Novel Anti-a2AP antibodies and uses thereof

Abstract

To provide an isolated antibody or antigen-binding fragment thereof binding to human A2AP.SOLUTION: The invention provides an isolated antibody or antigen-binding fragment thereof capable of binding to A2AP and inhibiting the activity of A2AP, where the isolated antibody or antigen-binding fragment thereof does not inhibit plasmin activity. In one aspect, the isolated antibody or antigen-binding fragment is characterized by i) cross-reacting with rabbit and / or cynomolgus A2AP, ii) not inhibiting human plasmin activity, and iii) increasing plasmin-mediated clot lysis in the presence of A2AP.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an isolated antibody or antibody complex that binds to human α2 antiplasmin (A2AP). The present invention provides an isolated antibody or antigen-binding fragment of the present invention. The ments were: i) rabbit and / or cynomolgus A2AP; ii) does not bind to human plasmin / does not inhibit human plasmin activity; iii) ) does not convert A2AP from a serine protease inhibitor to a serine protease substrate; and iv) Dissociation constant (KD) ≦100nM, ≦50nM, ≦25nM, ≦10nM, ≦1nM, or binds to human A2AP having a sequence of amino acids 40 to 491 of SEQ ID NO:1 at ≦0.5 nM; v) ≦500nM, ≦250nM, ≦100nM, ≦50nM, ≦25nM, ≦10nM , ≦1 nM, or ≦0.5 nM. vi) ≦500 nM, ≦250 nM, ≦100 nM, ≦50 EC50 of ≦25 nM, ≦10 nM, ≦1 nM, or ≦0.5 nM, 1, inhibiting the activity of human A2AP, which is a region of amino acids 40 to 491 of A2AP; and / or inhibiting the presence of A2AP. Increases plasmin-mediated clot lysis in the presence of

[0002] The present invention further provides isolated nucleic acid sequences encoding said antibodies or antigen-binding fragments. and vectors containing the same, isolated antibodies expressing said antibodies or antigen-binding fragments. The present invention also relates to a method for producing said antibody or antigen-binding fragment, ... and a method for producing said antibody or antigen-binding fragment. Pharmaceutical compositions and kits comprising the antigen-binding fragments are provided.

[0003] The antibody according to the present invention is useful for treating ischemic stroke, acute coronary syndrome, peripheral arterial disease, myocardial infarction, deep venous infarction, and other conditions. Partial or complete vascular thrombosis, such as venous thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis It can be used to treat diseases associated with ischemic events resulting from occlusion. [Background technology]

[0004] 2. Background of the Invention The formation of blood clots in blood vessels is a major cause of ischemic stroke, acute coronary syndrome, peripheral artery disease, myocardial infarction, and deep venous infarction. This can lead to multiple serious diseases such as venous thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis. The initiation and persistence of a clot depends on the rate of its formation by fibrin and platelets. The current standard of care is to treat thrombosis by The current focus of our research is on anticoagulation for chronic prevention of Despite all the improvements with oral anticoagulants (NOACs), there is still a risk of bleeding. As such, it has only an indirect effect on clot resolution.

[0005] The primary treatment goal for patients suffering from an ischemic or embolic event is the timely restoration of blood flow. Intravenous (IV) recombinant tissue plasminogen activator (tPA) and mechanical thrombosis Reperfusion therapy using thrombolysis, including endovascular treatments such as thrombolysis (MT), has been used to treat ischemic It is the only approved treatment for patients who have suffered a stroke. However, both Treatment options are limited, particularly with short-acting tPA, which is associated with a strong increased risk of bleeding. Its use is limited due to its addictive properties, neurotoxic effects, and limited time frame of effectiveness.

[0006] In contrast, inhibition of the major endogenous plasmin inhibitor α2-antiplasmin (a2Ap) It has been reported that it improves pathological parameters without the risk of bleeding. Therefore, inhibition of α2-antiplasmin may accelerate clot lysis and prevent (secondary) thrombotic events. This could be an innovative therapeutic option for the prevention of

[0007] α2-antiplasmin is a member of the serpin superfamily. It is It is the major physiological inhibitor of the serine protease plasmin. Plasmin, in turn, is involved in the synthesis of various It is an important enzyme involved in the degradation of other proteins and in fibrinolysis (Tone M, Kikuno R, Kume-Iwaki A, Hashimoto-Gotoh T, Structure of human alpha 2-plasmin inhib itor deduced from cDNA sequence, J Bioche m.1987;102(5):1033-1041;Silverman GA, Bir d PI, Carrell RW et al. The serpins are an expa nding superfamily of structurally simila r but functionally diverse proteins.Evol ution,mechanism of inhibition,novel func tions,and a revised nomenclature.J Biol Chem.2001;276(36):33293-33296).

[0008] α2-antiplasmin is a 491 amino acid sequence with a 27 amino acid signal peptide. The secreted form is synthesized as a precursor of the short propeptide (residues 28-39) and the mature The liver and kidney are the major sites of a2Ap production, but Other tissues, such as muscle, intestine, central nervous system, and placenta, also express its mRNA at moderate levels. Express.

[0009] The plasma concentration of α2-antiplasmin is approximately 1 micromolar (~70 micrograms / m l) and its half-life in plasma has been determined to be 2.6 days (Collen D, Wim an B, Turnover of antiplasmin, fast-acting plasmin inhibitor of plasma, Blood, 1979; 53(2):313-324).

[0010] Experimental therapeutic inactivation of α2-antiplasmin reduces microvascular thrombosis, ischemic brain injury, and cerebral Significantly reduces swelling, cerebral hemorrhage and death after thromboembolic stroke (Reed GL, H oung AK, Wang D, Microvascular thrombosis, fibrinolysis, ischemic injury, and death a cerebral thromboembolism are affecting ed by levels of circulating α2-antiplasm in, Arterioscler Thromb Vasc Biol, 2014;34 (12):2586-2593).

[0011] Mimuro et al. describe an A2AP antibody, JPTI-1. The binding activity of JPTI-1 to the adsorbed A2AP-plasmin complex was significantly higher than that of free A2AP. JPTI-1 inhibits the formation of the A2AP-plasmin complex. However, Mimuro et al. (Mimuro J et al., Blood , 1987;69:446-453).

[0012] One of the best characterized known antibodies in the prior art is described by Reed et al. 77A3 (Reed GL, Functional characterization tion of monoclonal antibody inhibitors o f alpha 2-antiplasmin that accelerate fi brinolysis in different animal plasmas, H ybridoma, 1997;16(3):281-286;WO98 / 12334, W O98 / 12329), an antibody derived from a classical mouse immunization approach. The fact that it is of murine origin has at least prompted a humanization campaign.

[0013] Other function-blocking anti-α2-antiplasmin antibodies have been reported using non-human antibodies, e.g., goat-derived Ser pin F2 / α2-antiplasmin antibody (R&D, Catalog No. AF1484-SP Due to their origin as IgG antibodies, their specificity, e.g., mouse-specific antibody clones, Sequences 27C9, 4H9, and CBYY-I0956 (MyBioSource, catalog Accession numbers MBS135095 and MBS135076, Creative Biolab s, Catalog No. CBMAB-I2124-YY) or because these antibodies are polyclonal. Due to the fact that it is a local 42) and is not suitable as a therapeutic agent.

[0014] Therefore, the previously unmet need for ischemic events resulting from partial or complete vascular occlusion is currently being addressed. There is a great need for new therapeutic A2AP antibodies useful in treating elephant-related diseases. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] WO 98 / 12334 [Patent Document 2] International Publication No. 98 / 12329 [Non-patent literature]

[0016] [Non-Patent Document 1] Tone M, Kikuno R, Kume-Iwaki A, Hashimoto-Gotoh T, J Biochem.1987;102(5):1033-1041 [Non-Patent Document 2] Silverman GA, Bird PI, Carrell RW et al. J Biol Chem. 2001;276(36):33293-33296 [Non-Patent Document 3] Collen D, Wiman B, Blood, 1979;53(2):313-324 [Non-Patent Document 4] Reed GL, Houng AK, Wang D, Arterioscler Thromb Vasc Biol, 2014;34(12):2586-2593 [Non-Patent Document 5] Mimuro J et al., Blood, 1987;69:446-453 [Non-Patent Document 6] Reed GL, Hybridoma, 1997;16(3):281-286 Summary of the Invention [Problem to be solved by the invention]

[0017] Subject of the Invention In view of the prior art, it is an object of the present invention to provide novel A2AP antibodies that overcome the shortcomings of the prior art. It is an object of the present invention to provide therapeutic A2AP antibodies. In particular, it is an object of the present invention to provide a method for the production of high affinity human A2AP antibodies. The present invention provides a novel A2AP antibody that is a compatible binder. They are cross-reactive to heron and / or cynomolgus monkey A2AP. Preferably, the antibodies are non-immunogenic in vivo, i.e., they are human or humanized. The A2AP antibody is selective for A2AP and, in particular, does not bind to human plasmin and They also do not inhibit plasmin-mediated clot lysis in the presence of A2AP. It is possible to do so.

[0018] Such novel A2AP antibodies may be useful in treating ischemic stroke, acute coronary syndrome, peripheral artery disease, and myocardial infarction. Partial or partial thrombosis, such as infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis or shunt thrombosis This represents a major advance in the treatment of diseases associated with ischemic events resulting from complete vascular occlusion. do. [Means for solving the problem]

[0019] Summary of the Invention These and other objects are accomplished by the teachings of the present invention. Has specific affinity for typlasmin and is capable of delivering a therapeutic benefit to a subject Based on the discovery of novel antibodies.

[0020] Thus, in a first aspect, the present invention provides an isolated antibody that binds to human A2AP. or an antigen-binding fragment thereof, wherein said isolated antibody or antigen-binding fragment thereof The combined fragment is i) cross-reacts with rabbit and / or cynomolgus monkey A2AP; ii) does not inhibit human plasmin activity; iii) does not convert A2AP from a serine protease inhibitor to a serine protease substrate; iv) Dissociation constant (KD) ≦100nM, ≦50nM, ≦25nM, ≦10nM, ≦1nM or ≦0.5 nM, binding to human A2AP of the sequence of amino acids 40 to 491 of SEQ ID NO:1. Combined; v) ≦500nM, ≦250nM, ≦100nM, ≦50nM, ≦25nM, ≦10nM , ≦1 nM, or ≦0.5 nM. Binds to human A2AP on the column; vi) ≦500nM, ≦250nM, ≦100nM, ≦50nM, ≦25nM, ≦10n M, ≦1 nM, or ≦0.5 nM. Inhibits the activity of human A2AP; and / or In the presence of A2AP, plasmin-mediated clot lysis is increased.

[0021] The isolated antibody or antigen-binding fragment according to the invention is a function-blocking anti-α2-antibody. Plasmin antibodies or antigen-binding fragments, which are useful in treating thrombolytic disorders and other thrombolytic disorders. typical of in vitro immunization without causing undesirable side effects such as bleeding. Induces accelerated clot lysis in vitro and in vivo Thus, the isolated antibody or antigen-binding fragment according to the invention is useful for treating ischemic Stroke, acute coronary syndrome, peripheral arterial disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis ischemic events resulting from partial or complete vascular occlusion, such as thrombosis or shunt thrombosis The isolated antibody or antigen-binding fragment of the present invention can be used to treat diseases associated with the The fragments may further be used in the diagnosis of A2AP-related disorders.

[0022] In a further embodiment, the present invention provides a method for the detection of A2AP-related inflammatory cytokines. The present invention relates to an isolated antibody or antigen-binding fragment thereof capable of inhibiting The isolated antibody or antigen-binding fragment thereof does not inhibit plasmin activity.

[0023] In a further aspect, the present invention provides a method for the preparation of a compound that binds to human A2AP and inhibits the activity of A2AP.

[0023] In a further aspect, an isolated antibody or antigen-binding fragment thereof is provided, comprising: wherein the isolated antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:1. Binds to an epitope of A2AP containing 402-408 (SRMSLSS).

[0024] In a further aspect, the present invention relates to the isolated antibody or is an isolated antibody or antigen-binding fragment thereof that competes with the antigen-binding fragment. Regarding.

[0025] In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. The present invention relates to an antibody conjugate comprising the ment.

[0026] In a further aspect, the present invention relates to a method for the preparation of a composition comprising an antibody or antigen-binding fragment according to the present invention. The present invention relates to an isolated nucleic acid sequence which

[0027] In a further aspect, the present invention relates to a vector comprising the nucleic acid sequence according to the invention.

[0028] In a further aspect, the present invention relates to an antibody or antigen-binding fragment according to the invention. An isolated vector expressing and / or comprising a nucleic acid according to the invention or a vector according to the invention. This relates to cells that have been cultured.

[0029] In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. A method for producing a marker comprising culturing a cell according to the invention and, optionally, culturing an antibody or or purifying an antigen-binding fragment thereof.

[0030] In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. The present invention also relates to a pharmaceutical composition comprising an antibody conjugate according to the present invention.

[0031] In a further aspect, the present invention relates to a compound according to the present invention for use in the treatment or prevention of a disease. or a conjugate according to the present invention; Or the pharmaceutical composition according to the present invention.

[0032] In a further aspect, the present invention relates to an isolated antibody according to the present invention for use as a diagnostic agent. The present invention relates to an antibody or antigen-binding fragment thereof or a conjugate thereof according to the present invention.

[0033] In a further aspect, the present invention provides a method for treating ischemic events resulting from partial or complete vascular occlusion. Associated disorders or diseases, such as ischemic stroke, acute coronary syndrome, peripheral arterial disease, myocardial infarction Used to treat or prevent deep vein thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis. an isolated antibody or antigen-binding fragment according to the invention for use in a method for treating a disease caused by infection with a rabies virus; The present invention relates to a conjugate or a pharmaceutical composition according to the present invention.

[0034] In a further aspect, the present invention relates to a method for treating, in particular, coagulation cascade inhibitors, anticoagulants, and hemolytic agents. Simultaneously, separately, and / or with one or more further therapeutically active compounds selected from platelet aggregation inhibitors or isolated antibodies or antigen-binding fragments according to the invention for use in sequential and combined applications. The present invention relates to a fragment, a conjugate according to the present invention or a pharmaceutical composition according to the present invention.

[0035] In a further aspect, the present invention provides an isolated antibody or antigen-binding fragment according to the present invention. The present invention also relates to a kit comprising a fragment or a conjugate according to the present invention and instructions for use. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention The present invention can be further understood by reference to the following detailed description of the invention and the examples contained therein. can be more easily understood.

[0037] definition Unless otherwise defined, all technical and scientific terms used herein are defined by the The term has the meaning commonly understood by a person skilled in the art to which the invention pertains. The following references will provide those of skill in the art to which this invention pertains with a general understanding of many of the terms used in this invention: Definitions may be provided, and such definitions may be consistent with commonly understood meanings in the art. The present invention may be used and referenced to any extent permitted by applicable law. Such references include, but are not limited to, those cited herein. Examples include, but are not limited to, Singleton et al., Dictionary of f Microbiology and Molecular Biology (Second edition, 1994);The Cambridge Dictionary of Scie nce and Technology (ed. Walker, 1988);Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3rd ed., 1999); and C ellular and Molecular Immunology, Eds.Abb as, Lichtman and Pober, 2nd Edition, WBSaunders C company. has the meaning commonly understood in the art and available to one of ordinary skill in the art Please consult any additional technical resources that provide definitions of terms used herein. For purposes of the present invention, the following terms are further defined. As used herein and in the appended claims, The singular forms "a" and "the" refer to plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a gene" includes one or more A reference to a gene includes equivalents thereof known to those skilled in the art, and so forth.

[0038] In the context of this specification, the words "comprises" or "comprises" are used interchangeably. The term "including, but not limited to" means This term means "not limited to." It does not identify the presence of stated features, elements, integers, steps or components. , the presence of one or more other features, elements, integers, steps, components, or groups thereof. does not exclude the presence or addition of any The more restrictive terms "consisting of" and "essentially consisting of" are used. "essentially consisting of" In the embodiments, the term "comprises" is used throughout this application and particularly in the claims. can be replaced with the term "consisting of."

[0039] In this context, the use of "about" or "approximately" )" refers to 80% to 120% or 90% to 110% of a given value or range. means a range of 95% to 105% inclusive.

[0040] The terms "polypeptide" and "protein" are used to refer to a polymer of amino acid residues. The terms are used interchangeably herein. Amino acid polymers that are artificial chemical mimics of the corresponding natural amino acids, as well as natural amino acid polymers. Applies to polymers and non-natural amino acid polymers. A peptide sequence also implicitly encompasses conservatively modified variants thereof.

[0041] As used herein, "A2AP" refers to "SerpinF2" (a member of the serpin family). Leadership Member 2), also known as AAP, API, PLI, or ALPHA-2-PI A2AP is a member of the serpin superfamily. It is a major physiological inhibitor of the serine protease plasmin. A2AP is expressed as a 491 amino acid precursor with a 27 amino acid signal peptide. The secreted form is synthesized as a short propeptide (residues 28-39) and a mature chain (residues 40- 491). The reference sequence of human A2AP is available from UniProtKB / Swiss-Pro The sequence of the sequence of the nucleic acid sequence of the present invention is available from the t database under the accession number P08697-1 (SEQ ID NO: 1) and is designated as SEQ ID NO: 1. The propeptide (residues 1-27), the propeptide (residues 28-39) and the mature chain (residues 40- 491) (numbering is according to the methionine at position 1).

[0042] Human A2AP (SEQ ID NO:1): MALLWGLLVLSWSCLQGPCSVFSPVSAMEPLGRQLTSGPNQEQVSPLTLLKLGNQEPGGQTALKSPPGVCSRDPTPEQTH RLARAMMAFTADLFSLVAQTSTCPNLILSPLSVALALSHLALGAQNHTLQRLQQVLHAGSGPCLPHLLSRLCQDLGPGAF RLAARMYLQKGFPIKEDFLEQSEQLFGAKPVSLTGKQEDDLANINQWVKEATEGKIQEFLSGLPEDTVLLLLNAIHFQGF WRNKFDPSLTQRDSFHLDEQFTVPVEMMQARTYPLRWFLLEQPEIQVAHFPFKNNMSFVVLVPTHFEWNVSQVLANLSWD TLHPPLVWERPTKVRLPKLYLKHQMDLVATLSQLGLQELFQAPDLRGISEQSLVVSGVQHQSTLELSEVGVEAAATSIA MSRMSLSSFSVNRPFLFFIFEDTTGLPLFVGSVRNPNPSAPRELKEQQDSPGNKDFLQSLKGFPRGDKLFGPDLKLVPPM EEDYPQFGSPK [Table 1]

[0043] As used herein, "plasmin" refers to "plasmin." Plasmin is For example, plasmin acts to dissolve fibrin in blood clots. Plasminogen is released from the liver into the systemic circulation as a proenzyme called plasminogen globulin (PLG). Two major glycoforms exist in humans - type I plasminogen has two glycoforms Contains a sylated moiety (N-linked to N289 and O-linked to T346) but is type II plus Plasminogen contains only a single O-linked sugar (O-linked to T346). Type II plasminogen Inogen is preferentially recruited to the cell surface over type I glycoforms. Plasminogen appears to be readily mobilized by blood clots. When bound to a clot or cell surface, the protein adopts a closed, activation-resistant conformation. Plasminogens are produced by a variety of enzymes, including, for example, tissue plasminogen activator (tPA). Fibrin has an open conformation that can be converted to active plasmin by enzymes. It is a cofactor for plasminogen activation by plasminogen activators. The conversion of plasmin to plasmin is mediated by Arg-561 and Val-562 (Wikipedia) This involves cleavage of the peptide bond between

[0044] The reference sequence of human plasmin is available from the UniProtKB / Swiss-Prot database. The nucleic acid sequence is available from the National Institute of Integrative Medicine under the accession number P00747-1 (numbering follows the methionine at position 1). do. [Table 2]

[0045] The terms "anti-A2AP antibody" or "anti-α2-antiplasmin antibody" and "α2-antiplasmin antibody" "Antibody that binds to typlasmin" or "Antibody that binds to A2AP" refers to α2-antiplasmin. This refers to an antibody that can bind α2-antibody with sufficient affinity to bind α-aminobutyric acid. These compounds are useful as diagnostic and / or therapeutic agents in targeting antiplasmin. In this state, anti-α2-antiplasmin to unrelated, non-α2-antiplasmin proteins The extent of binding of the α2-antibody was measured, for example, by standard ELISA procedures. The binding of the antibody to antiplasmin is less than about 10%, less than about 5%, or less than about 2%. In some embodiments, the antibody that binds α2-antiplasmin has a binding affinity of ≦1 μM, ≦100 nM , ≦10nM, ≦1nM, ≦0.1nM, ≦0.01nM, or ≦0.001nM (e.g. For example, 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 In certain embodiments, the anti-α2-antibody has a binding activity (EC50) of 100 M. α2-antiplasmin antibodies are conserved among α2-antiplasmins from different species. It binds to an epitope on antiplasmin.

[0046] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule. Antibodies consist of four polypeptide chains, two heavy (H) chains (approximately 50-70 kDa) and It may contain two light (L) chains (about 25 kDa), which are typically held together by disulfide bonds. In certain embodiments, the antibody is comprised of two identical pairs of polypeptide chains. The amino-terminal portion of each chain consists of approximately 100-110 amino acids that are primarily responsible for antigen recognition. A heavy chain variable region includes a "variable" region of 1 or more amino acids. and the light chain variable region is abbreviated herein as VL. The terminal portions define the constant regions primarily responsible for effector functions. The heavy chain constant region may, for example, The light chain constant region can include three domains, CH1, CH2 and CH3. The VH and VL regions are called framework regions (FR). Hypervariable regions called complementarity determining regions (CDRs) are interspersed with more conserved regions. Each VH and VL can be further subdivided into a region typically consisting of: At the carboxy terminus, for example, in the following order: FR1, CDR1, FR2, CDR2, FR3 It is composed of three CDRs and up to four FRs, arranged as follows: CDR1, CDR2, FR3.

[0047] As used herein, the term "complementarity determining region" (CDR; e.g., CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR10, CDR19, CDR110, CDR CDR2, and CDR3) are amino acid residues of the antibody variable domain whose presence is necessary for antigen binding. Each variable domain typically has CDR1, CDR2 and CDR3. Each complementarity determining region has three CDRs, each of which is identified by the following definition: Amino acid residues from the "complementarity determining region" (e.g., about 2 in the light chain variable domain) 3-36 (L1), 52-58 (L2) and 91-101 (L3) and heavy chain variable domain 31-35 (H1), 50-65 (H2) and 98-110 (H3) in the main; (K abat et al., Sequences of Proteins of Immunolog ical Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, M. D (1991)) and / or amino acid residues from the “hypervariable loops” (e.g., the light chain Approximately 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the variable domains and 26-32 (H1), 53-55 (H2), and 96-10 in the heavy chain variable domain. 1(H3)(Chothia and Lesk;J Mol Biol 196:90 1-917(1987)). In some instances, the complementarity determining region may comprise K may contain amino acids from both the CDRs and the hypervariable loops as defined according to abat .

[0048] "Framework" or FR residues are variable domain residues other than the hypervariable region residues. .

[0049] The phrase "constant region" refers to the portion of the antibody molecule that confers effector functions.

[0050] As used herein, the term "Fc region" refers to an immunoglobulin that contains at least a portion of the constant region. The term is used to define the C-terminal region of a native sequence Fc region and In one embodiment, the human IgG heavy chain Fc region comprises a mutant Fc region having a cysteine ​​residue at Cys226. or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine (Lys447) in the ribozyme region may be present or absent. Unless otherwise specified in the specification, the numbering of amino acid residues in the Fc region or constant region is as follows: Kabat et al., Sequences of Proteins of Immunolo gical Interest, 5th Edition, Public Health Service , National Institutes of Health, Bethesda; As described in MD, 1991, the EU numbering system (also known as the EU index) (called).

[0051] Immunoglobulins are divided into different classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains can be assigned to mu (μ), delta (Δ), gamma (γ), alpha (α), α) and epsilon (ε), which are classified as IgM, IgD, IgG, and Ig In certain embodiments, the isotype of the antibody is defined as IgA, IgB, and IgE. The most common antibodies are IgG antibodies. Some of these are subclasses or isotypes. types, e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 In a particular embodiment, the antibody according to the invention can be further divided into IgG1, IgG2 , IgG3 or IgG4 antibodies, more specifically IgG1 or IgG4 antibodies. Different isotypes may have different effector functions. Human light chains are kappa (K) and Within light and heavy chains, variable and constant regions are The heavy chains are joined by a "J" region of about 12 or more amino acids, and the heavy chains also have a "J" region of about 10 or more amino acids. The amino acid "D" region is generally ogy, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, NY (1 See U.S. Pat. No. 989).

[0052] "Functional Fragments" or "Antigen-Binding Antibody Fragments" of Antibodies / Immunoglobulins As used herein, a fragment of an antibody / immunoglobulin that retains the antigen-binding region (e.g. The "antigen-binding region" of an antibody is typically defined as the region that binds the antigen. in one or more hypervariable regions of the antibody, e.g., CDR1, -2, and / or -3 regions. however, variable "framework" regions also provide a scaffold for the CDRs. It can play an important role in antigen binding by, for example, The "antigen-binding region" includes at least the 4 to 103 regions of the variable light (VL) chain and the 4 to 103 regions of the variable heavy (VH) chain. 5 to 109 amino acid residues, more preferably 3 to 107 for VL and 4 to 111 for VH. Particularly preferred are the complete VL and VH chains (1-109 of VL and VH (amino acid positions 1 to 113 of the sequence; numbering according to WO97 / 08320).

[0053] Non-limiting examples of "functional fragments" or "antigen-binding antibody fragments" include , Fab, Fab', F(ab')2, Fv fragment, domain antibody (dAb), phase Complementarity determining region (CDR) fragments, single chain antibodies (scFv), single chain antibody fragments, Diabodies, triabodies, tetrabodies, linear antibodies (Zapata et al., Protein n Eng., 8(10):1057-1062(1995)); chelating recombinant antibodies , tribodies or bibodies, nanobodies, small modular immune preparations (SMIPs), antigens Binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or the like and CDR sequences, as long as the antibody retains the desired biological activity. At least a sufficient amount of immunoglobulin to confer specific antigen binding to the polypeptide, such as and bispecific and / or fusion proteins formed from antibody fragments. and trispecific antibodies (CAK Borrebaeck, editor (1995) An tibody Engineering(Breakthroughs in Mole) cular Biology), Oxford University Press;R .Kontermann & S. Duebel, editors (2001) Antibo dy Engineering(Springer Laboratory Manua "Bispecific" or "bifunctional" An antibody other than a "single-unit" antibody is understood to have each of its binding sites identical. F(ab') 2 Or Fab, C H1 Area and C L The intermolecular disulfide interactions occurring between the Papain digestion of antibodies can be used to reduce or completely remove two identical antigens. The binding fragments (called "Fab" fragments, each of which contains one antigen-binding site) the remaining "Fc" fragment (the name reflects its ability to crystallize readily). Pepsin treatment produces F(ab') which has two "Fv" fragments. The "Fv" fragment contains the complete antigen recognition and binding site. This region is the smallest antibody fragment that contains one heavy chain variable domain and one light chain variable domain. It consists of a dimer of two variable domains in tight, non-covalent association. The three CDRs of each VH-VL dimer interact to define an antigen-binding site on the surface of the VH-VL dimer. It is this configuration that determines the antigen binding of an antibody. However, a single variable domain (or three CDRs specific for an antigen) Even a single half of the Fv (containing only the IgG1 domain) has the ability to recognize and bind to antigen.

[0054] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the VH and VH of an antibody. and VL domains, and these domains are present in a single polypeptide chain.

[0055] Preferably, the Fv polypeptide is capable of forming the desired structure for antigen binding. and further comprising a polypeptide linker between the VH and VL domains that enables For a review of Fv, see Pluckthun in The Pharmacology Journal. of Monoclonal Antibodies, Vol. 113, Rosenbu R.G. and Moore, Springer-Verlag, New York, 2. See pages 69-315 (1994).

[0056] The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1 Fab fragments contain one or more systemic fragments from the antibody hinge region. The addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including the cysteine ​​residue, results in Fab'-SH fragments are different from Fab' fragments because they bind to the cysteine ​​residues in the constant domains. As used herein for Fab' in which the group(s) have a free thiol group. F(ab')2 antibody fragments are originally composed of two fragments with a hinge cysteine ​​between them. The antibody was produced as a pair of Fab' fragments containing 2-aminopropyl 1-phenylindole residues.

[0057] The terms "mutein" and "mutant" can be used interchangeably and refer to a variable or variable region. An antibody or a polypeptide having at least one amino acid substitution, deletion, or insertion in a portion equivalent to the variable region. refers to an antigen-binding fragment, provided that a mutein or variant has the desired binding affinity. or retain biological activity. Mutations of the fragments are molecules that retain the binding activity of the antibody or antigen-binding antibody fragment. It is.

[0058] A "chimeric antibody" or antigen-binding fragment thereof is defined herein as comprising a chimeric antibody having variable domains derived from a non-human source, with some or all of the constant domains derived from a human source. It is defined as:

[0059] "Humanized antibodies" include, for example, antibodies derived from human sequences along with any necessary framework backmutations. The CDR regions are derived from a non-human species, such as mouse, grafted onto native V regions. In most cases, humanized antibodies are made by integrating residues from the recipient's hypervariable regions with the desired specificity. Non-human animals such as mice, rats, rabbits, or non-human primates that have the sex, affinity, and ability to Human immunoglobulins that are replaced by residues derived from the hypervariable region of a human species (donor antibody) (recipient antibody). See, e.g., U.S. Patent No. 5,225,539; No. 85,089; No. 5,693,761; No. 5,693,762; No. 5,85 See, e.g., US Pat. No. 9,205, each of which is incorporated herein by reference. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues. (e.g., U.S. Pat. Nos. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, See Nos. 85,089; 5,693,761; and 5,693,762. Moreover, humanized antibodies contain residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance (e.g., to achieve desired affinity). Generally, humanized antibodies have all or substantially all of the hypervariable regions removed. All correspond to those of non-human immunoglobulins, and all or a substantial portion of the framework regions At least one, and typically two, substantially all human immunoglobulin sequences The humanized antibody optionally contains an immunoglobulin constant region ( Fc), typically that of a human immunoglobulin. See Jones et al., Nature 331:522-25 (1986); hmann et al., Nature 332:323-27 (1988); and Presta , Curr. Opin. Struct. Biol. 2:593-96 (1992) (each of which is incorporated herein by reference).

[0060] A "human antibody" or "fully human antibody" is an antibody that contains human-derived CDRs, i.e., CDRs of human origin. Fully human antibodies are available from the IMGT database (http: / / www.imgt.org / ). A small number of germline deviations compared to the closest human germline reference determined based on rg For example, a fully human antibody according to the present invention may comprise a sequence similar to that of the closest human germline reference. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 germline deviations in the CDRs Fully human antibodies can be produced using cloning techniques combined with cell enrichment or immortalization processes. However, fully human antibodies can be developed from human B cells using a combination of techniques. Most of these have been developed using transgenic immunized mice with the human IgG locus, or phage It is isolated from a refined combinatorial library by prey (Bruegge mann M., Osborn M.J., Ma B., Hayre J., Avis. S., Lundstrom B. and Buelow R., Human Anti body production in Transgenic Animals, Ar ch Immunol Ther Exp(Warsz.)63(2015), 101- 108;Carter PJ, Potent antibody therapy tics by design, Nat Rev Immunol 6(2006), 3 43-357;Frenzel A., Schirrmann T. and Hust M., Phage display-derived human antibodies es in clinical development and therapy, M Abs 8(2016), 1177-1194; Nelson AL, Dhimol ea E. and Reichert JM, Development tren. ds for human monoclonal antibody therapy utics, Nat Rev Drug Discov 9(2010), 767-77 4.)).

[0061] Several techniques are available for producing fully human antibodies (WO2008 / 1 (See Cambridge Antibody Technology, 12640A3). es(CAT) and Dyax are antibodies produced from peripheral B cells isolated from immunized humans. cDNA sequences were obtained and phage display assays were used to identify human variable region sequences of particular specificity. Briefly, antibody variable region sequences were synthesized using the M13 bacterioactivator. These antibodies are fused to either the gene III or gene VIII structures of the phage. The variable region sequences are expressed as Fab or single chain Fv (sFv) at the tip of the phage carrying the respective sequences. cFv) structures that exhibit different levels of antigen-binding conditions (stringency). Through rounds of the panning process using a Fab fragment (Fsp) that is specific for the antigen of interest, Alternatively, phage expressing the scFv structure can be selected and isolated. The antibody variable region cDNA sequences of the isolated phages are elucidated using standard sequencing procedures. These sequences can then be engineered using established antibody engineering techniques to produce the desired antibody. The method can be used to reconstitute complete antibodies with isotypes. The resulting antibody is considered to be fully human (including CDRs). Combinations of different heavy and light chains to improve antigen binding affinity and specificity In this case, deletions / additions / mutations in the CDR3 of the heavy and light chains (VJ and VDJ recombination), and random mutation (to mimic somatic hypermutation). An in vitro maturation process can be introduced, including: An example of a "fully human" antibody is the anti-tumor necrosis factor alpha antibody, Humira (adalimumab). .

[0062] "Human Engineered™" antibodies are described in detail in Studnicka et al., US Pat. By modifying the parent sequence according to the method described in Patent No. 5,766,886 is generated.

[0063] The antibodies of the present invention can be derived from a recombinant antibody gene library. Development of technologies to generate a repertoire of filamentous bacteriophages The display of encoded antibody fragments on the surface can be humanized, chimeric, murine, or The method is applicable to directly generate and select human antibodies, which can also be applied to mutein or mutein antibodies. The antibodies produced by phage technology have provided a recombinant means for the detection of antigens in bacteria. Produced as binding fragments (usually Fv or Fab fragments) and thus They lack effector functions. Effector functions are mediated by one of two strategies: The fragments can be inserted into a complete antibody for expression in mammalian cells. or bispecific antibodies having a second binding site capable of inducing an effector function. Typically, the Fd fragment of an antibody (VH- The CH1) and light chain (VL-CL) were cloned separately by PCR and combined randomly recombined in a trisomy phage display library and then Fab fragments can be selected for binding to an antigen. Fab fragments are expressed on the phage surface. , i.e., physically linked to the genes that encode them. Selection of Fabs by co-selection of the Fab-encoding sequences can then be amplified. Antibodies are identified by several rounds of antigen binding and reamplification, a procedure called panning. Fab specific for the antigen is enriched and finally isolated.

[0064] Various procedures have been described for the generation of human antibodies from phage display libraries. Such libraries are described by Carlsson and Soederlin d Exp.Rev.Mol.Diagn.1(1), 102-108 (2001), Soederlin et al., Nat. Biotech. 18, 852-856 (2000) and U.S. Pat. No. 6,989,250, various in vivo formed A single master frame into which the engineered (i.e., human-derived) CDRs can be recombined. Alternatively, such antibody libraries can be constructed in situ. Based on amino acid sequences encoded by nucleic acids designed in vitro and produced synthetically. In silico design of antibody sequences can be done, for example, by analyzing a database of human sequences and using the sequences obtained therefrom. This is accomplished by devising a polypeptide sequence using the data collected in silico. Methods for designing and obtaining sequences made in vitro are described, for example, in Knappik et al., J. Mol.Biol.(2000)296:57;Krebs et al., J.Immunol.M Ethods. (2001) 254:67; and U.S. Pat. No. 6,300,064. For an overview of phage display screening (e.g., Hoet RM et al. , Nat Biotechnol 2005;23(3):344-8) For this purpose, well-established hybridoma technology (e.g., Koehler et al. nd Milstein Nature.1975 Aug 7;256(5517): 495-7), or immunization of mice, particularly hMAb mice ( For example, VelocImmune mouse (registered trademark).

[0065] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies. The term "antibody" refers to an antibody obtained from a population, i.e., the individual antibodies that comprise the population may be present in small amounts. Identical except for possible variations, e.g., naturally occurring variations. Hence, the term "mono "Clonal" refers to the character of an antibody as not being a mixture of distinct antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different Each monoclonal antibody in a clonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations typically have similar properties to other immunoglobulins. The term "monoclonal" refers to any It should not be construed as requiring production of the antibody by any particular method. The monoclonal antibody is disclosed in Kohler et al., Nature, 256:495

[1975] . It can be produced by the hybridoma method first described by (See, e.g., U.S. Patent No. 4,816,567) "Monoclonal antibodies" also include, for example, recombinant, chimeric, humanized, human, Engineered™, or an antibody fragment.

[0066] An "isolated" antibody is one that has been identified and separated from components of the cell in which it is expressed. Contaminating components of cells are materials that interfere with the diagnostic or therapeutic use of the antibody, such as enzymes, phosphatase inhibitors, and phosphatase inhibitors. The solutes may include lumines, and other proteinaceous or non-proteinaceous solutes.

[0067] An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. The isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is not stained. It is present in vitro or at a chromosomal location different from its natural chromosomal location.

[0068] As used herein, an antibody is an antibody that is specific to an antigen of interest, e.g., A2AP. "binds to," "is specific for," or "specifically recognizes" with sufficient affinity Antibodies bind to antigens in a specific manner, so they target cells or tissues that express the antigen. These are useful as therapeutic agents in targeting orthologs and variants (e.g., (e.g., mutant forms, splice variants, or proteolytically truncated forms) As used herein, a specific polypeptide or a specific "Specifically recognizes" or "specifically binds to" an epitope on a given polypeptide target. The terms "specific for" and "specific for" refer to, for example, -4 Less than M or about 1 0 -5 Less than M or about 10 -6 Less than M or about 10 -7 Less than M or about 10 -8 Less than M or about 10 -9 Less than M or about 10 -10 Less than M or about 10 -11 Less than M or about 10 -12 Monovalent K for antigens less than or equal to M D of Such an antibody can be demonstrated by an antibody or an antigen-binding fragment thereof. If the body can distinguish such an antigen from one or more reference antigens, the antibody is , "specifically binds to," "is specific for," or "specifically recognizes" an antigen. In its most common form, "specific binding," "specifically binds to," "specifically binds to" "Is" or "specifically recognizes" is determined, for example, according to one of the following methods: This refers to the ability of an antibody to distinguish between an antigen of interest and unrelated antigens, such as antibodies. Although not limited to these, surface plasmon resonance (SPR), Western blot, ELI These include SA-, RIA-, ECL-, IRMA-tests and peptide scans. For example, A standard ELISA assay can be performed. Scoring is by standard color development. can be performed (e.g., secondary antibodies with horseradish peroxidase and hydrogen peroxide) The reaction in a particular well is measured by optical density, e.g., 450 The typical background (=negative reaction) is 0.1 OD. a typical positive reaction may be 1OD, which is a positive / negative difference of more than 5-fold, more than 10-fold, Typically, this means that the binding specificity is determined by the above-mentioned method. The determination is not based on a single reference antigen, but on approximately 3-5 different antigens, such as milk powder, BSA, and transferrin. This is done by using a set of related antigens.

[0069] "Binding affinity" or "affinity" refers to the relationship between a single binding site of a molecule and its binding partner. Unless otherwise specified, as used herein, the term "strength of the sum of non-covalent interactions between "Binding affinity" reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The dissociation constant, K D " is generally a molecule (such as an antibody) and its bond Affinity between a ligand and its target (such as an antigen), i.e., how strongly the ligand binds to a particular protein. Ligand-protein affinity is used to describe the binding between two molecules. Affinity is influenced by non-covalent intermolecular interactions. The amount of the protein can be measured by common methods known in the art, including, but not limited to, methods for measuring the protein content. In accordance with the present invention, D " or "K D Values ​​are based on a Biacore T200 instrument (GE Surface plasmon resonance assay using Healthcare Biacore, Inc. Other suitable devices include the BIACORE T100, the BIA CORE(R)-2000, BIACORe 4000, BIACORE(R)-300 0 (BIAcore, Inc., Piscataway, NJ), or ProteOn The instrument is an XPR36 (Bio-Rad Laboratories, Inc.).

[0070] As used herein, the term "epitope" refers to an immunoglobulin or T-cell antigen. Epitope definition includes any protein determinant capable of specific binding to a cell receptor. The functional groups are usually chemically linked to molecules such as amino acids or sugar side chains, or a combination of both. They consist of active surface groupings, usually with specific three-dimensional structural features, as well as specific charge characteristics. It has sexuality.

[0071] An antibody that binds to the same epitope as the reference antibody or competes for binding with the reference antibody. An "antibody that inhibits the binding of a reference antibody to its antigen in a competitive assay" is an antibody that inhibits the binding of a reference antibody to its antigen by 10%, 20%, 30%, or 40%. Blocks 0%, 40%, 50% or more of the antibody, whereas the reference antibody blocks 0%, 40%, 50% or more of the antibody in a competitive assay. Blocks antibody binding to antigen by 10%, 20%, 30%, 40%, 50% or more This refers to an antibody that

[0072] "Mature antibodies" or "mature antigen-binding fragments", such as mature Fab variants or The term "optimized" variant refers to a variant that is specific to a given antigen, such as the extracellular domain of a target protein. Antibodies or antibodies that exhibit stronger binding (i.e., binding with increased affinity) to Maturation includes the maturation of the antibody or antibody fragments that results in this increase in affinity. The maturation process is the process of identifying a small number of mutations within the six CDRs of a fragment. The present invention relates to a method for introducing mutations into antibodies and screening to identify improved binders. It is a combination of molecular biology methods for

[0073] "Percent sequence identity (%)" with respect to a reference polynucleotide or polypeptide sequence " aligns sequences to achieve maximum percent sequence identity and The sequence of the reference polynucleotide or polypeptide sequence, after introducing gaps if necessary. The nucleic acid or amino acid residues in the candidate sequence that are identical to the nucleic acid or amino acid residues in each of the candidate sequences are Conservative substitutions are defined as the percentage of each amino acid residue that is Amino acid sequence identity is not considered a part of the sequence. An ungapped alignment is preferred. Alignment for purposes of determining centroids can be accomplished in a variety of ways within the skill of the art, for example: BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) This can be accomplished using publicly available computer software, such as Those skilled in the art will recognize that in order to achieve maximum alignment over the entire length of the sequences being compared, Determine appropriate parameters for aligning sequences, including any algorithms required for It can be determined.

[0074] "Sequence homology" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. This indicates the index.

[0075] An "antagonist" or "blocking" antibody is one that blocks the biological activity of the antigen to which it binds. In certain embodiments, the present invention The antibody or antigen-binding fragment according to the present invention is an A2AP blocking antibody or antigen-binding fragment. It is.

[0076] The term "antibody conjugate" refers to an antibody conjugate that is conjugated to one or more molecules, including a drug. In this case, the antibody conjugate is called an "antibody-drug conjugate." These are called ADCs and are conjugated to large molecules such as peptides or proteins. This refers to a gated antibody.

[0077] Amino acids are referred to herein by their commonly known three letter symbols or by their IUPAC-IUB By the Biochemical Nomenclature Commission Nucleotides may be referred to by their recommended single-letter symbols. may be referred to by their commonly accepted single-letter codes.

[0078] The term "vector" as used herein refers to another nucleic acid to which it is linked. The term refers to a nucleic acid molecule that can propagate itself as a self-replicating nucleic acid structure. Vectors include vectors that are integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0079] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably. is used to refer to a cell into which at least one exogenous nucleic acid has been introduced, and to the progeny of such a cell Host cells are also referred to as "transformants" and "transformed cells," "transfectants" and "forms." "Transfected cells" and "transduced cells" refer to primary transformed / transfected cells, regardless of the number of passages. The term includes transfected / transduced cells and their derived progeny, which have exactly the same nucleic acid content as the parent cell. The mutation may not be unique, but may contain a mutation. Mutant progeny that have the same function or biological activity as the cloned or selected mutant. , which are incorporated herein by reference.

[0080] As used herein, the phrase "therapeutically effective amount" refers to the amount of and when administered in a therapeutically effective amount, the therapeutically effective amount of the compound is selected from the group consisting of acetaminophen, ... Suitable for inducing a desired therapeutic or prophylactic effect or response, including reducing the predisposition to a disease. "Amount of therapeutic or prophylactic antibody" is meant to refer to the amount of therapeutic or prophylactic antibody that would be effective.

[0081] The term "pharmaceutical formulation" / "pharmaceutical composition" refers to the biological activity of the active ingredient contained therein. in a form that allows the activity to be effective and to the subject to which the formulation is administered Refers to formulations that do not contain additional ingredients that are unacceptably toxic.

[0082] Antibodies according to the invention In one aspect, the present invention provides an isolated antibody or antigen binding site for human A2AP. and a combination fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof is Cross-reacts with rabbit and / or cynomolgus monkey A2AP. In certain embodiments, The isolated antibody or antigen-binding fragment of the present invention has an affinity for human A2AP. Sexuality is less than 100 times, especially less than 30 times, even more especially less than 15 times, most especially have affinities for rabbit A2AP that differ by less than 5-fold. The affinity is determined by measuring the affinity of human A2AP consisting of amino acids 40 to 491 of SEQ ID NO: 1 and 2 to amino acids 28-491 of rabbit A2AP. Thus, the isolated antibody or antigen-binding fragment of the present invention is directed to human A2AP. Affinity is less than 100-fold, particularly less than 30-fold, even more particularly less than 15-fold, most particularly In particular, such antibodies have affinities for cynomolgus monkey A2AP that differ by less than 5-fold. In one embodiment, the affinity is determined based on the amino acid sequence of human A2AP of SEQ ID NO:1 to amino acid sequence of human A2AP of SEQ ID NO:1. and against cynomolgus monkey A2AP consisting of amino acids 28 to 491 of SEQ ID NO:3.

[0083] In another embodiment, the present invention relates to a method for the detection of A2AP-related inflammatory cytokines. The present invention relates to an isolated antibody or antigen-binding fragment thereof capable of inhibiting The isolated antibody or antigen-binding fragment thereof does not inhibit plasmin activity.

[0084] In a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is ment inhibits A2AP activity by preventing A2AP from binding to plasmin do.

[0085] The endogenous plasma concentrations of A2AP are relatively high (1 μM and 70 μg / ml, respectively). Due to this fact, high concentrations of neutralizing antibodies are required to block A2AP activity. Therefore, it would be advantageous if A2AP antibodies could inhibit plasmin activity at high antibody concentrations. Surprisingly, the isolated antibody or antigen-binding fragment thereof according to the invention inhibited plasmin activity up to a concentration of 10 μM in an in vitro plasmin inhibition assay. antibody 77A3 inhibited plasma expression with an IC50 of 1.7 μM in the same assay. The inhibitor inhibited the activity of ribozyme A (see Example 11, Figure 16).

[0086] In a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is The ment is human plasmin, in particular SEQ ID NO: 118 (plasmin heavy chain A) and SEQ ID NO: 1 The activity of human plasmin containing plasmin light chain B (19) was examined in the presence of high micromolar concentrations of The present invention does not impede the above-mentioned measures even if they are implemented in a non-compliant manner.

[0087] In a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is The ment was tested at 1 μM, 2 μM, 5 μM or The concentration of the isolated antibody or antigen-binding fragment thereof was 10 μM. ation) does not inhibit plasmin activity.

[0088] In a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is The ment was tested at 1 μM, 2 μM, 5 μM or The concentration of the isolated antibody or antigen-binding fragment thereof was 10 μM. plasmin activity until the in vitro plasmin inhibition assay (a) Determines the inhibition of the proteolytic activity of plasmin.

[0089] Such an in vitro plasmin inhibition assay can be performed using plasmin inhibitors as described in Example 11. Such an assay may be an assay for determining inhibition of the proteolytic activity of rasmin. For the assay, plasmin and the fluorogenic substrate I-1275 (Bachem; MeOSuc -Ala-Phe-Lys-AMC trifluoroacetate salt; Catalog No. I-127 A labeled substrate for plasmin proteolytic activity such as 5) can be used.

[0090] In another aspect, the present invention relates to a method for the prevention and / or treatment of A2AP comprising the steps of: The present invention relates to an isolated antibody or antigen-binding fragment thereof, The antibody or antigen-binding fragment thereof comprises amino acids 402 to 408 (SR 402-408) of SEQ ID NO:1. It binds to an epitope on A2AP that contains the MSLSS.

[0091] A2AP is located in the reactive center loop of A2AP (amino acids 400 to 412 of SEQ ID NO: 1). The amino acid sequence SRMSLSS (amino acids 402 to 408 of SEQ ID NO: 1) is particularly In certain embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention The isolated antibody can bind to human A2AP and inhibit the activity of A2AP, The antibody or antigen-binding fragment thereof is a polypeptide comprising amino acids 402 to 408 of SEQ ID NO:1 ( 5. The isolated antibody or The antigen-binding fragment does not inhibit plasmin activity.

[0092] In certain embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention binds to human A2AP and can inhibit the activity of A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to an epitope of A2AP comprising amino acids 402-408 (SRMSLSS) of SEQ ID NO: 1, and the isolated antibody or its antigen-binding fragment does not inhibit plasmin activity up to a concentration of 1 μM, 2 μM, 5 μM or 10 μM of the isolated antibody or antigen-binding fragment thereof in an in vitro plasmin inhibition assay. In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof does not convert A2AP from a serine protease inhibitor to a serine protease substrate. In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a dissociation constant (KD) ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.5 nM. In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a KD ≤ 500 nM, ≤ 25

[0093] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a dissociation constant (KD) ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.5 nM. does not convert A2AP from a serine protease inhibitor to a serine protease substrate.

[0094] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a dissociation constant (KD) ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.5 nM. In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a KD ≤ 500 nM, ≤ 25 nM, ≤ 10 nM, ≤ 5 nM, ≤ 1 nM, or ≤ 0.5 nM. In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a KD ≤ 500 nM, ≤ 25 nM, ≤ 10 nM, ≤ 5 nM, ≤ 1 nM, or ≤ 0.5 nM.

[0095] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP, wherein the isolated antibody or antigen-binding fragment thereof binds to human A2AP having the sequence of amino acids 40-491 of SEQ ID NO: 1 with a KD ≤ 500 nM, ≤ 25 nM, ≤ 10 nM, ≤ 5 nM, ≤ 1 nM, or ≤ 0.5 nM. nM, ≤ 10 nM, ≤ 5 nM, ≤ 1 nM, or ≤ 0.5 nM. 0nM, ≤100nM, ≤50nM, ≤25nM, ≤10nM, ≤1nM, or ≤0. Binds with an EC50 of 5 nM.

[0096] In another aspect, the present invention provides an isolated antibody or an antigen thereof that binds to human A2AP. and a binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof is 1. The activity of human A2AP was measured at ≦500 nM, ≦250 nM, ≦100 nM, ≦50 nM, ≦ Inhibited with an EC50 of 25 nM, ≦10 nM, ≦1 nM, or ≦0.5 nM.

[0097] The in vitro A2AP functional blocking assay is an assay as described in Example 4. In such an assay, the test antibody is preincubated with A2AP. An A2AP substrate, such as trypsin, trypsin or chymotrypsin, is added to the assay, followed by addition of The activity of the added A2AP substrate (not blocked by A2AP) was measured, e.g., by the addition of an A2AP substrate For example, plasmin (A2AP) can be analyzed by using a labeled substrate for For the substrate, fluorescent substrate I-1275 (Bachem; MeOSuc-Ala-Ph e-Lys-AMC trifluoroacetate salt; Catalog No. I-1275) It is possible.

[0098] In a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is Mention is (i) Dissociation constant (KD) ≦100nM, ≦50nM, ≦25nM, ≦10nM, ≦1nM or ≦0.5 nM, binding to human A2AP of the sequence of amino acids 40 to 491 of SEQ ID NO:1. Combined; and (ii) In an in vitro A2AP function blocking assay, amino acids 40-4 of SEQ ID NO: 1 The activity of 91 human A2APs was measured at ≦500 nM, ≦250 nM, ≦100 nM, and ≦50 nM. , with an EC50 of ≦25 nM, ≦10 nM, ≦1 nM, or ≦0.5 nM.

[0099] In another aspect, the present invention provides an isolated antibody or an antigen thereof that binds to human A2AP. and a binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof is In particular, the single compound of the present invention increases plasmin-mediated clot lysis in the presence of A2AP. The isolated antibodies or antigen-binding fragments can be subjected to in vitro and / or in vivo protease activity. Increases plasmin-mediated clot lysis. Increases plasmin-mediated clot lysis in vitro. The ability of the antibody to induce plasmin-mediated cell death in vivo can be assessed as described in Example 7. The ability of the antibody to increase mediated clot lysis can be assessed as described in Example 8.

[0100] The isolated antibody or antigen-binding fragment according to the invention may have any combination of the above characteristics. It may show a match.

[0101] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , the interaction between A2AP and plasmin, particularly the interaction between human A2AP and human plasmin , in particular human A2AP of amino acids 40 to 491 of SEQ ID NO: 1 and human plasmin, in particular SEQ ID NO: Human plasmin comprising SEQ ID NO:118 (plasmin heavy chain A) and SEQ ID NO:119 (plasmin light chain B). In particular, the antibody or antigen-binding fragment according to the invention prevents the antibody from interacting with rasmin. is an A2AP blocking antibody or antigen-binding fragment.

[0102] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , at least 90%, at least 95%, at least 98% or less identical to SEQ ID NO:32 In a particular embodiment, the antibody of the present invention comprises a heavy chain variable domain that is 99% identical to the antibody of the present invention. The isolated antibody or antigen-binding fragment has a similar identity to SEQ ID NO:38, but at least 90% similar to SEQ ID NO:38. light chain variable domains that are at least 95%, at least 98% or at least 99% identical to each other In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention The sequence is at least 90%, at least 95%, at least 98% or at least a heavy chain variable domain that is at least 99% identical to SEQ ID NO: 38 and a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 39; and a light chain variable domain that is at least 95%, at least 98% or at least 99% identical to the Includes.

[0103] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , Array EX 1 YDSSGYYHLX 2 Heavy chain antibody comprising H-CDR3 containing Y (SEQ ID NO: 4) wherein X 1 is selected from the group consisting of Y, D and G; X 2 teeth, D, V, E and T. In certain embodiments, X 1 D and G; 2 is selected from the group consisting of V, E and T.

[0104] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is Array X 1 AWDX 2Light chain antigen binding comprising L-CDR3 comprising SLSGWV (SEQ ID NO:5) region, where X 1 is selected from the group consisting of A and W; X 2 D, N, L , W and V. In certain embodiments, X 1 consists of W selected from the group, X 2 is selected from the group consisting of N, L, W and V.

[0105] In particular, the isolated antibody or antigen-binding fragment according to the invention comprises: i) the sequence EX 1 Y DSSGYYHLX 2 A heavy chain antigen-binding region comprising H-CDR3 comprising Y (SEQ ID NO: 4). Here, X 1 is selected from the group consisting of Y, D and G; X 2 D, V, E and and T, and ii) the sequence X 1 AWDX 2 SLSGWV(sequence number 5) a light chain antigen-binding region comprising an L-CDR3 comprising X 1 A and W X is selected from the group consisting of 2 is selected from the group consisting of D, N, L, W and V.

[0106] In certain embodiments, two frame nucleotides immediately adjacent to the 5' end of the H-CDR3 region are Work residue X 1 X 2 (residue 96[X 1 ] and 97[ X 2 ]) is selected as follows:X 1 is selected from the group consisting of A and D Selected, especially X 1 is D, and X 2 is selected from the group consisting of R and S, X 2 is S.

[0107] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is H-CDR1 comprising SEQ ID NO: 6 or SEQ ID NO: 21 and SEQ ID NO: 7, SEQ ID NO: 8 or comprises a heavy chain antigen-binding region comprising an H-CDR2 comprising SEQ ID NO: 22. In accordance with the present invention, an isolated antibody or antigen-binding fragment thereof comprises a L sequence comprising SEQ ID NO:9. - a light chain antigen-binding region comprising CDR1 and L-CDR2 comprising SEQ ID NO: 10.

[0108] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , i) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 13 and an H-CDR4 comprising SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3; or ii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 11 and an H-CDR4 comprising SEQ ID NO: 17 a light chain antigen-binding region comprising an L-CDR3, iii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 11 and SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3 comprising iv) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 12 and an H-CDR4 comprising SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3, v) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 13 and an H-CDR3 comprising SEQ ID NO: 19 a light chain antigen-binding region comprising an L-CDR3; or vi) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 13 and a CDR comprising SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3, vii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 14 and SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3 comprising viii) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 14 and SEQ ID NO: 20 or ix) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 15 and an H-CDR4 comprising SEQ ID NO: 18 a light chain antigen-binding region comprising an L-CDR3, x) a heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO: 16 and an H-CDR3 comprising SEQ ID NO: 18 Light chain antigen-binding region including L-CDR3 Includes.

[0109] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is : i) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:8, and H-CDR3 comprising SEQ ID NO:1 3, and L-CDR1 comprising SEQ ID NO: 9. , a light chain comprising L-CDR2 comprising SEQ ID NO: 10, and L-CDR3 comprising SEQ ID NO: 18. an antigen-binding region, or ii) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 13, and an L-CDR3 comprising SEQ ID NO: 9. 1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO: 18. a chain antigen-binding region, or iii) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:11, and an L-CDR3 comprising SEQ ID NO:9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:17 a light chain antigen-binding region, or iv) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 11, and an L-CDR3 comprising SEQ ID NO: 9. 1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO: 18. a chain antigen-binding region, or v) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and H-CDR3 comprising SEQ ID NO:1 2, and L-CDR1 comprising SEQ ID NO:9. , a light chain comprising L-CDR2 comprising SEQ ID NO: 10, and L-CDR3 comprising SEQ ID NO: 18. an antigen-binding region, or vi) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 13, and an L-CDR3 comprising SEQ ID NO: 9. 1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO: 19. a chain antigen-binding region, or vii) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:14, and an L-CDR3 comprising SEQ ID NO:9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:18 a light chain antigen-binding region, or viii) H-CDR1 comprising SEQ ID NO: 6, H-CDR2 comprising SEQ ID NO: 7, and the sequence A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:14, and an LC comprising SEQ ID NO:9. L-CDR1, L-CDR2 comprising SEQ ID NO: 10, and L-CDR3 comprising SEQ ID NO: 20. a light chain antigen-binding region comprising ix) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 15, and an L-CDR3 comprising SEQ ID NO: 9. 1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO: 18. a chain antigen-binding region, or x) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and H-CDR3 comprising SEQ ID NO:1 6, and L-CDR1 comprising SEQ ID NO: 9. , a light chain comprising L-CDR2 comprising SEQ ID NO: 10, and L-CDR3 comprising SEQ ID NO: 18. an antigen-binding region, or xi) H-CDR1 comprising SEQ ID NO: 21, H-CDR2 comprising SEQ ID NO: 8, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:13, and an L-CDR3 comprising SEQ ID NO:9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:18 a light chain antigen-binding region, or xii) H-CDR1 comprising SEQ ID NO: 6, H-CDR2 comprising SEQ ID NO: 22, and the sequence A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:13, and an LC comprising SEQ ID NO:9. L-CDR1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:18. The light chain antigen-binding region comprises

[0110] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , 30S, 31S, 53S, 56S, 97K, and At least one, at least two, at least three or more of the heavy chain variable domain frameworks or at least four or at least five of the amino acid positions are set forth in SEQ ID NO:3. 2, and the amino acid positions of the framework and H-CDR 1 and H-CDR2 amino acid residues.

[0111] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is : i) a variable heavy chain domain comprising SEQ ID NO: 32 and a variable light chain domain comprising SEQ ID NO: 38; or ii) a variable heavy chain domain comprising SEQ ID NO: 23 and a variable light chain domain comprising SEQ ID NO: 37 ;or iii) a variable heavy chain domain comprising SEQ ID NO:24 and a variable light chain domain comprising SEQ ID NO:38 or iv) a variable heavy chain domain comprising SEQ ID NO: 25 and a variable light chain domain comprising SEQ ID NO: 38 ;or v) a variable heavy chain domain comprising SEQ ID NO:26 and a variable light chain domain comprising SEQ ID NO:38; or vi) a variable heavy chain domain comprising SEQ ID NO: 27 and a variable light chain domain comprising SEQ ID NO: 39 ;or vii) a variable heavy chain domain comprising SEQ ID NO: 27 and a variable light chain domain comprising SEQ ID NO: 38 or viii) a variable heavy chain domain comprising SEQ ID NO: 28 and a variable light chain domain comprising SEQ ID NO: 38 in; or ix) a variable heavy chain domain comprising SEQ ID NO: 28 and a variable light chain domain comprising SEQ ID NO: 40 ;or x) a variable heavy chain domain comprising SEQ ID NO:29 and a variable light chain domain comprising SEQ ID NO:38; or xi) a variable heavy chain domain comprising SEQ ID NO: 30 and a variable light chain domain comprising SEQ ID NO: 38 ;or xii) a variable heavy chain domain comprising SEQ ID NO: 31 and a variable light chain domain comprising SEQ ID NO: 38 or xiii) a variable heavy chain domain comprising SEQ ID NO: 33 and a variable light chain domain comprising SEQ ID NO: 38 in; or xiv) a variable heavy chain domain comprising SEQ ID NO: 34 and a variable light chain domain comprising SEQ ID NO: 38 or xv) a variable heavy chain domain comprising SEQ ID NO: 35 and a variable light chain domain comprising SEQ ID NO: 38 ;or xvi) a variable heavy chain domain comprising SEQ ID NO: 36 and a variable light chain domain comprising SEQ ID NO: 38 hmm Includes.

[0112] In a particular embodiment, the isolated antibody according to the invention is an IgG antibody. In such embodiments, the isolated antibody according to the invention is an IgG1, IgG2, Ig Most particularly, the isolated antibodies according to the invention are IgG3 or IgG4 antibodies. G1 or IgG4 antibody.

[0113] In a particular embodiment, the isolated antibody according to the invention comprises: i) a heavy chain comprising SEQ ID NO:55 and a light chain comprising SEQ ID NO:57; or ii) a heavy chain comprising SEQ ID NO:41 and a light chain comprising SEQ ID NO:56; or iii) a heavy chain comprising SEQ ID NO:42 and a light chain comprising SEQ ID NO:57; or iv) a heavy chain comprising SEQ ID NO:43 and a light chain comprising SEQ ID NO:57; or v) a heavy chain comprising SEQ ID NO:44 and a light chain comprising SEQ ID NO:57; or vi) a heavy chain comprising SEQ ID NO:45 and a light chain comprising SEQ ID NO:58; or vii) a heavy chain comprising SEQ ID NO:45 and a light chain comprising SEQ ID NO:57; or viii) a heavy chain comprising SEQ ID NO: 46 and a light chain comprising SEQ ID NO: 57; or ix) a heavy chain comprising SEQ ID NO:46 and a light chain comprising SEQ ID NO:59; or x) a heavy chain comprising SEQ ID NO:47 and a light chain comprising SEQ ID NO:57; or xi) a heavy chain comprising SEQ ID NO:48 and a light chain comprising SEQ ID NO:57; or xii) a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:57; or xiii) a heavy chain comprising SEQ ID NO:50 and a light chain comprising SEQ ID NO:57; or xiv) a heavy chain comprising SEQ ID NO:51 and a light chain comprising SEQ ID NO:57; or xv) a heavy chain comprising SEQ ID NO:52 and a light chain comprising SEQ ID NO:57; or xvi) a heavy chain comprising SEQ ID NO:53 and a light chain comprising SEQ ID NO:57; or xvii) a heavy chain comprising SEQ ID NO: 54 and a light chain comprising SEQ ID NO: 57 Includes.

[0114] In certain embodiments, the antigen-binding fragment according to the invention is an scFv, Fab, It is a Fab' fragment or a F(ab')2 fragment.

[0115] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , a monoclonal antibody or an antigen-binding fragment.

[0116] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is , human, humanized or chimeric antibodies or antigen-binding fragments, more particularly fully human It is an antibody or an antigen-binding fragment.

[0117] In certain embodiments, the isolated antibody or antigen-binding fragment according to the invention is In certain other embodiments, the isolated antibodies according to the invention are or antigen-binding fragment thereof binds to A2AP and at least one additional antigen. A multispecific antibody, for example a bispecific, trispecific or tetraspecific antibody.

[0118] In a further aspect, the present invention relates to an isolated antibody according to the present invention for binding to A2AP. An isolated antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment Regarding segments.

[0119] The amino acid sequences of preferred antibodies according to the invention are listed in Table 1.

[0120] Table 1: Amino acid sequences of preferred antibodies according to the invention [Table 3] TIFF2024063234000004.tif59152

[0121] The nucleic acid sequences of preferred antibodies according to the invention are listed in Table 2.

[0122] Table 2: Nucleic acid sequences of preferred antibodies according to the invention [Table 4] TIFF2024063234000006.tif151155

[0123] Peptide variants The antibodies or antigen-binding fragments of the present invention may be prepared using the specific peptide sequences provided herein. The present invention is not limited to the sequence of these polypeptides. Rather, the present invention also embodies variants of these polypeptides. With reference to the disclosure and prior art and references available therein, one of skill in the art will appreciate that Functional variants of the antibody that binds to A2AP can be prepared, tested, and utilized. It will be appreciated that such variants which retain the ability to bind are within the scope of the present invention.

[0124] Variants include, for example, at least one change to the peptide sequences disclosed herein. The complementarity determining regions (CDRs) (hypervariable) and / or framework (FR) (possibly The antibody may include antibodies having variable domains / locations.

[0125] By modifying one or more amino acid residues in the CDR or FR regions, For example, commercial manufacturers can routinely generate mutated or diversified antibody sequences, These can be screened against antigens for new or improved properties. Cut.

[0126] In a further preferred embodiment of the present invention, the VH and VL sequences are selected as shown in Table 1. The antibody or antigen-binding fragment is an antibody or antigen-binding fragment that is capable of binding to a target antigen. Variants of the peptides can be designed that fall within the scope of the invention. Variants include one or more Preferably, the variant is constructed by varying the amino acids in the CDR regions of may also have one or more altered framework regions. For example, the peptide FR domain may be altered by changing the residues may be changed if there is a deviation in

[0127] Alternatively, the skilled artisan can refer, for example, to Knappik A. et al., JMB 2000, 296:5 The amino acid sequences disclosed herein may be synthesized using the procedures described in The same analysis can be performed by comparing the sequence of the antibody with known sequences of the same class, such as can.

[0128] Furthermore, the mutants allow for the use of one antibody as a starting point for further optimization. Thus, one or more amino acid residues in the antibody, preferably one or more amino acid residues in the CDRs, By diversifying the bases, and for mutants with improved properties, The mutants can be obtained by screening a collection of mutants. What is needed is a diversity of one or more amino acid residues in the CDR3 of the VL and / or VH. Diversification can be achieved, for example, by using trinucleotide mutagenesis (TRIM) technology to create a DNA sequence. This can be done by synthesizing a collection of NA molecules (Virnekaes B. et al., Nucl. Acids Res. 1994, 22:5600). The antigen-binding fragment of the present invention may, for example, have an altered half-life (e.g., a modified Fc portion or a P EG), altered binding affinity or altered ADCC or molecules with modifications / mutations, including but not limited to modifications that confer CDC activity. Includes.

[0129] Conservative amino acid variants Polypeptide variants that preserve the overall molecular structure of the antibody peptide sequences described herein Taking into account the properties of individual amino acids, some rational substitutions can be made. Amino acid substitutions, i.e., "conservative substitutions," may be made, for example, by The polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the donor residues This can be done based on similarity.

[0130] For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, and isoleucine. , valine, proline, phenylalanine, tryptophan, and methionine. (b) polar neutral amino acids: glycine, serine, threonine, cysteine, tyrosine, (c) positively charged (basic) amino acids. acids include arginine, lysine, and histidine; and (d) negatively charged The (acidic) amino acids that are substituted include aspartic acid and glutamic acid. Typically, it can be made within groups (a) to (d). In addition, glycine and proline can be substituted for one another based on their ability to disrupt α-helices. Certain amino acids, such as alanine, cysteine, leucine, methionine, and glutamic acid , glutamine, histidine and lysine are more commonly found in α-helices. On the other hand, valine, isoleucine, phenylalanine, tyrosine, tryptophan and Threonine is more commonly found in β-pleated sheets. Glycine, Serine Aspartic acid, asparagine, and proline are commonly found alternating. Preferred substitutions may be made within the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. The known genetic code, as well as recombination and In light of the above and synthetic DNA techniques, one of skill in the art can easily create DNA that encodes conservative amino acid variants. It can be easily constructed.

[0131] Glycosylation variants If the antibody contains an Fc region, the carbohydrate attached to it can be modified. Natural antibodies produced by mammalian cells typically contain the Kaba domain of the CH2 domain of the Fc region. t Typically attached by an N-bond to Asn297 using EU numbering Contains branched and biantennary oligosaccharides; see, e.g., Wright et al., Trends Biotech. See chnol.15:26-32(1997).

[0132] In certain embodiments, the antibodies provided herein are directed to a method for the treatment of cancer, comprising administering to the patient a therapeutically effective amount of the antibody, wherein the antibody is glycosylated. The addition of glycosylation sites to the antibody or The deletion may be achieved by modifying the expression system (e.g., the host cell) and / or by modifying one or more by altering the amino acid sequence to create or remove glycosylation sites on the This can be conveniently achieved by

[0133] In one embodiment of the invention, aglycosyl antibodies with reduced effector function or or antibody derivatives are prepared by expression in prokaryotic hosts. These include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, as well as Pseudomonas and Streptomyces. Examples of suitable strains include, but are not limited to, the genus Staphylococcus ... and various species within the genus Staphylococcus.

[0134] In one embodiment, antibody variants are provided that have reduced effector function, The present invention relates to a method for the preparation of a method for the preparation of a medicament for the treatment of a pulmonary artery disease, comprising the steps of: In one embodiment of the invention, the modification comprises a mutation in the heavy chain glycosylation site. , preventing glycosylation at that site. Thus, one preferred embodiment of the present invention In the present invention, aglycosyl antibodies or antibody derivatives are obtained by mutating heavy chain glycosylation sites, prepared by mutation of N297 using the Kabat EU numbering system, It is expressed in a suitable host cell.

[0135] In another embodiment of the invention, the aglycosyl antibody or antibody derivative has a reduced effector. in the CH2 domain of the Fc portion of said antibody or antibody derivative, Modifications at the conserved N-linked site in the CH2 domain result in the removal of the CH2 domain glycan, i.e., deglycosylation. These aglycosyl antibodies are produced by conventional methods and then Methods for enzymatic deglycosylation of antibodies are known in the art. These methods are well known in the art (see, e.g., Winkelhake & Nicolson (1999) 76), J Biol Chem. 251(4):1074-80).

[0136] In another embodiment of the invention, deglycosylation is achieved by the use of a glycosylation inhibitor, tunicamycin. Nose & Wigzell (1983), Proc Natl Acad Sc. i USA 80(21):6632-6). Glycosylation at the conserved N-linked site in the CH2 domain of the Fc portion of the antibody The aim is to prevent the

[0137] In one embodiment, the carbon atom lacking fucose is attached (directly or indirectly) to the Fc region. Antibody variants having hydrate structures are provided. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of the source may be, for example, MALDI- All sugar structures (e.g., complexes) attached to Asn297 as determined by TOF mass spectrometry The total number of glycans at Asn297 (combined, hybrid and high mannose structures) It is determined by calculating the average amount of fucose in the chain. Asn297 is the This refers to the asparagine residue located at approximately position 297 in the Fc domain (Eu numbering of Fc domain residues). However, Asn297 may vary upstream of position 297 due to slight sequence variation in the antibody. or about ±3 amino acids downstream, i.e., between positions 294 and 300. The fucosylation variants may have improved ADCC function.

[0138] Examples of published "defucosylated" or "fucose-deficient" antibody variants include O kazaki et al., J Mol.Biol.336:1239-1249(2004);Y Amane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004 ) are mentioned.

[0139] Examples of cell lines capable of producing defucosylated antibodies include those lacking protein fucosylation. Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. s.249:533-545(1986); and WO2004 / 056312), if and alpha-1,6-fucosyltransferase gene, FUT8, knockout C Knockout cell lines such as HO cells (e.g., Yamane-Ohnuki et al., Biochem. ech.Bioeng.87:614(2004);Kanda, Y. et al., Biotec hnol. Bioeng., 94(4):680-688(2006) Some examples include:

[0140] The antibody mutants include, for example, those in which the biantennary oligosaccharides attached to the Fc region of the antibody are replaced with GlcNAc. Thus, bisected oligosaccharides are further provided. Such antibody variants may include those having fucosylated Examples of such antibody variants may have reduced cleavage and / or improved ADCC function. See, e.g., WO2003 / 011878; U.S. Pat. No. 6,602,684; and US 2005 / 0123546.

[0141] Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region Such antibody variants may have improved CDC function. Such antibody mutants are described, for example, in WO1997 / 30087; WO1998 / 58964; and It is described in WO1999 / 22764.

[0142] FC domain mutations In certain embodiments, one or more amino acid modifications (e.g., substitutions) are made to the polypeptides described herein. The Fc region of the antibody provided herein (e.g., human IgG1, IgG2, IgG3 or IgG G4Fc region), thereby generating an Fc region variant.

[0143] In certain embodiments, the present invention relates to a method for determining whether or not the half-life of an antibody in vivo is important. For applications where effector functions of the Antibody variants with some, but not all, effector functions are desirable candidates for To confirm the reduction / depletion of CDC and / or ADCC activity, In vitro and / or in vivo cytotoxicity assays can be performed. For example, Fetal muscle receptor (FcR) binding assays demonstrate that the antibody lacks FcγR binding (and therefore likely To ensure that the antibodies (which lack ADCC activity) retain FcRn binding ability, In some embodiments, the antibody is capable of binding C1q and / or complement dependent cytotoxicity ( Changes are made in the Fc region that result in alterations (i.e., either improvements or decreases) in CDC. It can be done.

[0144] In certain embodiments, the present invention provides antibody variants with increased or decreased half-lives. The aim is to develop a method to increase the half-life of the neonatal Fc receptor ( Antibodies with improved binding to FcRn (Guyer et al., J Immunol. 117:5 87 (1976) and Kim et al., J Immunol. 24:249 (1994) These antibodies are described in US 2005 / 0014934 (Hinton et al.). and an Fc region having one or more substitutions that improve binding of the Fc region to FcRn. .

[0145] In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. The present invention relates to an antibody conjugate comprising the ment.

[0146] Antibody production The antibodies of the present invention are based on amino acid sequences isolated from the antibodies of a large number of healthy volunteers. They may be derived from recombinant antibody libraries, for example using n-CoDeR® technology. Using this technique, fully human CDRs can be recombined into new antibody molecules (Carlson & Soe derlind, Expert Rev Mol Diagn.2001 May;1( 1):102-8). Alternatively, see, for example, Hoet RM et al., Nat Biotechn ol 2005;23(3):344-8) Isolation of A2AP-specific antibodies using an antibody library as a spray library The antibodies or antibody fragments isolated from the human antibody library can be , are considered herein to be human antibodies or human antibody fragments.

[0147] Human antibodies are antibodies that have intact human antibodies or human variable regions in response to antigenic challenge. The immunogen is administered to a transgenic animal that has been engineered to produce intact antibodies. Such animals can be further prepared by transfecting animals with endogenous immunoglobulins. They either replace gene loci, are extrachromosomal, or are randomly inserted into animal chromosomes. The present invention relates to a method for producing a human immunoglobulin gene comprising the steps of: Immunization of genetically engineered mice, particularly hMAb mice (e.g., VelocImm Immunize the mouse (XENOMOUSE® or XENOMOUSE®) can be done.

[0148] Further antibodies can be obtained using hybridoma technology (e.g., Koehler and Milst See Nature.1975 Aug 7;256(5517):495-7 (see below) and can be converted, for example, into a chimeric or humanized antibody. Humanized antibodies and methods for making them are described in US Pat. For example, Almagro and Fransson, Front.Biosci.13 :1619-1633 (2008) and, for example, Riechmann et al. Nature 332:323-329 (1988); Queen et al., Proc. Nat. l Acad.Sci.USA 86:10029-10033(1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087, No. 409; Kashmiri et al., Methods 36:25-34 (2005) (special (Describes sex-determining region (SDR) grafting); Padlan, Mol., Immunol. ol., 28:489-498 (1991) (describing "ReSurfacing"); All'Acqua et al., Methods 36:43-60 (2005) ("FR Schafer and Osboum et al., Methods 36:61-68. (2005) and Klimka et al., J. Cancer, 83:252-260 (200 0) (which describes a "guided selection" approach to FR shuffling). It is being done.

[0149] Examples are provided for the production of antibodies using recombinant antibody libraries.

[0150] DNA molecules according to the invention The present invention also relates to an isolated nucleic acid encoding an antibody or antigen-binding fragment according to the present invention. The present invention relates to an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof. The nucleic acid sequences are described, for example, in Sambrook et al., 1989, and Ausubel et al., 1989. 9 or, alternatively, by chemical synthesis. (e.g., Oligonucleotide Synthesis (1984, Gal (Techniques described in IEEE Trans. Eng., 1999, 1999, IRL Press, Oxford). The DNA sequences identified are shown in Table 2. These sequences are used in the specific case for mammalian expression. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but may be any of the following: The DNA variants within the present invention include those that exhibit similar or similar properties in hybridization. It can be described by reference to physical properties. One of skill in the art can easily identify a DNA and its complement. and because DNA is double-stranded, nucleic acid hybridization Hybridization techniques will be used to recognize equivalents or homologues. It will also be appreciated that redization can occur at less than 100% complementarity. However, given the appropriate selection of conditions, hybridization techniques can be used , to distinguish DNA sequences based on their structural relatedness to a particular probe. For guidance on such conditions, see Sambrook et al., 1989 See above, and Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D., Sedman, J.G. In Smith, J. A., & Struhl, K. (eds.) (1995), Molecular Biology See the current protocol, New York: John Wiley and Sons. Light.

[0151] Structural similarity between two polynucleotide sequences is the degree to which the two sequences hybridize to each other. As used herein, the term "stringency" can be expressed as a function of the "stringency" of the conditions under which the reaction is carried out. In the case of hybridization, the term "stringency" refers to the degree to which conditions are unfavorable for hybridization. Stringent conditions are those that strongly disfavor hybridization and are the most Only structurally related molecules will hybridize to each other under these conditions. Stringent conditions are those that favor the hybridization of molecules exhibiting lesser degrees of structural relatedness. Therefore, hybridization stringency is favored by two It directly correlates with the structural relationships of nucleic acid sequences.

[0152] Hybridization stringency is determined by the overall DNA concentration, ionic strength, temperature, It is a function of many factors, including the degree of binding, probe size, and the presence of agents that disrupt hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, and low These include lower temperatures, longer probe sizes, and the absence of agents that disrupt hydrogen bonds. Hybridization is typically carried out in two phases: a "binding" phase and a "washing" phase. do.

[0153] Functionally equivalent DNA variants Yet another class of DNA variants within the scope of the present invention are These functionally equivalent polynucleotides can be described as These are characterized by the fact that they all encode the same peptide sequence for the same purpose.

[0154] Variants of the DNA molecules provided herein can be constructed in a number of different ways. It will be appreciated that they may be constructed as completely synthetic DNA, for example. Methods for efficient synthesis of oligonucleotides are widely available. See section 2.11, Supplement 21 (1993). The oligonucleotides are described in Khorana et al., J. Mol. Biol., 72:209-21. 7 (1971); See also El et al., supra, Section 8.2. Synthetic DNA is preferably prepared by To facilitate cloning into suitable vectors, manipulations were made at the 5' and 3' ends of the gene. The plasmid is designed with convenient restriction sites engineered into it.

[0155] As indicated, the method of making the mutants includes cloning one of the DNAs disclosed herein. The first step is to start with the sequence of the nucleotide sequence and then perform site-directed mutagenesis. Ausubel et al., supra See Chapter 8, Supplement 37 (1997). clones the target DNA into a single-stranded DNA bacteriophage vehicle. The DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide change. The complementary strand is synthesized and the double-stranded phage is introduced into the host. Some of the resulting progeny are The progeny contain the desired mutations, which can be confirmed using DNA sequencing. There are various methods available that increase the probability that a gene is the desired mutation. These methods include Those skilled in the art are familiar with, and kits are commercially available for, generating such mutants.

[0156] Recombinant DNA constructs and expression The present invention further relates to a recombinant DNA vector comprising one or more of the nucleotide sequences according to the invention. The recombinant constructs of the invention are provided as follows: A vector, e.g., a plant, into which a DNA molecule encoding the polypeptide or a mutant thereof has been inserted. It can be used in conjunction with a smid, phagemid, phage or viral vector. do.

[0157] Thus, in one aspect, the present invention relates to a vector comprising a nucleic acid sequence according to the invention. do.

[0158] The antibodies, antigen-binding portions, or variants thereof provided herein can be expressed in host cells. The present invention is prepared by recombinant expression of nucleic acid sequences encoding the light and heavy chains or portions thereof. To recombinantly express an antibody, antigen-binding portion, or variant thereof, The light and / or heavy chains or portions thereof may be used to express the light and heavy chains in a host cell. and (b) expressing the recombinant DNA fragments in a host cell using one or more recombinant expression vectors carrying the DNA fragments encoding the recombinant DNA fragments. The nucleic acids encoding the heavy and light chains can be prepared and / or transfected. or by inserting these nucleic acids into a recombinant expression vector and infecting the vector into a host cell. To introduce, Sambrook, Fritsch, and Maniatis (eds.), Molecular Cloning;A Laboratory Manual, Part 2 Edition, Cold Spring Harbor, NY, (1989), Ausubel , F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1 989) and the method described in U.S. Pat. No. 4,816,397 to Boss et al. Standard recombinant DNA methods, such as those described above, are used.

[0159] Additionally, the nucleic acid sequences encoding the heavy and / or light chain variable regions can be derived from, e.g., full length antibodies. The nucleic acid sequence encoding the Fab fragment can be converted to a nucleic acid sequence encoding a Fab fragment, or a scFv. The DNA fragment encoding the VL or VH is (composed of two DNA fragments such that the amino acid sequences encoded by the can be operably linked to another DNA fragment encoding a flexible linker The sequences of human heavy and light chain constant regions are known in the art (e.g., Kabat et al., J. Am. Soc. 1999, 143:1311-1315). , EA et al. (1991) Sequences of Proteins of Im Munological Interest, 5th Edition, USDepartment of Health and Human Services, NIH Publica (see Ref. No. 91-3242), DNA fragments encompassing these regions can be obtained by standard PCR amplification.

[0160] To generate a polynucleotide sequence encoding an scFv, VH and VL are encoded The nucleic acid to be encoded comprises a VH and VL sequence linked by a flexible linker. and VH regions, and is flexible so that it can be expressed as a continuous single chain protein. The fragment may be operably linked to another fragment encoding a linker (e.g., B Ird et al. (1988) Science 242:423-426; Huston et al. (19 88) Proc.Natl.Acad.Sci.USA 85:5879-5883; See McCafferty et al., Nature (1990) 348:552-554).

[0161] Standard recombinant methods can be used to express the antibody, antigen-binding fragment thereof, or variants thereof. Alternatively, DNA expression methods can be used (e.g., Goeddel; Gene Expression ssion Technology.Methods in Enzymology 1 85, Academic Press, San Diego, Calif (1990)) For example, DNA encoding the desired polypeptide is inserted into an expression vector and then The vector can be transfected into any suitable host cell. Suitable host cells include prokaryotic cells and Examples of prokaryotic host cells are eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are Yeast, insects and insect cells, plants and plant cells, transgenic animals, or mammals. The host cells are mammalian cells. The introduction of the recombinant construct into the host cells is carried out by calcium phosphate transfection. ion, DEAE-dextran-mediated transfection, electroporation, morphology This can be done using standard techniques such as transduction or phage infection.

[0162] In some embodiments, the DNA encoding the heavy and light chains are inserted into separate vectors. In another embodiment, DNA encoding the heavy and light chains are inserted into the same vector. The design of the expression vector, including the selection of regulatory sequences, depends on the choice of host cell, the desired transcription factor, and the The expression level of the protein and whether expression is constitutive or inducible are factors that influence the expression of the protein. It is understood that it will be affected.

[0163] Thus, in a further aspect, the present invention relates to an antibody or antigen-binding fragment according to the invention. and / or a nucleic acid according to the invention or a vector according to the invention expressing a fragment thereof The present invention relates to an isolated cell comprising:

[0164] The isolated cell can be virtually any cell for which an expression vector is available. The cells can be, for example, higher eukaryotic host cells, such as mammalian cells, lower eukaryotic host cells, such as yeast cells, or the like. It may be a cell, which may be a prokaryotic cell, such as a bacterial cell.

[0165] In a further aspect, the present invention relates to a method for the preparation of ... cell according to the present invention, comprising culturing the cell according to the present invention. The present invention relates to a method for producing an isolated antibody or antigen-binding fragment thereof. In this embodiment, the cells according to the present invention are cultured under conditions suitable for antibody expression, and the antibody or antigen binding is produced. In certain embodiments, the antibody or antigen-binding fragment is recovered. The compound is preferably purified to at least 95% homogeneity by weight.

[0166] Bacterial expression Useful expression vectors for bacterial use contain a DNA sequence encoding a desired protein. , an operable reader having a functional promoter with appropriate translation initiation and termination signals. The vector is constructed by inserting it into the readout phase. The vector ensures the maintenance of the vector and If desired, one or more phenotypic selectable markers and / or vectors may be used to provide for amplification in the host. Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Murine Fusobacteria, as well as various strains within the genera Pseudomonas, Streptomyces, and Staphylococcus These include, but are not limited to, various species.

[0167] Bacterial vectors can be, for example, bacteriophage, plasmid, or phagemid based. These vectors can contain a selectable marker and the well-known cloning vector pBR The plasmid is derived from a commercially available plasmid that typically contains elements of ATCC 322 (ATCC 37017). The vector may contain a bacterial origin of replication that is in turn required for the transformation of a suitable host strain and for the preparation of a suitable cellular Following growth of the host strain to cell density, the selected promoter may be expressed by appropriate means (e.g., temperature, The expression level is then de-repressed / induced by temperature change or chemical induction, and the cells are cultured for an additional period. Cells are typically harvested by centrifugation and disrupted by physical or chemical means. The crude extract obtained is then saved for further purification.

[0168] Bacterial systems have a number of expression vectors available, depending on the intended use of the expressed protein. For example, if large amounts of such a protein are produced, For example, for the generation of antibodies or for screening peptide libraries, When vectors are desired which direct the expression of high levels of fusion protein products which are easily purified, There is a match.

[0169] Thus, one embodiment of the present invention is a gene encoding a novel antibody of the present invention. This is the current vector.

[0170] The antibody or antigen-binding fragment or variant thereof of the present invention may be purified from naturally occurring Products, products of chemical synthesis procedures, and products of, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and Various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably Includes products produced by recombinant techniques from prokaryotic hosts, including E. coli cells.

[0171] Mammalian Expression Preferred regulatory sequences for mammalian host cell expression include those that regulate high levels of ribozyme expression in mammalian cells. Viral elements that direct protein expression, such as cytomegalovirus (CMV) V) (CMV promoter / enhancer, etc.), Simian Virus 40 (SV40) ( SV40 promoter / enhancer, etc.), adenovirus (e.g. Major late promoter (AdMLP) and promoters derived from polyoma Expression of the antibody may be constitutive or regulated. Alternatively, the addition of a small molecule inducer such as tetracycline in conjunction with the Tet system may be used. (Inducible by addition or removal of the viral regulatory elements and their sequences) For a description of this, see, for example, US Pat. No. 5,168,062 by Stinski, US4,510,245 by ll et al. and US4 by Schaffner et al. See, e.g., 968,615. The recombinant expression vector also contains an origin of replication and a selection marker. (e.g., US 4,399,216, 4,634,665 and (See US Pat. No. 5,179,017.) Suitable selectable markers include G418 , puromycin, hygromycin, blasticidin, zeocin / bleomycin, or genes that confer resistance to drugs such as methotrexate or glutamine synthetase. A selection marker that utilizes an auxotrophy such as taurate (Bebbington et al., Biotechnol. 2013). chnology(NY)1992 Feb;10(2):169-75) For example, the dihydrofolate reductase (DHFR) gene is introduced into the host cell. confers resistance to methotrexate, and the neo gene confers resistance to G418. And, bs derived from Aspergillus terreus The d gene confers resistance to blasticidin and puromycin N-acetyltransferase. The enzyme confers resistance to puromycin, and the Sh ble gene product inhibits Resistance to eosin is conferred by the hygromycin gene in Escherichia coli. Resistance to glutamine is conferred by the DHFR or glutamine Selectable markers such as ribonuclease are also useful in amplification techniques in conjunction with MTX and MSX. It is useful.

[0172] Transfection of expression vectors into host cells can be achieved by electroporation, nuclease Rheofection, calcium phosphate precipitation, lipofection, polyethyleneimine (P EI)-based transfection and DEAE-dextran transfection This can be done using standard techniques such as polycation-based transfection. This can be done.

[0173] To express the antibodies, antigen-binding fragments thereof, or variants thereof provided herein, Suitable mammalian host cells for this purpose include CHO-K1, CHO-S, CHO-K1SV, etc. Chinese hamster ovary (CHO) cells [Urlaub and Chasin, (19 80) Proc. Natl. Acad. Sci. USA 77:4216-4220 and and Urlaub et al., Cell. 1983 Jun;33(2):405-12. For example, RJ Kaufman and PA Sharp (1982) DHFR selectable marker described in ol. Biol. 159:601-621 and Fan et al., Biotechnol Bio Other knockouts exemplified in eng.2012 Apr;109(4):1007-15 myeloma cells, NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, These include BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0174] Expression was also demonstrated in HEK293, HEK293T, HEK293-EBNA, HEK293 E, HEK293-6E, HEK293-Freestyle, HKB11, Expi2 93F, 293EBNALT75, CHO Freestyle, CHO-S, CHO- K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (e.g., Durocher et al., Nucleic Acids Receptor s.2002 Jan 15;30(2):E9) It can be stable.

[0175] In some embodiments, the expression vector is capable of inducing the expression of a protein that is expressed in a host cell. The antibody, antigen-binding fragment thereof or its derivative is designed to be secreted into the medium in which the antibody grows. The mutant can be recovered from the culture medium using standard protein purification methods.

[0176] purification The antibody or antigen-binding fragment or variant thereof of the present invention may be prepared by the method comprising the steps of: Or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography topography, anion or cation exchange chromatography, phosphocellulose chromatography Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography -, hydroxylapatite chromatography and lectin chromatography It may be recovered and purified from recombinant cell culture by well-known methods, including, but not limited to, High performance liquid chromatography ("HPLC") can also be used for purification. For example, Colligan, Current Protocols in Immunology. unology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997- 2001), see, for example, Chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated by reference in its entirety.

[0177] The antibodies or antigen-binding fragments or variants thereof of the present invention include naturally occurring The products of synthetic procedures and those derived from, for example, yeast, higher plants, insects and mammalian cells. Recombinant production procedures include those produced by recombinant techniques from eukaryotic hosts, including cells. Depending upon the host used in sequence, the antibody of the present invention may be glycosylated or Such methods are described in Sambrook, supra, Sectio ns17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20 are described in many standard laboratory manuals.

[0178] In a preferred embodiment, the antibodies are analyzed by, for example, the Lowry method, UV-Vis spectroscopy, or S DS-Capillary gel electrophoresis resin (e.g., Caliper LabChip G XII, GX 90 or Biorad Bioanalyzer instruments) As determined, 95% by weight or more of the antibody, and in a more preferred embodiment, 99% by weight (2) enough to obtain at least 15 residues of the N-terminal or internal amino acid sequence or (3) reduced or non-reduced staining with Coomassie blue or, preferably, silver staining. The antibody is purified to homogeneity by SDS-PAGE under conditions that minimize the natural environment of the antibody. Since neither one component is present, an isolated naturally occurring antibody can be expressed as an antibody produced by recombinant cells. However, isolated antibodies are usually less common. Both are prepared by a single purification step.

[0179] Treatment method The method of treatment involves administering to a subject in need of treatment a therapeutically effective amount of a compound contemplated by the present invention. The present invention includes administering an antibody or an antigen-binding fragment thereof, or a variant thereof, to the patient. A "therapeutically effective" amount is used herein as a single dose or as a multiple dose regimen. The drug, alone or in combination with other agents, reduces plasmin-mediated vasopressin in subjects. is defined as the amount of antibody or antigen-binding fragment sufficient to increase mochi lysis. The subject is given an amount that provides relief from the adverse condition but is toxicologically acceptable. Human or non-human animals (e.g., rabbits, rats, mice, dogs, monkeys or other lower primates) (type).

[0180] Thus, in one aspect, the present invention provides a method for treating or preventing a disease comprising administering to said patient a An isolated antibody or antigen-binding fragment according to the invention, or an isolated A conjugate comprising the antibody or antigen-binding fragment thereof, or an isolated antibody or antigen-binding fragment thereof according to the present invention. The present invention relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof.

[0181] The isolated antibodies or antigen-binding fragments of the present invention can be used to treat partial or complete vascular In various A2AP-related disorders and / or diseases associated with ischemic events due to occlusion It can be used as a therapeutic or diagnostic tool.

[0182] An ischemic event can result from partial or complete occlusion of one blood vessel, but it can also result from several Two or more blood vessels may be partially occluded and some blood vessels may be completely occluded. This may be the result of partial or complete blockage of the ventricle.

[0183] Thus, in a further aspect, the present invention relates to a method for treating ischemic stroke, acute coronary syndrome, peripheral arterial disease, and the like. Cardiac disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis. Treatment of any disorder or disease associated with an ischemic event resulting from partial or complete vascular occlusion or for use in prophylaxis. or a conjugate comprising the isolated antibody or antigen-binding fragment according to the invention. or a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to the invention. Pertaining to things.

[0184] In a further aspect, the present invention relates to a method for treating diseases, particularly ischemic stroke, acute coronary syndrome, peripheral arterial disease, and the like. Cardiac disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis Treatment of disorders or diseases associated with ischemic events resulting from partial or complete vascular occlusion or an isolated antibody or antigen-binding fragment thereof according to the present invention for prophylaxis. A conjugate comprising an isolated antibody or antigen-binding fragment according to the invention; or The present invention also relates to a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to the present invention.

[0185] In a further aspect, the present invention relates to a method for treating diseases, particularly ischemic stroke, acute coronary syndrome, peripheral arterial disease, and the like. Cardiac disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis Treatment of disorders or diseases associated with ischemic events resulting from partial or complete vascular occlusion The present invention also provides an isolated antibody or antigen-binding fragment thereof in a method of prevention. or a conjugate comprising an isolated antibody or antigen-binding fragment according to the invention; or the use of a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to the invention. Regarding use.

[0186] In a further aspect, the present invention relates to a method for treating diseases, particularly ischemic stroke, acute coronary syndrome, peripheral arterial disease, and the like. Cardiac disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis Prevention or treatment of disorders or diseases associated with ischemic events resulting from partial or complete vascular occlusion The present invention relates to an isolated antibody against a medicament for the preparation of a pharmaceutical composition, preferably a medicament, for treatment. or an isolated antibody or antigen-binding fragment according to the present invention. Conjugates comprising fragments or isolated antibodies or antigen-binding fragments according to the invention. The present invention relates to the use of pharmaceutical compositions comprising the conjugated fragments.

[0187] In a further aspect, the present invention provides an isolated antibody or antigen-binding fragment according to the present invention. or a conjugate comprising an isolated antibody or antigen-binding fragment according to the invention. or a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to the present invention. The composition is used in an effective amount to treat ischemic stroke, acute coronary syndrome, peripheral artery disease, myocardial infarction, deep Partial or complete hematoma, such as venous thrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis The present invention relates to a method for treating or preventing a disorder or disease associated with an ischemic event resulting from vascular occlusion. The above-mentioned disorders have been well characterized in humans, but also in other animals, including mammals. These diseases have the same etiology and can be treated by administering the pharmaceutical composition according to the present invention. This can be done.

[0188] The antibodies or antigen-binding fragments or variants thereof according to the present invention can be used in the same manner as known pharmaceuticals. In some cases, the antibody or antigen-binding fragment thereof may itself be modified. For example, the antibody or antigen-binding fragment thereof or variant thereof may be To potentially further increase It can be adjuvanted.

[0189] The antibody or antigen-binding fragment or variant thereof of the present invention can be used as the sole pharmaceutical agent. or in combination with one or more additional therapeutic agents, wherein The combination does not cause unacceptable adverse effects.

[0190] Thus, in a further aspect, the present invention provides one or more further therapeutically active The isolated compounds according to the invention for use in combination with the compound, either simultaneously, separately or sequentially. or a conjugate according to the invention, The present invention relates to a pharmaceutical composition.

[0191] Therapeutic active compounds for use in combination with the antibodies or antigen-binding fragments according to the invention Non-limiting examples of products are: i) Inhibitors of the coagulation cascade such as plasminogen activators (thrombolytic / fibrinolytic agents) lytic agents), and compounds that increase thrombolysis and / or fibrinolysis (tissue plus t-PA, streptokinase, reteplase, and urokinase plasminogen activator inhibitors (PAIs) or thrombin time inhibitors (THAILs). inhibitors of thrombin-activatable fibrinolysis inhibitor (TAFI); ii) Unfractionated heparin, low molecular weight heparin, heparinoids, hirudin, bivalirudin and and / or anticoagulants such as argatroban; direct oral anticoagulants / factor Xa inhibitors, etc. non-vitamin K anticoagulants, such as apixaban, edoxaban and rivaroxaban, as well as thrombin inhibitors, such as dabigatran.

[0192] iii) aspirin, clopidogrel, cilostazol, prasugrel, ticagrelor, Platelet aggregation inhibitors such as cangrelor.

[0193] The combination therapy comprises an antibody or antigen-binding fragment or variant thereof according to the invention and Administration of a single pharmaceutical dosage formulation containing one or more additional therapeutic agents, as well as an antibody according to the invention, or antigen-binding fragment and each additional therapeutic agent in its own separate pharmaceutical dosage form. For example, the antibody or antigen-binding fragment thereof of the present invention or The variant and the therapeutic agent may be administered to the patient together in a single liquid composition; Alternatively, each agent may be administered in a separate dosage formulation.

[0194] When separate dosage formulations are used, the antibody or antigen-binding fragment or or a variant thereof and one or more additional therapeutic agents are administered essentially simultaneously (e.g., simultaneously (c The doses may be administered concurrently) or at staggered intervals (eg, sequentially).

[0195] The antibody or antigen-binding fragment or variant thereof according to the invention may be used in the treatment of mechanical embolectomy. Surgical interventions such as, but not limited to, thrombectomy, thrombectomy devices, and cerebral revascularization It may be used in combination with

[0196] Diagnostic methods Furthermore, the antibodies or antigen-binding fragments according to the invention may be used by themselves or in compositions In particular, they may be utilized in research and diagnosis, or as analytical standards, and the like.

[0197] The anti-A2AP antibody or antigen-binding fragment thereof is used to detect the presence of A2AP. Thus, in a further aspect, the present invention relates to a method for the preparation of a medicament for use as a diagnostic agent. An isolated antibody or antigen-binding fragment according to the invention or a method for treating a chronic obstructive pulmonary disease (COPD) using the invention The present invention relates to an antibody conjugate according to the present invention.

[0198] Pharmaceutical Compositions and Administration In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. The present invention also relates to a pharmaceutical composition comprising the antibody conjugate according to the present invention for treating the aforementioned disorders. The pharmaceutical compositions for use in accordance with the present invention may comprise one or more biomarkers for the treatment of any of the following: The composition may be formulated in any conventional manner using physiologically acceptable carriers, excipients, or auxiliaries. Further details regarding techniques for formulation and administration can be found in Remington's Pharmaceutical Sciences (ed. Maack Publi (Easton, Pa.), most recent edition.

[0199] The antibodies or antigen-binding fragments according to the invention may be administered in a variety of ways depending on the type of disorder being treated. The compound can be administered by any suitable means that can effectively inhibit the release of the compound. Possible routes of administration include oral administration. These include oral, parenteral, and topical administration. Methods of parenteral delivery include intraarterial, intramuscular, subcutaneous, In addition, the present invention also provides methods for administering the present invention, including intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, or intranasal administration. The antibody or antigen-binding fragment may be administered, for example, by pulse injection with decreasing doses of the antibody. Preferably, administration is by injection, most preferably intravenous or subcutaneous. The effect is based on the dose, depending in part on whether the administration is brief or chronic. The amount administered depends on a variety of factors, including clinical symptoms, the weight of the individual, and whether other drugs are being administered. Those skilled in the art will appreciate that the route of administration will vary depending on the disorder or condition being treated. They will recognize it.

[0200] The pharmaceutical composition according to the invention comprises an antibody or antigen-binding fragment according to the invention alone. or in combination with at least one other agent, such as a stabilizing compound. The antibody or antigen-binding fragment may be dissolved in saline, buffered saline, dextrose, and water. In certain embodiments, the pharmaceutical composition according to the invention may be administered in combination with one or more additional pharmaceutical agents. and, in particular, compounds which are therapeutically active in treating A2AP-associated disorders and / or partial or complete vascular occlusion. and one or more further pharma- ceutical active agents suitable for treating disorders associated with an ischemic event due to Any of these agents may be used alone or in combination with other agents or drugs. In a pharmaceutical composition in combination with an excipient(s) or a pharma- ceutically acceptable carrier. In certain embodiments, the pharma- ceutically acceptable carrier can be It is pharmacologic-inactive.

[0201] Pharmaceutical compositions for oral administration may be prepared using any of the pharmaceutical compositions known in the art in dosages suitable for oral administration. The composition can be formulated using a carrier that is acceptable for the patient. For the purpose of the present invention, the pharmaceutical composition may be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurry, etc. It can be formulated into a liquid, suspension, etc.

[0202] Pharmaceutical preparations for oral use consist of a combination of the active compound with solid excipients, optionally resulting in Grinding the resulting mixture and, if desired, mixing the granules after adding suitable auxiliaries. The mixture can be processed to obtain tablets or dragee cores. The agent may be a carbohydrate or protein filler, e.g., lactose, sucrose, mannitol, or sugars containing sorbitol; from corn, wheat, rice, potato, or other Starch of vegetable origin; methylcellulose, hydroxypropyl methylcellulose, or Cellulose such as sodium carboxymethylcellulose; gum arabic and tragacanth and proteins such as gelatin and collagen. Depending on the situation, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be used. Disintegrating or solubilizing agents, such as any salt thereof, may be added.

[0203] The dragee core contains gum arabic, talc, polyvinylpyrrolidone, carbopol gel, Polyethylene glycol and / or titanium dioxide, lacquer solution and suitable Apply a suitable coating such as a concentrated sugar solution that may contain an organic solvent or mixture of solvents. Dyes or pigments can be used for product identification or to indicate the amount of active compound, i.e. They may be added to the tablets or dragee coatings, i.e. to characterize dosage.

[0204] Pharmaceutical preparations that can be administered orally include gelatin push-fit capsules. and coatings such as gelatin and glycerol or sorbitol. The push-fit capsules include soft, sealed capsules containing lactose or Fillers or binders such as starch, lubricants such as talc or magnesium stearate The soft capsule may contain the active ingredient mixed with a stimulant, a stimulant, and optionally a stabilizer. In this formulation, the active compound is dissolved in fatty oils, liquid paraffin, liquid glycerol, or glycerol, with or without stabilizers. It is dissolved or suspended in a suitable liquid, such as polyethylene glycol.

[0205] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds. The pharmaceutical compositions of the invention may be prepared in aqueous solutions, preferably in physiologically compatible buffers, such as Hanks' The formulation may be formulated in saline, Ringer's solution, or physiologically buffered saline. Suspensions may be made with sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of active compounds may contain agents which increase the viscosity of the suspension, such as Suitable oily injection suspensions can be prepared. A suitable lipophilic solvent or vehicle is sesame oil. or synthetic fatty acid esters such as ethyl oleate or triglycerides. Optionally, the suspension also allows for the preparation of highly concentrated solutions. In order to make the compound more soluble, it may contain suitable stabilizers or agents that increase the solubility of the compound. be.

[0206] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0207] The pharmaceutical compositions of the present invention can be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, micronizing, or other suitable methods. by means of a vigating, emulsifying, encapsulating, entrapment or freeze-drying process. It can be prepared by methods known in the art.

[0208] The pharmaceutical compositions may be provided as salts, such as hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, phosphoric acid, etc. Salts can be formed with acids including, but not limited to, acetic acid, succinic acid, etc. They tend to be more soluble in aqueous or other protic solvents than their corresponding free base forms. In other cases, preferred preparations contain 1 mM to 50 mM histidine or phosphate or in Tris, 0.1% to 2% sucrose and / or 2% to 7% mannitol Alternatively, the composition may be a lyophilized powder having a pH in the range of 4.5 to 7.5, and may be optionally diluted with a buffer solution prior to use. It contains additional substances such as polysorbates that are mixed in.

[0209] After a pharmaceutical composition containing a compound of the present invention formulated in an acceptable carrier is prepared, They can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of an anti-A2AP antibody or antigen-binding fragment thereof, such labeling may be used. includes the amount, frequency and method of administration.

[0210] Therapeutically Effective Dose Pharmaceutical compositions suitable for use according to the invention include those that are capable of delivering the intended purpose, e.g. partially or completely in an amount effective to effect treatment of a particular disease state characterized by ischemic events due to a vascular occlusion. Compositions in which the active ingredient is contained are included.

[0211] Determination of an effective dose is well within the capabilities of those skilled in the art. Determining a therapeutically effective amount of an antigen-binding fragment or variant thereof will depend primarily on the specific It depends on the characteristics of the patient, the route of administration, and the nature of the disorder being treated. For example, the International Conference on Harmonization armonization) publications and REMINGTON'S PHARMACE UTICAL SCIENCES, Chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pu. b.Co., 1990). More specifically, determining a therapeutically effective amount The efficacy of the drug depends on factors such as toxicity and efficacy. Toxicity can be determined by methods well known in the art. The efficacy can be determined using the methods described in the above references. The same guidance may be used in conjunction with the methods described below to determine.

[0212] For any compound, the therapeutically effective dose can be determined in cell culture assays or in animal models. The first estimation can be done in mice, rabbits, dogs, pigs or monkeys. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. It can be determined.

[0213] A therapeutically effective dose is an amount of an antibody or antigen-binding fragment thereof that ameliorates a symptom or condition. The therapeutic efficacy and toxicity of such compounds can be determined in cell cultures or experimental animals. Conventional pharmaceutical procedures in, e.g., ED 50 (therapeutic effective in 50% of the population Dose) and LD 50(the dose that is lethal to 50% of the population) The dose ratio between therapeutic and toxic effects is the therapeutic index, and the ED 50 / LD 50 Ratio of Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from clinical and animal studies should be used in formulating a range of dosages for human use. The dosage of such compounds is preferably such that it poses little or no harmful effects. No ED 50 The dose will vary depending on the dosage form used, the patient's This will vary within this range depending on sensitivity and route of administration.

[0214] The exact dosage is chosen by the individual physician in view of the patient to be treated. and administration should be such as to provide sufficient levels of the active moiety or to maintain the desired effect. Further factors that may be considered are the severity of the disease state, the age, weight and diet, time and frequency of administration, drug combinations, reaction sensitivities, and treatment Long-acting pharmaceutical compositions include those that are resistant / responsive to the drug. Depending on the rate of relapse, for example, every 3-4 days, every week, every 2 weeks, or every 3 weeks It is possible.

[0215] Usual doses range from 0.1 to 100,000 micrograms, up to about 1 mg, depending on the route of administration. The total dose may vary from 0.5 to 10 g. Guidance regarding specific doses and methods of delivery is available in the literature. Nos. 4,657,760, 5,206,344, and 5,22 See No. 5,212.

[0216] kit In a further aspect, the present invention relates to an isolated antibody or antigen-binding fragment according to the present invention. The present invention also relates to a kit comprising a composition or a conjugate according to the present invention and instructions for use. In one embodiment, the kit comprises one or more of the components of the aforementioned compositions of the invention. In connection with such container, the container or containers may include a container or containers filled with A pharmaceutical or biological product that reflects approval by a regulatory agency for the manufacture, use, or sale of the product for or (b) a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of the product. It is possible.

[0217] BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0218] [Figure 1] Figure 1: Panning strategies to find species-specific neutralizing anti-α2-antiplasmin antibodies. Four main strategies for selection against biotinylated antigen are shown. Where indicated, we included a depletion step of related or non-α2-antiplasmin biotinylated proteins before each round of selection. [Diagram 2] Figure 2: ELISA-based analysis of the binding of Fab 431A-M080-C01 to human and rabbit α2-antiplasmin. Specific binding of Fab 431A-M080-C01 to human (black columns) and rabbit (grey columns) α2-antiplasmin, assessed in an ELISA assay, is shown. The antigen was coated on microtiter plates at a final concentration of 1 μg / ml. For this, the supernatant of transfected cells was diluted in phosphate-buffered saline (PBS) with the following factors: 1:1,5, 1:4,5, 1:13,5, 1:40,5, 1:121,5, 1:364,5, 1:1093,5. Relative fluorescence units (RFU, ordinate) are plotted against the diluted Fab (abscissa). For details, see Example 3. [Diagram 3] Figure 3: Analysis of the function blocking activity of Fab 431A-M080-C01. The function blocking activity of Fab 431A-M080-C01 is shown as measured in a plasmin-α2-antiplasmin biochemical assay. For this, the supernatant from mammalian cells containing the Fab of interest was preincubated with human or rabbit α2-antiplasmin, followed by the addition of human plasmin and a fluorogenic plasmin substrate. The relative fluorescence units resulting from the cleavage of the substrate by plasmin were measured. The data obtained are shown as a percentage of inhibition. The light grey columns on the left represent the neutralization of human α2-antiplasmin, and the dark grey columns on the right represent the neutralization of rabbit α2-antiplasmin. For a detailed description of the biochemical assay, see Example 4. [Figure 4] Figure 4: Binding activity of antibody TPP-12 387 to human and rabbit α2-antiplasmin. Antibody TPP-12 387 was tested for its ability to bind human and rabbit α2-antiplasmin in a dose-dependent manner according to the method described in Example 3. The binding activity to human α2-antiplasmin is shown in the left panel and the binding activity to rabbit α2-antiplasmin is shown in the right panel of this figure. The binding activity was calculated as EC50 at M values. One dose-response curve is shown as an example of two to three independent experiments performed in quadruplicate: EC50 (human A2AP) was 1.2E-07M; EC50 (rabbit A2AP) was 6.0E-09M. [Diagram 5] Figure 5: Neutralizing activity of antibody TPP-12 387 against human and rabbit α2-antiplasmin. Antibody TPP-12 387 was tested for its ability to block the activity of human and rabbit α2-antiplasmin in a dose-dependent manner according to the methods described in Example 4. The neutralizing activity against human α2-antiplasmin is shown in the left panel and the neutralizing activity against rabbit α2-antiplasmin is shown in the right panel of this figure. The functional blocking activity was calculated as EC50 in M ​​values. One dose-response curve is shown as an example of two to three independent experiments performed in quadruplicate: EC50 (human A2AP) was 1.7E-07M; EC50 (rabbit A2AP) was 1.4E-09M. [Figure 6] Figure 6: Binding and function blocking activity of antibody TPP-1238 7 against cynomolgus monkey α2-antiplasmin. Antibody TPP-12387 was tested for its ability to block the activity of cynomolgus monkey α2-antiplasmin in a dose-dependent manner according to the methods described in Examples 3 and 4. The binding activity of the antibody against cynomolgus monkey α-2 antiplasmin is shown in Figure 6.1, and its neutralizing activity is shown in Figure 6.2. Activity was calculated as EC50 in M ​​values. One dose-response curve is shown as an example of two to three independent experiments performed in quadruplicate: EC50 (cynomolgus monkey A2AP binding) was 9.9E-08M; EC50 (cynomolgus monkey A2AP activity blocking) was 1.6E-07M. [Figure 7] Figure 7: Binding and function-blocking activity of TPP-12387 variants against human α2-antiplasmin. According to the methods described in Examples 3 and 4, antibodies TPP-14323 (7.17, 7.18), TPP-14318 (7.15, 7.16), TPP-14314 (7.13, 7.14), TPP-14313 (7.11, 7.12), TPP-14308 (7.9, 7.10), TPP-14305 (7.7, 7.8), TPP-14303 (7.5, 7.6), TPP-14298 (7.3; 7.4) and TPP-14293 (7.1; 7.2) were tested for their ability to bind and block the activity of human α2-antiplasmin in a dose-dependent manner. The binding activities against human α2-antiplasmin are shown in 7.1, 7.3, 7.5, 7.7, 7.9, 7.11, 7.13, 7.15, 7.17, and the neutralizing activities are shown in 7.2, 7.4, 7.6, 7.8, 7.10, 7.12, 7.14, 7.16, 7.18. The binding and function blocking activities were calculated as EC50 at M values ​​(Table 3.3). For each antibody, one dose-response curve is shown as an example of two to three independent experiments performed in quadruplicate.

[0219] [Figure 8]Figure 8: Testing germline variants of TPP-14308 for binding and neutralization of human alpha2-antiplasmin. Forty-seven antibodies resulting from the germline approach of TPP-14308 were tested for their ability to bind and block the activity of human alpha2-antiplasmin in a dose-dependent manner compared to TPP-14308. Six antibodies resulting from the germline approach of TPP-14308 (TPP-17041, TPP-17044, TPP-17045, TPP-17048, TPP-17051, TPP-17053) show improved binding and / or neutralizing activity. Binding activity against human alpha2-antiplasmin is shown in 8.1, 8.3, 8.5, 8.7, 8.9, 8.11 and neutralizing activity is shown in 8.2, 8.4, 8.6, 8.8, 8.10, 8.12. Binding and function blocking activity was calculated as EC50 at M values ​​(Table 3.5). For each antibody, one dose-response curve is shown as an example from 2-3 independent experiments performed in quadruplicate (squares = TPP17308, circles = TPP-17041, TPP-17044, TPP-17045, TPP-17048, TPP-17051 or TPP-17053). [Figure 9]Figure 9: Neutralizing activity of TPP-170 44 IgG1 antibody against human α2-antiplasmin from different species. Testing of TPP-17044 for function blocking activity against human (9.1), cynomolgus monkey (9.2), and rabbit (9.3) α2-antiplasmin by the method described in Example 4 is shown. Neutralizing activity was calculated as EC50 in M ​​values. For this antibody, one dose-response curve is shown as an example of 2-3 independent experiments performed in quadruplicate. The function blocking activity of TPP-17044 against human α2-antiplasmin was 4.4E-10 M (shown in Figure 9.1), 5.4E-10 M in the second experiment, and 5.0E-10 M in the third experiment. For inhibition of cynomolgus monkey α2-antiplasmin the values ​​were 4.6E-10M (Figure 9.2), for the second experiment 4.9E-10M and for the third experiment 5.1E-10M. Rabbit α2-antiplasmin had its activity blocked by TPP-17044 with IC50 values ​​of 2.7E-08M (Figure 9.3), for the second experiment 3.6E-08M and for the third experiment 2.9E-08M. [Figure 10] Figure 10: Neutralizing activity of TPP-1 7928 IgG4 antibody against human α2-antiplasmin from different species. Testing of TPP-1 7928 for function blocking activity by the method described in Example 4 against human (10.1), cynomolgus monkey (10.2), and rabbit (10.3) α2-antiplasmin is shown. Neutralizing activity was calculated as EC50 in M ​​values. For this antibody, one dose-response curve is shown as an example of 2-3 independent experiments performed in quadruplicate. The function blocking activity of TPP-17928 against human α2-antiplasmin was 1.1E-10 M (shown in Figure 10.1) and in the second experiment 1.6E-10 M. For inhibition of cynomolgus monkey α2-antiplasmin, the values ​​were 2.6E-10 M (Figure 10.2), 3.4E-10 M in the second experiment, and 2.9E-10 M in the third experiment. Rabbit α2-antiplasmin had its activity blocked by TPP-17928 with IC50 values ​​of 1.5E-08M (Figure 10.3) in one experiment, 1.9E-10M in the second, and 1.6E-10M in the third. [Figure 11] Figure 11: Reduction of clot lysis time by TPP-17928. Antibody TPP-17928 reduces tPA-induced clot lysis time in human (triangles) and rabbit (squares) plasma in a dose-dependent manner, respectively. Activity was calculated as IC50 at M value. Curves represent the mean (+ / -SD) from three independent experiments. For experimental details, see Example 7. IC50 (human plasma) was 2.5E-07M and EC50 (rabbit plasma) was 2.3E-07M. [Figure 12] Figure 12: In vivo effect of TPP-17928 on clot lysis. Animals received fluorescently labeled plasma clots 30 min before the measurements, and each treatment was administered at 0 min. Plasma samples were taken over 360 min to measure the amount of plasma fluorescence as an indirect parameter of clot lysis. The effect of different concentrations of TPP-17928 (Figure 12.1, control circles; 3.75 mg / kg open circles; 7.5 mg / kg open squares; 15 mg / kg open triangles), as well as the effect of different concentrations of tPA (Figure 12.2, control circles, 0.125 mg / kg triangles, 0.25 mg / kg squares, 1 mg / kg diamonds) or a combination of both (Figure 12.1, 15 mg / kg + 0.125 mg / kg tPA (open diamonds)) on clot lysis was measured. Relative fluorescence units (rFU, ordinate) are plotted against the time points at which the plasma samples were taken (abscissa). Values ​​are mean + / - SD. TPP-17928 alone has a dose-dependent effect on clot lysis. After application of the antibody of the present invention, the activity maximum is reached at about 60 minutes, which then leads to a sustained effect over the entire experimental time (360 minutes). tPA treatment also shows a dose-dependent effect on clot lysis. tPA shows a rapid increase in clot lysis (maximum after 15 minutes), but does not show a longer-lasting effect, as observed with TPP-17928. Co-administration of TPP-17928 to low-dose tPA leads to faster clot lysis than administration of a single compound. For details, see Example 8. [Figure 13] Figure 13: Measurement of tPA and TPP-17928 induced ear bleeding time. Concurrent with the plasma fluorescence measurements (shown in Figure 12), ear bleeding time was measured at 0 minutes after compound administration. Bleeding time (seconds) is shown for each treatment group. Values ​​are mean + / - SEM.

[0220] Column 1: Control Column 2: 0.125 mg / kg tPA; Column 3: 0.25 mg / kg tPA Column 4: 1 mg / kg tPA Column 5: 3.75 mg / kg TPP-17928 Column 6: 7.5 mg / kg TPP-17928 Column 7: 15 mg / kg TPP-17928 Column 8: 15 mg / kg TPP-17928 + 0.125 mg / kg tPA [Figure 14] FIG. 14: Effect of 77A3 and antibodies of the invention on plasmin. Results of the A2AP functional blocking assay described in Example 10 are shown.

[0221] The following antibodies and antibody concentrations were used: Figure 14.1: 6.1E-11~3.0E-08M 77A3 (left) and 6.11E- 11~1.0E-06M 77A3 (right) Figure 14.2: 6.11E-11~1E-06M 77A3 (circle) and 6.1E-11~ 1.0E-06M TPP-17041 (triangle) Figure 14.3: 6.1E-11~1.0E-06M 77A3 (circle) and 6.1E-11~ 1.0E-06M TPP-17044 (square) Figure 14.4: 6.1E-11~1Ee-06M 77A3 (circle) and 6.1E-11~ 1.0E-06M TPP-17045 (triangle) Figure 14.5: 6.1E-11~1.0E-06M 77A3(circle) and 6.1E-11 ~1.0E-06M TPP-17048 (diamond) Figure 14.6: 6.1E-11~1.0E-06M 77A3(circle) and 6.1E-11 ~1.0E-06M TPP-17051 (triangle). Figure 14.7: 6.1E-11~1.0E-06M 77A3(circle) and 6.1E-11 ~1.0E-06M TPP-17053 (square).

[0222] Increasing concentrations of 77A3 up to 0.03 μM inhibited the plasmin-mediated inhibition of the fluorescent substrate I-1275 However, further increase in the concentration of 77A3 resulted in an increase in the fluorescent signal due to cleavage of An increase in the concentration of 0.03 μM resulted in a decrease in the fluorescent signal. Testing of antibody 77A3 in the biochemical assay described in Further increases in 77A3 antibody concentration resulted in a decrease in plasmin activity, We show that a concentration of 77A3 of M results in complete inhibition of plasmin activity (Figure 14.1). Surprisingly, in comparison to this finding, the test antibodies of the present invention up to 1 μM did not significantly reduce the fluorescent signal. No reduction in plasmin activity was observed, indicating that the test antibody of the present invention does not affect plasmin activity. This shows that (Figure 14.2-14.7). [Figure 15] Figure 15: Amino acid sequences of preferred antibodies according to the invention. The amino acid sequences of VH, H-CDR1, H-CDR2, H-CDR2, H-CDR3, VL, L-CDR1, L-CDR2, L-CDR3, heavy chain and light chain of preferred antibodies according to the invention are shown. [Figure 16] Figure 16: Effect of 77A3 and antibodies of the invention on the proteolytic activity of plasmin. Plasmin activity as a function of the antibody concentration used as described in Example 11 for 77A3 (squares) and TPP-17928 (circles). One dose-response curve from 2-3 independent experiments performed in duplicate is shown as an example. 77A3 shows a concentration-dependent inhibitory effect on plasmin activity (IC50 1.7 μM), whereas TPP-17928 surprisingly does not inhibit plasmin activity up to a concentration of 10 μM (see also the tabular summary of IC50 values ​​in Example 11).

[0223] Sequence Listing Attached is a sequence listing disclosing the following sequences: [Table 5] TIFF2024063234000008.tif228168 [Table 6] TIFF2024063234000010.tif11166 [Table 7] [Table 8] TIFF2024063234000013.tif254166TIFF2024063234000014.tif254166TIFF2024063234000015.tif253166TIFF202 4063234000016.tif254166TIFF2024063234000017.tif254168TIFF2024063234000018.tif254166TIFF20240632340 00019.tif253167TIFF2024063234000020.tif253166TIFF2024063234000021.tif254166TIFF2024063234000022.t if254168TIFF2024063234000023.tif252168TIFF2024063234000024.tif253167TIFF2024063234000025.tif185167 [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] TIFF2024063234000032.tif252166TIFF2024063234000033.tif252168TIFF2024063234000034.tif253167TIFF2024063234000035.tif57166 [Table 15] TIFF2024063234000037.tif254166TIFF2024063234000038.tif255167 TIFF2024063234000039.tif253166TIFF2024063234000040.tif48166 [Table 16] TIFF2024063234000042.tif253167TIFF2024063234000043.tif253168TIFF2024063234000044.tif253167 TIFF2024063234000045.tif252168TIFF2024063234000046.tif251167TIFF2024063234000047.tif252167 TIFF2024063234000048.tif253168TIFF2024063234000049.tif254167TIFF2024063234000050.tif253168 TIFF2024063234000051.tif253166TIFF2024063234000052.tif252166TIFF2024063234000053.tif183167 [Table 17] TIFF2024063234000055.tif239168 [Table 18] EXAMPLES

[0224] Working Example Example 1: Generation of Antibody TPP-12387 from the BioInvent Antibody Library Fully human antibody phage display library (BioInvent n-CoDe R Fab lambda library) to identify human and rabbit-derived human α2-alpha Human monoclonal antibodies were isolated by selection against a soluble biotinylated antigen, antiplasmin. The body was isolated.

[0225] Human α2-antiplasmin was obtained from a commercial source (Antibody Online; catalog number ABIN 2544306), but the rabbit antigen was produced in-house by recombinant expression and purification. For this purpose, the cDNA from rabbit α2-antiplasmin was inserted into the standard expression vector Cloned HEK293 cells into 293fectin transfectants according to the manufacturer's instructions. The ELISA kit was prepared using a ELISA kit (Invitrogen, Catalog No. 12347-019). The construct was transiently transfected to express rabbit α2-antiplasmin. Purified from cell culture supernatant via Ni-IMAC and size-exclusion chromatography did.

[0226] Sulfo-NHS-LC-Biotin kit (Thermo Scientific The antigen was biotinylated using 100% Fibrinogen (Catalogue No. A39257). Free biotin was then added to the appropriate The reaction was removed by dialysis against the appropriate buffer.

[0227] For the panning procedure the following protocol was applied: streptavidin-bound D Ynabeads M-280 (Invitrogen, Catalog No. 11205D) , biotinylated antigen (1 tube) and biotinylated off-target (3 tubes), respectively. The Dynabeads were washed and then coated with 1000 mM NaCl for 1 hour at room temperature (RT). Blocking was performed for 1 h at RT with band rotation. The phage library was then loaded onto a blocked off-target loading dye. The beads were added and incubated for 10 min at room temperature with end-over-end rotation. This depletion step was repeated twice. The depleted phage library was then purified by centrifugation using a blocked target. The ink was added to the loaded Dynabeads and incubated at RT for 60 min with end-over-end rotation. After stringent washing (3x in blocking buffer, 9x 0.05% in PBS with Tween-20 (150 mM NaCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; pH adjusted to 7.4-7.6), coated target Dynabeads carrying Fab-phages that specifically bind to Escherichia coli (E The cells were infected with HB101, a scherichia coli strain. The phage (Invitrogen, Cat. No. 18311019) was used to The phages are amplified in E. coli strain HB101. In the following selection rounds, the target concentration is lowered. This increased the selection pressure for high affinity binders.

[0228] During panning of this library, four different selection strategies were implemented: Strategy I used full-length human α2-antiplasmin and rabbit α2-antiplasmin, both of which lack the N-terminus. The study was designed to identify antibodies that exhibited binding activity against antiplasmin. A depletion step was included using otinated irrelevant proteins.

[0229] Strategy II aimed to develop antibodies that recognize the plasmin-binding site of α2-antiplasmin. Similar to strategy I, to increase the probability of success, we used α-antigens that lack plasmin-binding sites. A depletion step was included using the 2-antiplasmin mutant.

[0230] In two further strategies (Strategies III and IV), the so-called α2-antiplasmin Biotinylated linear and cyclic peptides representing various reactive center loops (RCLs) were prepared using the In both cases, an irrelevant biotinylated protein was used for the depletion step. .

[0231] A detailed overview of the panning strategy is shown in Figure 1.

[0232] Example 2: Recombinant DNA constructs and expression, purification and quantification of Fab and full-length antibodies quantification Production in HEK293-6E cells Mammalian cell culture using transiently transfected HEK293-6E cells Fab and full length antibodies were produced by the method described above. Heavy and light chains were expressed in appropriate expression vector systems. The cells were incubated for 3-4 days. The supernatant was collected and Fab and and antibodies were purified as described.

[0233] Fab and antibody purification and quantification Antibodies were purified using Protein A chromatography (ThermoFischer, catalog no. The product was purified according to the manufacturer's instructions using the same procedure as in Example 1 (No. A26455).

[0234] Antibodies, antigen-binding portions, or portions thereof may be isolated using standard protein purification techniques. The derivatives were recovered from the culture medium.

[0235] Fab is produced from sterile-filtered mammalian cell supernatant using a three-step laboratory downstream process. The capture step was carried out using the "Capture Se" equilibrated in PBS pH 7.4. "lect IgG-CH1" affinity column (ThermoFisher, Catalog The washing buffer (PBS pH 7.4) was used for 10 min. After washing with 1000 cc of glycine, elution of Fab was performed using glycine 0.1 M pH 3.0 (6 CV). Neutralization with Tris Base resulted in size exclusion chromatography (GE Healthcare, Superdex 200, Catalog Number GE29321905 ) was used to exchange the buffer into DPBS pH 7.4 and remove aggregates. Size exclusion chromatography demonstrated the absence of dimers in the resulting batch. .

[0236] For quantification of full-length antibodies, anti-human IgG Fc-specific antibody (Sigma, catalog no. No. I2136) in a 384-well microtiter plate (Nunc) at 4°C overnight. The solution containing the IgG of interest was coated at different concentrations. The mixture was incubated at room temperature for 1 hour. gma) and Amplex Red was added as a substrate. Fluorescence was measured using SpectraF The results were monitored at 535 / 590 nm using a luorplus reader (Tecan).

[0237] For quantification of antibody variants such as Fab, use the Human Kappa ELISA K It (Abcam, Cat. No. ab157709) was used according to the manufacturer's instructions. Ta.

[0238] Example 3: Enzyme-linked immunosorbent assay to test anti-α2-antiplasmin binding activity Sei (ELISA) A standard ELISA format was used to detect human and rabbit α-2-antiplasmin. The binding affinity of the Fab of the present invention to each of the antigens was analyzed. Isorp microtiter plates (Nunc; Cat. No. 460518) were coated with Coating buffer (Candor Bioscience; catalog no. The plates were incubated overnight at 4°C. After overnight incubation, the plates were washed with PBS + 0.05% Tween 20. The plates were washed 2X with 50 μl / well. Then, 50 μl / well of blocking buffer was added. Buffer solution (Smart Block; Candor Bioscience; Cat. no. 1 13500) was added and the plate was incubated at room temperature for 1 hour. , washed 3X with 50 μl / well of PBS+0,05% Tween 20 buffer The Fabs of the present invention were added at different concentrations in a final volume of 30 μl / well. The plate was incubated at room temperature for 1 hour. After this incubation step, the plate was Wash 3X with μl / well of PBS+0.05% Tween 20 buffer. For detection of bound Fab and full-length antibodies, anti-human lambda light chain (bound and free)-peptide was used. Antibody to ribozyme (Sigma; Cat. No. A5175) in 10% blocking buffer Add 30 μl / well of this diluted detection antibody to the plate. The plates were incubated at room temperature for 1 hour. After this incubation step, the plates Wash 3X with 50 μl / well of PBS+0.05% Tween 20 buffer. As a substrate, 30 μl / well of 1:1000 diluted Amplex Red (Invitrogen) was used. rogen; Catalog No. 12222; 10 mM stock solution in DMSO) and 1:10.0 Add a mixture of 0.1 ml of hydrogen peroxide (Merck; catalog number 107209; 30% stock solution) The plate was then incubated in the dark for 20 minutes.

[0239] The measurements were performed using an Infinite f500 reader (Tecan). Do: Fluorescence; top reading; Ex 535nm; Em 590nm.

[0240] Screened 2 x 10 10 From the total number of Fab mutants, 2944 mutants were selected. Potential candidates to be tested for binding to human and rabbit α2-antiplasmin, respectively was selected as a complement.

[0241] HEK293 cells were transiently transfected as described in Example 2. The supernatants were further analyzed to test their ability to bind human and rabbit antigens. From these 2944 Fabs, different sequences were identified and used directly without further purification or dilution. Eighty-eight candidates shown exhibited the required cross-species binding activity for the two antigens.

[0242] Conformation-wise, 88 mutants of the invention were retested for their binding ability. For this purpose, the supernatant of the transfected cells was diluted with phosphate-buffered saline (PBS) and BS), 1:1,5, 1:4,5, 1:13,5, 1:40,5, 1:121,5, The diluted samples were diluted with a factor of 1:364.5 and 1:1093.5. The binding of rabbit α-2-antiplasmin was tested.

[0243] One dose-response curve is shown as an example of two to three independent experiments performed in quadruplicate in Figure 4. The binding of TPP-1 to human α2-antiplasmin was confirmed in three independent experiments. The EC50 values ​​for the binding activity of 2387 were as follows: 1.2E -07M (as shown in Figure 4), 1.0E-07M, and 1.3E-07M. Rabbit α2 - The binding activity to antiplasmin was 6.0E-09M (as shown in Figure 4), respectively. ), 6.07E-09M and 6.2E-09M.

[0244] Example 4: Bioassays for Testing Selected Candidates for α2-Antiplasmin Function Blocking Activity Chemical Assays To test the functional blocking activity of the anti-α2 antiplasmin molecule, Fabs or whole The long antibody was incubated with 1 nM human α2-antiplasmin (Antibody Online; Catalog No. ABI N2544306), or in-house produced rabbit α2-antiplasmin or in-house produced Cynomolgus monkey α2-antiplasmin and 50 mM TRIS-HCl (GIBCO; Catalog No. 15567-027 (50 mM)), 100 mM NaCl (Sigma; Catalog number S7653), 5 mM CaCl2 (Sigma; Catalog number 21115- 100ML), 0,1% Albumin 0,1% (Sigma; BSA, Cat. No. A45 Preincubate for 20 min at 37°C in a buffer consisting of 03-100g) at pH 7.4. I installed it.

[0245] Then, human plasmin (Haematologic Tech. nologies INC; Catalog No. HCPM0140) and a final concentration of 50 μm Fluorescent substrate I-1275 (Bachem; MeOSuc-Ala-Phe-Lys-AMC Trifluoroacetate salt; Catalog No. I-1275 (Stock: 10 mM in DMSO) )) was added and the reaction was incubated at 37°C for 1 hour. Performed in clotting plates (Nunc; Cat. No. 262260). Fluorescence generation The signal was measured under the following conditions: Mode Fluorescence Top Reading, Ex 360 nM , Em 465nm, Ex bandwidth 5nm, Em bandwidth 5nm.

[0246] To determine the dose-dependence of Fab and / or full-length antibody function blocking activity, The concentrations of the molecules were measured as above. Starting with a defined concentration, the concentration was then increased to 1:3 or Different concentrations of Fab and / or full-length antibodies were added, followed by a 1:4 dilution step, up to 1 nM each. Preincubated with human, cynomolgus monkey, or rabbit α2-antiplasmin .

[0247] 88 Fab candidates were assayed in single-point assays with human and rabbit α2-antiplasmin. The function-blocking activity of the IL-11A1-binding domain in transiently transfected HEK29 cells was tested. This means that the maximum possible volume of supernatant from 3 cells was added to the activity assay. This resulted in the identification of 17 Fas that showed at least a 30% reduction in α2-antiplasmin activity. b was identified.

[0248] In the next step, these 17 Fabs were refolded into a full-length IgG1 antibody format. Of these, 12 showed reasonable expression rates.

[0249] These 12 antibodies were synthesized using human and rabbit α2-antibodies according to the method described in Example 3. The ability of the antibody to bind antiplasmin in a dose-dependent manner was tested. The binding activity of the antibody was analyzed using raphPadPrism software. The results were calculated as follows: Two to three independent experiments were performed in quadruplicate.

[0250] In the next step, these 12 full-length antibodies were synthesized using human and rabbit α2-antibodies. Blocking activity against rasmin was retested in a dose-dependent manner.

[0251] Its binding and functional blocking activities for human and rabbit α2-antiplasmin antibodies Based on the efficacy and safety of TPP-12387, TPP-12387 was selected for further testing and optimization. is the T for human and rabbit α2-antiplasmin determined in Example 3. FIG. 3 shows the binding activity of the Fab fragment corresponding to PP-12387. The activity of TPP-1238 against human and rabbit α2-antiplasmin was determined in FIG. 4 shows the function blocking activity of the Fab fragment corresponding to 7. I for human and rabbit α2-antiplasmin as determined in binding assays Figure 5 shows the binding activity of the gG1 antibody TPP-12387. IgG1 against human and rabbit α2-antiplasmin as determined in the IgG1 assay Figure 1 shows the functional blocking activity of the antibody TPP-12387. A dose-response curve for the neutralizing activity of TPP-12387 against rasmin was performed in quadruplicate. Figure 5 shows an example of two to three independent experiments performed in duplicate. The IC5 value of TPP-12387 for blocking activity against human α2-antiplasmin was The 0 values ​​were as follows: 1.7E-07M (as shown in FIG. 5), 1.8 E-07M, and 1.8E-07M. Binding activity to rabbit α2-antiplasmin The values ​​were 1.4E-08M (shown in Figure 4), 1.3E-08M, and 1.5E-08M. Ta.

[0252] In the next step, TPP-12387 was tested against cynomolgus monkey α2-antiplasmin. The binding activity of cynomolgus monkeys was tested, as well as their function blocking activity. α2-antiplasmin was prepared in the same manner as rabbit α2-antiplasmin. Binding activity and function of TPP-12387 to monkey α2-antiplasmin The inhibitory activity values ​​are shown in Figure 6. One dose-response curve for the binding and function blocking activity of 12387 was performed in quadruplicate. As an example of two or three independent experiments, Fig. 6 shows the results of the cynomolgus monkey α2 -The EC50 value for the binding activity of TPP-12387 to antiplasmin was 9.0E-08M (shown in Figure 6.1) and 9.0E-08M in two independent experiments The IC50 for blocking the function of cynomolgus monkey α2-antiplasmin was In two experiments, the results were 1.6E-07M (shown in Figure 6.2) and 1.6E-07M .

[0253] Example 5: Affinity optimization of lead antibody TPP-12387 The antibody TPP-12387 was developed with the aim of optimizing its affinity and increasing its functional efficiency. The compound was subjected to a lead optimization procedure.

[0254] Affinity maturation involves an initial round of single mutation collection followed by selection of the most affinity and potent mutants. Recombination of amino acid exchanges to increase the expression of β-lactamase, followed by germlining and sequence-optimized capsid NNK (N=A or G or C or T, K=G or T) For the mutations that collect the following, randomization at individual amino acid positions is performed using residues AAW DDSLSGWV (residues 91-101 including CDR-L3) and AREYYDSSGY YHLDY (CDR-H3 residues 98-110 and its N-terminal region adjacent to the CDR) A synthetic oligonucleotide containing NNK codon diversification (residues 96–110) containing two additional amino acids It was generated by site-directed mutagenesis using nucleotides.

[0255] The resulting single NNK library was sequenced and CDR-L3 and 156 CDR- We identified 139 amino acid exchange variants of TPP-12387 against H3 (Table 3.1 and 3.2).

[0256] Table 3.1: List of exchanges in CDRL3 of TPP-12387 [Table 19]

[0257] Table 3.2: List of exchanges of the two N-terminal adjacent amino acids in CDRH3 and TPP-12387. to [Table 20]

[0258] All mutants were transfected into the mammalian cell line HEK293 by transient transfection. and the resulting expression supernatants were assayed for their binding ability to human α2-antiplasmin. as well as directly tested for their function blocking activity.

[0259] We identified mutants that showed higher binding and function-blocking activity than the antibody TPP-12387 in HEK29 mice. The antibodies were expressed one or more times in 3 cells, and the antibodies were purified and quantified as described in Example 2. In direct comparison with the mutant TPP-12387, their activity against human α2-antiplasmin were re-tested for their binding ability and their ability to block function.

[0260] This resulted in 5 exchanges in CDR-L3 and 7 exchanges in CDR-H3 that were identical to the parent antibody. The compound was identified as having improved activity compared to TPP-12387. Improved affinity and These 11 single substitution mutants with functional efficiency were identified as 1 The selected mutations or the corresponding wild-type mutants were recombined in one recombinant library. Oligonucleotides were generated to introduce the amino acid sequence at each selected position. Library construction was performed using successive rounds of long PCR. The final PCR products were It was ligated into an IgG1 expression vector and the mutants were sequenced.

[0261] The antibodies generated in this recombinant library were TPP-14290, TPP-1 4291, TPP-14292, TPP-14293, TPP-14294, TPP-1 4295, TPP-14296, TPP-14297, TPP-14298, TPP-1 4299, TPP-14300, TPP-14301, TPP-14302, TPP-1 4303, TPP-14304, TPP-14305, TPP-14306, TPP-1 4307, TPP-14308, TPP-14309, TPP-14310, TPP-1 4311, TPP-14312, TPP-14313, TPP-14314, TPP-1 4315, TPP-14316, TPP-14317, TPP-14318, TPP-1 4319, TPP-14320, TPP-14322, TPP-14323, TPP-1 The number was 4324.

[0262] The antibodies were purified from the supernatant and their concentrations were determined as described in Example 2. The antibody was then subjected to a test for its ability to bind to human α2-antiplasmin and to detect human α2-antiplasmin. The compounds were tested for their ability to block the IL-11 receptor agonist (described in Examples 3 and 4).

[0263] Antibodies TPP-14293, TPP-14298, TPP-14303, TPP-143 05, TPP-14308, TPP-14313, TPP-14314, TPP-143 18. TPP-14323 inhibits the binding of human α2-antiplasmin and human α2-antiplasmin. The most potent blockade of tiplasmin activity was identified as TPP-12387. also represent improved recombinant variants. Binding and activity data for these antibodies are shown in Figure 7.

[0264] According to the methods described in Examples 3 and 4, TPP-14323, TPP-143 18, TPP-14314, TPP-14313, TPP-14308, TPP-143 05, TPP-14303, TPP-14298, and TPP-14293 in a dose-dependent manner. The ability of these compounds to selectively bind to and block the activity of human α2-antiplasmin was tested. For each antibody, two or three independent runs performed in quadruplicate are shown as examples in Figures 7.1 to 7.18. A dose-response curve from a single experiment is shown (see Table 3.3). Table 3.3: Binding activity EC[M] and function blocking activity IC50[M ] [Table 21] * Shown in Figure 7 Example 6: Sequence-based immunogenicity risk reduction Antibody TPPs for further optimization to reduce the risk of sequence-based immunogenicity -14308 was chosen because it replaced an amino acid different from the closest germline sequence. The corresponding cDNA was synthesized and transiently transfected into HEK293 cells. The antibody was quantified but not purified, and bound to human α2-antiplasmin. -Tested for its ability to block the function of antiplasmin.

[0265] This approach resulted in 47 antibodies.

[0266] Most germline exchanges show only modest improvements in both functionalities.

[0267] Only amino acids within the framework sequences and in CDRH1 and CDRH2 were exchanged. Surprisingly, it showed even higher binding activity and Six mutants were identified that exhibited reduced and / or improved function-blocking activity.

[0268] Dose-response curves for the binding and function-blocking activity of these mutants are shown in FIG.

[0269] Table 3.5: Binding activity EC[M] and function blocking activity IC50 from 2-3 independent experiments [M] [Table 22] *Shown in Figure 8.

[0270] In the next step, the most active antibody, TPP-17044, was expressed in larger quantities and purified. The purified and quantified antibodies were used to detect human, cynomolgus monkey, and rabbit α2-α. Antiplasmin was retested for its function blocking activity. The results are shown in Figure 9. Dose-response curves obtained from two or three independent experiments are shown as examples. The function blocking activity of TPP-17044 against antiplasmin was 4.4E-10M (Figure 9 .1), the second experiment was 5.4E-10M, and the third experiment was 5E-10 For inhibition of cynomolgus monkey α2-antiplasmin, the value was 4.6E-1 0M (Figure 9.2), 4.9E-10M for the second experiment, and 5. The rabbit α2-antiplasmin was 2.7E-08M, the second TPP had an IC50 value of 3.6E-08M in one experiment and 2.9E-08M in the third experiment. Its activity was blocked by -17044 (Figure 9.3).

[0271] Finally, to further minimize the theoretical risk of immunogenic reactions, TPP-1704 4 was recloned into a human IgG4Fc version of the human antibody. The resulting antibody, TPP -17928 against human, cynomolgus monkey, and rabbit α2-antiplasmin The function blocking activity of was tested again. The results are shown in Figure 10. Dose-response curves obtained from independent experiments are shown as examples. The functional blocking activity of TPP-17928 against amine was 1.1E-10M (Figure 10.1). In the second experiment, it was 1.6E-10M. For inhibition of sumin, the value was 2.6E-10M (Fig. 10.2) and in the second experiment 3. 4E-10M in the first experiment, and 2.9E-10M in the third experiment. Rabbit α2-antiplus The activity of the amine was reduced by TPP-17928 to 1.5E-08M (Figure 10.3), and the second in the first experiment and 1.6E-10M in the third experiment.

[0272] Example 7: In vitro clot lysis Human blood was collected by venipuncture from healthy subjects who had not taken any medication for the past 10 days (ethics By the committee "Aerztekammer Nordrhein", #2017029 (Approved procedure). Blood was diluted with 1 / 10 volume of 3.8% trisodium citrate. Platelet poor plasma (PPP) was collected in a plastic tube at 2500g for 10 min. The mixture was centrifuged immediately at 4°C for 1 h and stored at -20°C.

[0273] For all experiments, plasma from at least n=3 independent donors was used. The lysis assay was performed as follows: Frozen plasma was thawed (37°C for 30 min) and 96 μl In a well plate, a test compound at a specified concentration [varies between 0.015 μM and 1 μM] or was mixed with the solvent only as a control. In a second 96-well plate, CaCl2 (Sig ma; Catalog No. 21115 ~ 100ML) [final concentration: 12.5mM] to induce clots The agent was low-dose tPA (Actilyse®, Boehringer Inst. gelheim) [final concentration 0.3 μg / ml] was prepared as a dissolution initiator. After a short incubation in plasma (5 min at 37° C.), the mixture was transferred to a second plate. In addition, a microplate reader (Tecan infinite 200 Pro) The absorbance (405 nm, 37°C, 1 / min) was measured over a period of 3 hours. For the final determination of time reduction, the tPA-induced lysis time was determined as 100% (individually for each donor plasma). ) and the dissolution time reduction was calculated for each test compound in a dose-response curve.

[0274] For rabbit plasma analysis, whole blood from male New Zealand White rabbits was collected by venipuncture. and prepared and used as described above for human plasma.

[0275] As shown in FIG. 11, antibody TPP-17928 exhibited anti-inflammatory properties in human and rabbit plasma. In human plasma, each of them dose-dependently decreased the tPA-induced clot lysis time. The functional blocking activity of TPP-17928 was 2.5E-07M in rabbits. For plasma it was 2.3E-07M.

[0276] Example 8: In vivo study of TPP-17928 in acute pulmonary embolism in rabbits The effect of the anti-α2-antiplasmin antibody of the present invention on clot lysis in pulmonary embolism was studied. To study this, a rabbit in vivo model was used.

[0277] Male New Zealand white rabbits were administered xylazine / ketamine (5mg / kg + 40mg / kg The rats were anesthetized with an intramuscular injection of 1000 mg ... The ears, neck and left hind leg (femoral triangle area) were shaved. The rabbits were kept anesthetized with Xylazine / ketamine (80ml + 800ml, 60ml NaCl 0.9%) for 5 ml The rabbits were placed on a heating plate and kept at 37°C for the entire duration of the experiment. The left femoral vein was cannulated for compound administration and blood sampling, and the right jugular vein was cannulated for clot injection. The patient was cannulated for

[0278] For the preparation of fluorescently labeled clots, rabbit platelet-poor plasma was incubated with ALEXA488 fluorescently labeled human plasma. Tofibrinogen (Thermo Fisher Scientific, catalog no. F13191) and 2.5 μl of batroxobin [20 U / ml] (LOXO, Catalog No. 101-04) and 2.5 μl of CaCl2 [0.1 mM] in 45 μl Clotting was initiated by adding 75 μg of erythrocyte sediment to the plasma mixture; the final clot volume was 50 μl. It contains fluorescently labeled fibrinogen.

[0279] After maturation of the clot (30 min at 37°C), 2 clots / kg body weight were injected into the jugular vein of anesthetized rabbits. This resulted in embolization of blood clots in the lungs. Thirty minutes later, saline, antibody TPP-17928 bolus injection of either tPA, tPA, or a combination of the antibody TPP-17928 and tPA Embolization therapy was started by intravenous injection. Over a period of 360 minutes, blood was collected from the rabbits via the vein. Fluid samples were taken and analyzed for plasma fluorescence as an indirect parameter of clot lysis. Ear bleeding times were determined immediately after the start of treatment and 300 minutes after treatment. To do this, make a 3-5 mm incision in the ear with a scalpel blade parallel to the outer edge of the ear (close to the external ear vein). Every 30 seconds, gently tap the area close to the incision with a small filter tip. This was used to demonstrate whether the incision was still bleeding.

[0280] As shown in Figure 12.1, the antibody TPP-17928 was used to detect pulmonary embolism in rabbits. Treatment resulted in increased clot lysis that appeared to be comparable to tPA treatment over time. TPP-17928 dose-dependently increased plasma fluorescence as an indirect measure of clot lysis. (2.1-fold increase in AUC compared to control). Combining 928 with low-dose tPA (0.125 mg / kg) promoted faster clot lysis. tPA treatment also shows a dose-dependent effect on clot lysis (Figure 12.2).

[0281] Similar results with increased plasma fluorescence as a parameter of clot lysis were observed in further rabbit lung In embolization experiments, 30 mg / kg TPP-12387 (AUC 2. 6-fold increase) and 15 mg / kg TPP-17044 (2. 1-fold increase) was observed.

[0282] Ear bleeding time measurement In the above experiments, ear bleeding time was measured at the same time. Immediately after treatment application (0 min), ear bleeding time was The time between the ear bleeding and the ear was measured as follows: An incision was made with a scalpel blade parallel to the outer edge of the ear (close to the external ear vein). Every 30 seconds, the incision was Ensure the incision is still bleeding by gently tapping with a small filter tip just adjacent to the incision. Ear bleeding time was significantly longer with anti-a2AP antibody than with tPA treatment. The study revealed an excellent safety profile of the treatment. Although there was no detectable increase in bleeding time for any of the concentrations, tPA treatment, especially at 1 mg The highest concentration of 100 mg / kg shows an immediate effect on prolonging ear bleeding time. 15 mg / kg TPP-179 in combination with tPA (0.125 mg / kg) The combination of 28 did not result in a significant increase in bleeding time. In these cases, anti-a2AP antibody therapy was shown to be superior to tPA therapy in terms of bleeding. The results are shown in Figure 13. The bleeding time was prolonged with tPA [1 mg / kg] 2. 03x, TPP-17928[15mg / kg]0.95x, tPA[0.125mg / kg] + TPP-17928 [15 mg / kg] 1.56 times. In the blood experiment, similar results were obtained with 30 mg / kg of TPP-12387 (0.91x) and 15 mg / kg TPP-17044 (1.0 5-fold) was observed.

[0283] Example 9: Determination of the binding affinity of antibodies of the invention Binding assays were performed on a Biacore T200 instrument using a Protein G sensor chip and The assay was performed at 25°C using the assay buffer HBS-EP+. Antibody capture was approximately 150 RU. Analytes were used at concentrations ranging from 1.56 to 200 nM for all kinetics. Human, cynomolgus monkey and rabbit α2-antiplasmin were used as donors. Regeneration was performed with glycine-HCl at pH 1.5. The kinetic parameters were calculated from the experimental The sensorgrams were derived by fitting to a 1:1 Langmuir binding model. is shown in Table 4.

[0284] Table 4: Affinity values ​​of antibodies of the invention. [Table 23]

[0285] Example 10: Direct comparison of the function blocking activity of 77A3 and antibodies of the invention To compare the blocking activity of the test antibody 77A3 against A2AP activity with the test antibodies of the present invention, To determine whether or not a functional blockade assay is required, a functional blockade assay was used, as described in detail in Example 4. The antibodies were incubated at concentrations of 6.1E-11 to 3.0E-08M and .0E-06M in A2, respectively. Preincubated with AP. Plasmin and I-1275 (plasmin serine protease inhibitor). After addition of a fluorogenic substrate for plasminase activity to the assay, the serine protease of plasmin was The fluorescence signal as a measure of the enzyme activity was determined.

[0286] Increasing the concentration of 77A3 to 0.03 μM increased the uptake of the fluorescent substrate I-1275 by plasmin. This resulted in an increase in the fluorescent signal due to cleavage. However, further increases in 77A3 concentration This resulted in a decrease in the fluorescent signal, as described in Example 4 up to a concentration of 0.03 μM. Testing of antibody 77A3 in the biochemical assay described above demonstrated that it also blocks α2-antiplasmin. Further increases in 77A3 antibody concentration resulted in a decrease in plasmin activity, with a 1 μM We show that a concentration of 77A3 results in complete inhibition of plasmin activity (Figure 14.1). The following IC50 values ​​for A2AP function blocking activity were generated for the antibodies: PP-17041-6.4E-10M, TPP-17044-3.9E-10M, TPP -17045-1.4E-10M, TPP-17048-1.2E-09M, TPP-1 7051-3.3E-10M, TPP-17053-1.5E-10M. Up to 1μM concentration None of the antibodies of the present invention tested in this study resulted in a decrease in the fluorescent signal, which indicates that It is shown that the test antibody of the invention does not affect plasmin activity (Figure 14.2-1 4.7).

[0287] Example 11: Antibodies of the invention for inhibiting the proteolytic activity of plasmin Biochemical assays to test To further explore the surprising results obtained for antibody 77A3 in Example 10 The anti-α2-antiplasmin antibody of the present invention and the antibody 77A3 were then subjected to biochemical plasminometry. The inhibitory activity was tested in a serotonin assay. For this, starting at a defined concentration, Different concentrations of antibodies, followed by 1:2 or 1:3 dilution steps, were added to the Haematologi c 400 pM human plasmin obtained from Technologies INC.; Catalog number HCPM140) and the fluorogenic substrate I-1275 (B achem;MeOSuc-Ala-Phe-Lys-AMC trifluoroacetate salt Catalog No. I-1275 (Stock: 10 mM in DMSO)) at 37°C for 1 hour. The reaction was incubated for 1 h at 37 °C for 1 h. The reaction was incubated for 1 h at 37 °C ... in 384-well microtiter plates (Nunc; The fluorescence signal was measured under the following conditions: Mode: Fluorescence top reading, Ex 360nM, Em 465nm, Ex Bandwidth 5 nm, Em bandwidth 5nm.

[0288] As shown in FIG. 16, TPP-17928 (exemplary for all antibodies of the present invention) (shown in the figure) had no effect on biochemical activity, whereas 77A3 had an IC50 value of 1.7 μM. Blocks the proteolytic activity of plasmin.

[0289] Table 5: Inhibition of plasmin proteolytic activity by 77A3 and test antibodies of the invention. [Table 24] *Two to three independent studies performed in duplicate for 77A3 and TPP-17928 Dose-response curves from the experiments performed are shown as examples in FIG.

[0290] Example 12: Epitope mapping of TPP-12387 and 77A3 Epitope mapping was performed using the PEPperCHIP® Peptide Mic By using the roarray platform, PEPperPRINT ( The study was carried out by the National Institute of Standards and Technology (Heidelberg, Germany).

[0291] For linear epitope mapping, the antigen sequence was split into 14 amino acid peptide-peptide fragments. The resulting peptide microarrays were translated into overlapping linear 15 amino acid peptides. The array contained 491 different peptides printed in duplicate.

[0292] For conformational epitope mapping, the antigen sequence was divided into peptides of 6, 9, and 12 amino acids. The peptides were translated into overlapping 7, 10 and 13 amino acid peptides with peptide-peptide overlaps. After peptide synthesis, all peptides were synthesized by cleaving the C-terminal cysteine ​​side chain and appropriately modified N-terminal The peptide was cyclized via a thioether bond between the two. The resulting conformational peptide microarray contained 1,488 different cyclic constrained peptides printed in duplicate. .

[0293] Microarrays were blocked with Rockland Blocking Buffer MB-070 (first assay) Block the antibody incubation by using PBS (30 min prior to I). Incubation buffer consisting of 0.0% 5% (linear epitope mapping) or 0.005% (conformational epitope mapping) ) The incubation was performed with Tween 20 at pH 7.4.

[0294] After incubation, the array was washed with 0.05% (linear epitope mapper) PBS. ing) or 0.005% (conformational epitope mapping) Tween 20 at pH 7. 4. The arrays were washed for 3 × 1 min (linear epitope mapping) or 2 × 10 s (standby). (Conformational epitope mapping) was washed.

[0295] Antibodies were incubated in incubation buffer at 1 μg / ml, 10 μg / ml and 100 μg / ml. The incubation time was 4 h. The mixture was incubated at 4° C. for 16 hours with shaking at 140 rpm.

[0296] As a control, mouse monoclonal anti-HA (12CA5) DyLight800 was used at 1: The detection antibody was incubated at room temperature in incubation buffer for 45 min. Stained and incubated on the microarray.

[0297] The secondary antibody was goat anti-human IgG (Fc) DyLight680 at a dilution of 1:5000. The detection antibody was used for staining in incubation buffer at room temperature for 45 minutes. The cells were incubated on the microarray.

[0298] Scan offset was measured using the LI-COR Odyssey Imaging System. 0.65mm, resolution 21μm, scan intensity 7 / 7 (red=700nm / green=800nm ) signal was detected.

[0299] Quantification of spot intensities and peptide annotations were performed using 24-bit colorized tiff 16-bit grayscale at 7 / 7 scan intensity showing higher dynamic range than the file Microarray image analysis was performed using PepSlide (registered trademark). The analysis was performed using a software algorithm to determine the concentration of each spot. The fluorescence intensity was decomposed into raw, foreground and background signals, and the mean median and spot values ​​of the foreground intensity were calculated. The spot-to-spot variation of the replicates was calculated. The intensity map was based on the average median foreground intensity. The interactions in the peptide map are displayed in red for high spot intensity and red for low spot intensity. The intensity is highlighted by a color code with white for the chromatic intensity. The spot intensities were plotted against the antibody samples for the antigen sequence from the N-terminus to the C-terminus, and the overall The relative spot intensity and signal-to-noise ratio were visualized. The intensity plots were Peptide and intensity maps, as well as microarrays, to identify somatic epitopes Correlated with visual inspection of the scans.

[0300] Secondary goat anti-human IgG(Fc) DyLight680 antibody (1:5000) and control Mouse monoclonal anti-HA (12CA5) DyLight800 antibody (1:2000) Prestaining of linear and conformational peptide microarrays with Fab was performed to determine the linear or cyclic binding of antigens. In contrast, the antibody of interest showed no background interaction with the bundle peptide. Incubation resulted in the following observations: As shown in Table 6, TPP-12387 has the consensus motif SRMSLSS The peptide showed a high signal-to-noise ratio, which indicates that the A2AP reactive center Amino acid 402 of SEQ ID NO: 1 located in the loop (amino acids 400 to 412 of SEQ ID NO: 1) Corresponds to ~408.

[0301] In contrast, antibody 77A3 contains the basic consensus motif RPTKVRLPK. A very weak signal to noise ratio was identified for a peptide having the sequence SEQ ID NO:1. It corresponds to amino acids 330 to 338. There is no clear description of this part of A2AP. .

[0302] Table 6: A2AP binding sites of TPP-12387 and 77A3 [Table 25]

Claims

1. An isolated antibody or antibody capable of binding to A2AP and inhibiting the activity of A2AP. An antigen-binding fragment, comprising the isolated antibody or antigen-binding fragment thereof. The isolated antibody or antigen-binding fragment thereof, which does not inhibit plasmin activity.

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, The isolated antibody or antigen-binding fragment thereof has a dissociation constant (KD) of ≦100 nM , ≦50 nM, ≦25 nM, ≦10 nM, ≦1 nM, or ≦0.5 nM, and and binds to human A2AP having the sequence of amino acids 40 to 491 of The isolated antibody or antigen-binding fragment thereof is an in vitro A2AP function blocking antibody. In the assay, ≦500 nM, ≦250 nM, ≦100 nM, ≦50 nM, ≦25 nM , amino acid 40 of SEQ ID NO: 1 with an EC50 of ≦10 nM, ≦1 nM, or ≦0.5 nM. Inhibits the activity of human A2AP of sequence ~491; The isolated antibody or antigen-binding fragment thereof.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, The isolated antibody or antigen-binding fragment thereof is an in vitro plasmin inhibitor. In the above, 1 μM, 2 μM, 5 μM or 10 μM of the isolated antibody or does not inhibit plasmin activity up to the concentration of the antigen-binding fragment; The isolated antibody or antigen-binding fragment thereof.

4. An isolated antibody or antigen-binding fragment thereof according to claim 1, 2 or 3. hand, The plasmin is human plasmin, in particular the plasmin is selected from the group consisting of SEQ ID NO: 118 and and human plasmin comprising SEQ ID NO: 119; The isolated antibody or antigen-binding fragment thereof.

5. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 4. So, i) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:8, and SEQ ID NO:1 3, and L-CDR1 comprising SEQ ID NO:

9. , a light chain comprising an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:

18. An antigen-binding region; or ii) an H-CDR1 comprising SEQ ID NO:6, an H-CDR2 comprising SEQ ID NO:7, and an H-CDR3 comprising SEQ ID NO:8; 13, and an L-CDR3 comprising SEQ ID NO:

9.

1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO:

18. chain antigen-binding region; or iii) an H-CDR1 comprising SEQ ID NO:6, an H-CDR2 comprising SEQ ID NO:7, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:11, and an L-CDR3 comprising SEQ ID NO:

9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:

17. a light chain antigen-binding region; or iv) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 11, and an L-CDR3 comprising SEQ ID NO:

9.

1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO:

18. chain antigen-binding region; or v) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO:1 2, and L-CDR1 comprising SEQ ID NO:

9. , a light chain comprising an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:

18. An antigen-binding region; or vi) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 13, and an L-CDR3 comprising SEQ ID NO:

9.

1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO:

19. chain antigen-binding region; or vii) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:14, and an L-CDR3 comprising SEQ ID NO:

9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:

18. a light chain antigen-binding region; or viii) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and the sequence A heavy chain antigen-binding region comprising H-CDR3 comprising SEQ ID NO:14, and an L-CDR3 comprising SEQ ID NO:

9. L-CDR1 comprises SEQ ID NO:10, L-CDR2 comprises SEQ ID NO:20, and a light chain antigen-binding region comprising the ix) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO: 15, and an L-CDR3 comprising SEQ ID NO:

9.

1. A light antibody having an L-CDR2 comprising SEQ ID NO: 10 and an L-CDR3 comprising SEQ ID NO:

18. chain antigen-binding region; or x) H-CDR1 comprising SEQ ID NO:6, H-CDR2 comprising SEQ ID NO:7, and SEQ ID NO:1 6, and L-CDR1 comprising SEQ ID NO:

9. , a light chain comprising an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:

18. An antigen-binding region; or xi) H-CDR1 comprising SEQ ID NO:21, H-CDR2 comprising SEQ ID NO:8, and A heavy chain antigen-binding region comprising an H-CDR3 comprising SEQ ID NO:13, and an L-CDR3 comprising SEQ ID NO:

9. R1, L-CDR2 comprising SEQ ID NO:10, and L-CDR3 comprising SEQ ID NO:

18. a light chain antigen-binding region; or xii) an H-CDR1 comprising SEQ ID NO:6, an H-CDR2 comprising SEQ ID NO:22, and the sequence A heavy chain antigen-binding region comprising H-CDR3 comprising SEQ ID NO: 13, and an L-CDR4 comprising SEQ ID NO:

9. L-CDR1 comprises SEQ ID NO:10, and L-CDR3 comprises SEQ ID NO:

18. Light chain antigen-binding region The isolated antibody or antigen-binding fragment comprising:

6. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 5. So, xiii) a variable heavy chain domain comprising SEQ ID NO: 32 and a variable light chain domain comprising SEQ ID NO: 38 in; or xiv) a variable heavy chain domain comprising SEQ ID NO: 23 and a variable light chain domain comprising SEQ ID NO: 37 or xv) a variable heavy chain domain comprising SEQ ID NO: 24 and a variable light chain domain comprising SEQ ID NO: 38 ;or xvi) a variable heavy chain domain comprising SEQ ID NO:25 and a variable light chain domain comprising SEQ ID NO:38 or xvii) a variable heavy chain domain comprising SEQ ID NO: 26 and a variable light chain domain comprising SEQ ID NO: 38 in; or xviii) a variable heavy chain domain comprising SEQ ID NO:27 and a variable light chain domain comprising SEQ ID NO:39 Main; or xix) a variable heavy chain domain comprising SEQ ID NO:27 and a variable light chain domain comprising SEQ ID NO:38 or xx) a variable heavy chain domain comprising SEQ ID NO: 28 and a variable light chain domain comprising SEQ ID NO: 38 ;or xxi) a variable heavy chain domain comprising SEQ ID NO:28 and a variable light chain domain comprising SEQ ID NO:40 or xxii) a variable heavy chain domain comprising SEQ ID NO:29 and a variable light chain domain comprising SEQ ID NO:38 in; or xxiii) a variable heavy chain domain comprising SEQ ID NO: 30 and a variable light chain domain comprising SEQ ID NO: 38 Main; or xxiv) a variable heavy chain domain comprising SEQ ID NO: 31 and a variable light chain domain comprising SEQ ID NO: 38 in; or xxv) a variable heavy chain domain comprising SEQ ID NO: 33 and a variable light chain domain comprising SEQ ID NO: 38 or xxvi) a variable heavy chain domain comprising SEQ ID NO: 34 and a variable light chain domain comprising SEQ ID NO: 38 in; or xxvii) a variable heavy chain domain comprising SEQ ID NO: 35 and a variable light chain domain comprising SEQ ID NO: 38 Main; or xxviii) a variable heavy chain domain comprising SEQ ID NO: 36 and a variable light chain domain comprising SEQ ID NO: 38 domain The isolated antibody or antigen-binding fragment comprising:

7. 7. The isolated antibody of any one of claims 1 to 6, The isolated antibody which is an IgG antibody, in particular an IgG1 or IgG4 antibody.

8. 8. The isolated antibody of any one of claims 1 to 7, xxix) a heavy chain comprising SEQ ID NO:55 and a light chain comprising SEQ ID NO:57; or xxx) a heavy chain comprising SEQ ID NO:41 and a light chain comprising SEQ ID NO:56; or xxxi) a heavy chain comprising SEQ ID NO:42 and a light chain comprising SEQ ID NO:57; or xxxii) a heavy chain comprising SEQ ID NO:43 and a light chain comprising SEQ ID NO:57; or xxxiii) a heavy chain comprising SEQ ID NO:44 and a light chain comprising SEQ ID NO:57; or xxxiv) a heavy chain comprising SEQ ID NO:45 and a light chain comprising SEQ ID NO:58; or xxxv) a heavy chain comprising SEQ ID NO:45 and a light chain comprising SEQ ID NO:57; or xxxvi) a heavy chain comprising SEQ ID NO:46 and a light chain comprising SEQ ID NO:57; or xxxvii) a heavy chain comprising SEQ ID NO:46 and a light chain comprising SEQ ID NO:59; or xxxviii) a heavy chain comprising SEQ ID NO:47 and a light chain comprising SEQ ID NO:57; or xxxix) a heavy chain comprising SEQ ID NO:48 and a light chain comprising SEQ ID NO:57; or xl) a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:57; or xli) a heavy chain comprising SEQ ID NO:50 and a light chain comprising SEQ ID NO:57; or xlii) a heavy chain comprising SEQ ID NO:51 and a light chain comprising SEQ ID NO:57; or xliii) a heavy chain comprising SEQ ID NO:52 and a light chain comprising SEQ ID NO:57; or xliv) a heavy chain comprising SEQ ID NO:53 and a light chain comprising SEQ ID NO:57; or xlv) a heavy chain comprising SEQ ID NO:54 and a light chain comprising SEQ ID NO:57 The isolated antibody comprising:

9. 7. The antigen-binding fragment of any one of claims 1 to 6, scFv, Fab, Fab' fragment or F(ab')2 fragment. The antigen-binding fragment.

10. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 9. So, The isolated antibody or antigen is a monoclonal antibody or antigen-binding fragment. Combined fragments.

11. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 10. There was, The isolated antibody is a human, humanized or chimeric antibody or antigen-binding fragment. or an antigen-binding fragment.

12. The isolated antibody or antibody of any one of claims 1 to 11 for binding to A2AP. or antigen-binding fragment thereof, 。

13. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 12.

4. An antibody conjugate comprising:

14. A method encoding the antibody or antigen-binding fragment of any one of claims 1 to 12 , an isolated nucleic acid sequence.

15. A vector comprising the nucleic acid sequence of claim 14.

16. Expressing the antibody or antigen-binding fragment according to any one of claims 1 to 12. and / or a nucleic acid according to claim 14 or a vector according to claim 15, Isolated cells.

17. 17. The isolated cell of claim 16, wherein the cell is a prokaryotic or eukaryotic cell. The isolated cell.

18. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 12. A method for producing a medicament for the treatment of a pulmonary edema, comprising culturing a cell according to any one of claims 16 or 17. and optionally purifying the antibody or antigen-binding fragment.

19. The isolated antibody or antigen-binding fragment of any one of claims 1 to 12. or the antibody conjugate of claim 13, and optionally one or more pharma- ceutically acceptable A pharmaceutical composition comprising an excipient as described above.

20. An isolated compound according to any one of claims 1 to 12 for use in the treatment or prevention of a disease. or a conjugate according to claim 13. Or the pharmaceutical composition of claim 19.

21. The isolated antibody according to any one of claims 1 to 12 for use as a diagnostic agent. or an antigen-binding fragment, or an antibody conjugate according to claim 13.

22. Ischemic stroke, acute coronary syndrome, peripheral artery disease, myocardial infarction, deep vein thrombosis, pulmonary embolism, Ischemia due to partial or complete vascular occlusion, such as venous thrombosis or shunt thrombosis A compound according to claims 1 to 12 for use in the treatment or prevention of disorders or diseases related to sexual events.

14. The isolated antibody or antigen-binding fragment of any one of claims 13 to 14.

20. The conjugate according to claim 19, or the pharmaceutical composition according to claim 19.

23. One or more further therapeutically active compounds, in particular inhibitors of the coagulation cascade, anticoagulants and blood The present invention relates to a method for treating platelet aggregation by administering to a patient a compound selected from the group consisting of: An isolated antibody or antigen-binding molecule according to any one of claims 1 to 12 for use in a method for treating a chronic illness. or a conjugate according to claim 16 or a pharmaceutical composition according to claim 19. Pharmaceutical composition.

24. The isolated antibody or antigen-binding fragment of any one of claims 1 to 12. or the conjugate according to claim 13, or the pharmaceutical composition according to claim 19, and instructions for use.

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