signal was mediated (Figure 15). The mVH / mVL, A-D5, A-D5.4, and A-D5.16, all contain human CD4 It showed strong binding to 47+ HL60 cells (Fig. 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 improved. It showed that it was being 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 evaluate 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 designed to measure potential "fits" and interactions with MHC class II molecules. The peptide scores calculated by the software ranged from 0 to 1. Produces high average binding scores (>0.55 by the iTope® scoring function). Peptides with >50% (i.e., 17 of 34 alleles) M HC class II binding peptides have high binding affinity (score > 0.6), and such peptides 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 (The number does not exceed 0.6.) Further analysis of the sequences was performed using TCED®. This sequence was used to perform a BLAST search on TCED®, performed by Abzena Ltd. Irrelevant T-cell responses stimulated in previous in vitro T-cell epitope mapping studies were High sequence homology between peptides (T cell epitopes) from various proteins / antibodies was identified. .

[0230] The peptides were divided into four classes: high affinity foreign ("HAF" - highly immunogenic) Low Affinity ("LAF" - Low Immunogenicity Risk), TECD+ (previously TCED (registered trademark) epitopes identified in the target ("GE"-M) database), and germline epitopes ("GE"-M Human germline peptide sequence with high HC class II binding affinity. Germline epitope The 9-mer peptide has been validated in previous studies using a wide range of germline peptides. As such, these peptides are unlikely to be immunogenic due to T cell tolerance (i.e., (Recognized as "self" by the host). Importantly, such germline v-domain endogenous The pitope (further supported by similar sequences in human antibody constant regions) is essential for 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 efficient 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 requiring murine residues in the framework. We successfully selected antibodies with the same binding affinity (Table 2). Multiple HAF and LAF epitopes found within the framework of the domains were all The GE epitope was absent from the library-derived and designer leads (Figure 17). The amount of leukocytes also increased significantly (3 to 14 or more for all leads), especially for the lead cluster. In the VH region of the lone, GE content increased from 0 to 9 in all reads, and TCED+ epitopes The number of groups decreased from 3 to 2 for all leads (Table 8). The epitope was also eliminated by germline mutations found in the CDRs of the lead clone For example, the TCED+ peptide found in LCDR-1 of mVH / mVL, "LVH SNGNTY" (SEQ ID NO: 116) (and therefore any previously humanized by CDR grafting) The VxP037 form of the nucleotide sequence was found to contain the mutation V>L at position 2 and the mutation V>L at position 3 in most lead clones. The LC of the mVH / mVL sequence was also excluded by N>Y at position 7 (Tables 4 and 5). DR2 consists of three foreign molecules spanning the VL framework 2-LCDR2-framework 3. These encoded epitope peptides, including 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 completely addressed this issue. The whole sequence from mVH / mVL "LLIYKVSYRFSGVPDR" (sequence No. 120) maintains 100% identity in the IGKV2-28 grafted sequence (Table 2). For clones A-D5, A-D5.4, and A-D5.16, there was one H AF and two LAF peptides were still found, but the HAF sequence “YRFSGVPDR” ( SEQ ID NO: 118) was deleted by the mutation Y>N at position 1. However, surprisingly , 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 ameliorate all predicted foreign epitopes in this region. (HAF, LAF, and TECD+ peptides are not predicted), but two additional GE sequences are also present. The lead clone A-D5 and all 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. This disrupts the loop structure and was found to eliminate the immunogenic risk of this peptide.

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

[0233] Cancer cell phagocytosis CD47 inhibition in driving phagocytosis of HL60 human cancer cells by human primary macrophages As shown in Figure 20A, 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 efficacy 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 compared to mVH / mVL. This phenomenon was then examined in human macrophages from four different donors. IgG4 A-D5 and mVH / mVL were then examined (Figure 20B). This confirms the higher potency shown in Figure 20A, with A-D5 being the most potent in all donors. It was significantly more potent at 1000 kJ / kg / day (Fig. 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 invention may be readily understood by those skilled in the art. and that various modifications and changes can be made thereto without departing from the scope thereof; and will recognize that such modifications are expressly contemplated herein. LIMITATIONS ON THE PARTICULAR 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, wherein the antibody molecule or antigen-binding portion comprises 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 NTHTTPR (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) 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 NTHTTPR (SEQ ID NO: 93) (LCDR3), respectively.

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

4. 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 claim 1 , wherein the VH is: (a) amino acid residues 1-23 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. An antibody molecule or antigen-binding portion thereof comprising a sequence having at least 90% sequence identity to any of the above.

8. 8. The antibody molecule or antigen-binding portion of claim 7, wherein the VH is: (a) amino acid residues 1-23 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 1-24 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. An antibody molecule or antigen-binding portion thereof comprising a sequence having at least 90% sequence identity to any of the above.

10. 10. The antibody molecule or antigen-binding portion of claim 9, wherein the VL comprises: (a) amino acid residues 1-24 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. 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. The antibody molecule or antigen-binding portion of any one of claims 1 to 12, comprising an immunologically inert constant region.

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

15. An immunoconjugate comprising an antibody molecule or 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 that result 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. 20. A composition for enhancing an immune response in a subject, the 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, or a pharmaceutical composition as defined in claim 20.

22. 20. Use of an antibody molecule or 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 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 a composition comprising an immunoconjugate as defined in claim 15, or a composition comprising a nucleic acid molecule as defined in claim 16, or a composition 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, stomach cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, 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 gland cancer, osteosarcoma, chondrosarcoma, and cancer of the blood tissue.

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

26. Use of an antibody molecule or 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 ischemia-reperfusion injury, an autoimmune disease, or an inflammatory disease.

27. Use of an antibody molecule or 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 disease or a fibrotic disease.

28. 28. The use of 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. 20. 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 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.

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