Antigen-binding molecule specifically binding to a2AP and medical use thereof

Abstract

Provided are an antigen-binding molecule that specifically binds to A2AP and the medical use thereof, particularly, the use of the antigen-binding molecule that specifically binds to A2AP in the treatment of ischemic event-related disorders or diseases.

Description

Antigen-binding molecules that specifically bind to A2AP and their pharmaceutical applications Technical Field

[0001] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules that specifically bind to A2AP and their pharmaceutical uses. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] Stroke is one of the leading causes of death in China and globally, characterized by high mortality and disability rates. Globally, there are approximately 15 million new stroke cases each year, with ischemic stroke accounting for about 85%. Ischemic stroke occurs when blood vessels in the brain are blocked by thrombi, leading to a severe reduction in cerebral blood flow and resulting in ischemic damage or necrosis of brain tissue. The main biological mechanism of thrombolysis in vivo involves plasminogen activator (tPA or uPA) converting plasminogen into active plasmin. Plasmin then degrades fibrin on the surface of the thrombus, achieving thrombolysis. Simultaneously, plasmin activity in the body is inhibited by endogenous A2AP (α2 anti-plasmin, α2AP). As a major inhibitor of plasmin in vivo, A2AP's arginine residue at position 376 of its RCL region can form a 1:1 covalent complex with the active site of plasmin, rendering plasmin inactive and inhibiting thrombolysis. Patients with A2AP gene deletion have normal coagulation systems, and patients with homozygous A2AP deletion do not have symptoms of spontaneous cerebral hemorrhage.

[0004] Currently, revascularization treatment for acute ischemic stroke can be divided into two main categories: intravenous thrombolytic drugs and endovascular thrombectomy. When a patient meets the criteria for both treatments, bridging therapy can be used. Currently, revascularization treatment still follows the principle of prioritizing intravenous thrombolysis, which is the preferred method. To date, alteplase is the only intravenous thrombolytic drug approved by the FDA for ischemic stroke, but its thrombolysis time window is significantly limited, and the number of patients who benefit is very limited. Statistics show that during 2019-2020, only 5.64% of ischemic stroke patients in my country received thrombolytic therapy. The revascularization rate and safety of thrombolytic drugs still need improvement. Although tenecteplase (TNK), as a new generation of thrombolytic drug genetically modified from alteplase, has advantages such as higher fibrin binding specificity and single-dose administration, its effectiveness is still limited. However, analysis of the biological mechanism of thrombolysis in vivo reveals that alteplase and tenecteplase are both plasminogen activators. They both enhance the thrombolytic effect by converting inactive plasminogen into active plasmin, thereby increasing the production of endogenous plasmin. However, they cannot avoid the risk of bleeding caused by excessive enhancement of the fibrinolytic system due to the production of large amounts of plasmin in the body.

[0005] WO2022058261A2, WO2016106186A1, and other publications disclose a variety of antigen-binding molecules that specifically bind to A2AP, but there is still a need for antigen-binding molecules that specifically bind to A2AP to meet clinical needs. Summary of the Invention

[0006] This disclosure provides an antigen-binding molecule that specifically binds to A2AP, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0007] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 86, and HCDR3 contains the amino acid sequence of SEQ ID NO: 87; the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 88, LCDR2 contains the amino acid sequence of SEQ ID NO: 89, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10, wherein:

[0008] HIX1X2DX3DKX4YNPALKS SEQ ID NO: 86;

[0009] RGAX5YGSSFGWYFDV SEQ ID NO: 87;

[0010] SAX6X7X8IX9X 10 X 11 YLH SEQ ID NO: 88;

[0011] RASX 12 LAS SEQ ID NO: 89;

[0012] X1 is W or H, preferably W; X2 is W or S, preferably W; X3 is N, D or E, preferably N; X4 is S or G, preferably S.

[0013] X5 can be Y or H, with Y being preferred;

[0014] X6 is T or Q, T is preferred; X7 is S or Q, S is preferred; X8 is S or R, S is preferred; X9 is S, R, or T, S is preferred; X 10 S, K, or T, with S being the preferred choice; X 11 It can be N or D, with N being preferred;

[0015] X 12 It can be N or D, with N being preferred.

[0016] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein:

[0017] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 86, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 88, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10, wherein:

[0018] HIX1X2DX3DKX4YNPALKS SEQ ID NO: 86;

[0019] SAX6X7X8IX9X10 X 11 YLH SEQ ID NO: 88;

[0020] X1 is W; X2 is W; X3 is N, D, or E; X4 is S;

[0021] X5 can be Y or H, with Y being preferred;

[0022] X6 is T; X7 is S or QS; X8 is S; X9 is S; X 10 S; X 11 Let N be the number of elements in the array.

[0023] This disclosure provides an antigen-binding molecule that specifically binds to A2AP, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0024] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, 22, 19, 20, 21, 23, 24, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, 68, 26, 27, 28, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 69; or

[0025] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12.

[0026] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0027] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 73, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66; or

[0028] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 73, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 68, 69, or 57; or

[0029] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79, or 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25, 26, 27, 28, 55, 64, 65, or 66; or

[0030] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, 19, 20, 21, or 23, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12, 25, 26, 27, or 28.

[0031] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0032] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 22, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, or 68; or

[0033] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55 or 68; or

[0034] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12.

[0035] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0036] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55.

[0037] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0038] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 68.

[0039] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0040] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25.

[0041] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0042] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12.

[0043] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the Kabat numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the IMGT numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the Chothia numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the AbM numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the Contact numbering rules.

[0044] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0045] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, 18, 6, 45, 46 or 48, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7 or 54. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, 33, 32, 34, 35, 36, 37, 38 or 39, LCDR2 contains the amino acid sequence of SEQ ID NO: 9 or 43, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0046] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0047] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, 18 or 6, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8 or 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0048] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0049] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47 or 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8 or 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0050] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0051] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0052] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0053] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0054] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0055] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0056] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0057] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 6, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10.

[0058] In some embodiments, the antigen-binding molecules that specifically bind to A2AP as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0059] In some embodiments, the antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, is an antibody. In some embodiments, the antigen-binding molecule that specifically binds to A2AP is a monoclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, or a fusion protein. In some embodiments, the antigen-binding molecule that specifically binds to A2AP is a monoclonal antibody. In some embodiments, the antigen-binding molecule that specifically binds to A2AP is a monospecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to A2AP is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antigen-binding molecule that specifically binds to A2AP is a humanized antibody. In some embodiments, the antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, includes a frame region (FR) of a human antibody.

[0060] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments, wherein the heavy chain variable region has FR1, FR2, FR3 derived from IGHV2-70*04 and FR4 derived from IGHJ6*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 30N, 82I and / or 1E; and / or the light chain variable region has FR1, FR2, FR3 derived from IGKV4-1*01 or IGKV7-3*01 and FR4 derived from IGKJ2*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 4F, 22T, 43S, 71Y, 60A and / or 81E.

[0061] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments, wherein the heavy chain variable region has FR1, FR2, FR3 fragments derived from IGHV2-70*04 and FR4 fragments derived from IGHJ2*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 30N and / or 10T; and / or the light chain variable region has FR1, FR2, FR3 fragments derived from IGKV3D-20*02 and FR4 fragments derived from IGKJ4*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1D, 58V, 4M and / or 13V.

[0062] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the heavy chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 55N, 85T, 49T, 52H, 53S, 58G, 82V, 82bQ, 85E, 89S and / or 98H; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 20Q, 27Q, 26Q, 27aR, 29R, 29T, 30K, 30T, 31D, 47D, 48E, 49G and / or 53D. In some embodiments, the above-mentioned variable regions are defined according to the Kabat numbering rules.

[0063] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, HCDR2 comprises the amino acid sequence of SEQ ID NO: 47 or 18, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the FR of the heavy chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 49T, 82V, 82bQ, 85E, 85T, 89S, 1E, 30N, 82I, and / or 10T; and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 8 or 33, LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8. The amino acid sequence of NO: 10, and the FR of the light chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 20Q, 47D, 48E, 49G, 4F, 22T, 43S, 60A, 71Y, 81E, 1D, 58V, 4M, and / or 13V. In some embodiments, the above-mentioned variable region and CDR are defined according to the Kabat numbering rules.

[0064] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0065] The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82), and the light chain variable region comprises SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82. The amino acid sequence of SEQ ID NO: 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63 or 67 or an amino acid sequence having at least 80% (e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63 or 67.

[0066] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0067] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66; or

[0068] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, 69, or 57 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 68, 69, or 57; or

[0069] The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79, or 82, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79, or 82, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 25, 26, 27, 28, 55, 64, 65, or 66, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79, or 82, and ... NO: 25, 26, 27, 28, 55, 64, 65 or 66 has an amino acid sequence identity of at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0070] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0071] The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 11, 19, 20, 21 or 23 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 11, 19, 20, 21 or 23, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66.

[0072] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0073] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 12.

[0074] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0075] The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80 or 82, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63 or 67.

[0076] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0077] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66; or

[0078] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, 69 or 57; or

[0079] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79 or 82, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25, 26, 27, 28, 55, 64, 65 or 66; or

[0080] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, 19, 20, 21 or 23, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66.

[0081] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0082] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or

[0083] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68; or

[0084] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or

[0085] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12.

[0086] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments is humanized, reversed mutation, affinity maturation, dehydrophobicity reduced, T-cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced. In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is an antibody fragment. In some embodiments, the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, Fv, scFv, and dsFv.

[0087] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region is a light chain constant region of human κ chain, λ chain, or variants thereof. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region or a variant thereof, and the light chain constant region is a human κ light chain constant region or a variant thereof. In some embodiments, the human IgG4 heavy chain constant region variant comprises one or more mutations selected from S228P, F234A, and L235A on the human IgG4 heavy chain constant region, and the light chain constant region is a human κ light chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15.

[0088] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein the antigen-binding molecule that specifically binds to A2AP comprises a heavy chain and a light chain, wherein:

[0089] The heavy chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 84 or 29, and the light chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 83, 85, or 30; or

[0090] The heavy chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 17, and the light chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 16.

[0091] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0092] The heavy chain contains the amino acid sequence of SEQ ID NO: 84 or 29, and the light chain contains the amino acid sequence of SEQ ID NO: 83, 85 or 30.

[0093] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0094] The heavy chain contains the amino acid sequence of SEQ ID NO: 17, and the light chain contains the amino acid sequence of SEQ ID NO: 16.

[0095] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0096] The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 83 or 85; or

[0097] The heavy chain contains the amino acid sequence of SEQ ID NO: 29, and the light chain contains the amino acid sequence of SEQ ID NO: 83 or 30.

[0098] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0099] The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 83; or

[0100] The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0101] The heavy chain contains the amino acid sequence of SEQ ID NO: 29, and the light chain contains the amino acid sequence of SEQ ID NO: 30.

[0102] In some embodiments, an antigen-binding molecule that specifically binds to A2AP, as described in any of the preceding embodiments, wherein:

[0103] The heavy chain comprises amino acid residues 1 to 451 as shown in SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 83; or

[0104] The heavy chain comprises amino acid residues 1 to 451 as shown in SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0105] The heavy chain comprises amino acid residues from position 1 to position 451 of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 30.

[0106] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to A2AP, which competes with the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims for binding to human A2AP.

[0107] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to A2AP, which, together with the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, binds to the same human A2AP antigenic epitope.

[0108] On the other hand, this disclosure provides a pharmaceutical composition comprising an antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, a unit dose of the pharmaceutical composition contains 0.1-3000 mg or 1-1000 mg of the antigen-binding molecule that specifically binds to A2AP as described above.

[0109] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, or the conjugate as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 1%-99% of the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 2%-98% of the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims.

[0110] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable carriers, diluents, or excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable carriers, diluents, or excipients.

[0111] On the other hand, this disclosure provides a nucleic acid that encodes an antigen-binding molecule that specifically binds to A2AP as described in the previous one.

[0112] On the other hand, this disclosure provides a carrier containing the nucleic acid as described above.

[0113] On the other hand, this disclosure provides a host cell containing the nucleic acid as described above, or the vector as described above.

[0114] On the other hand, this disclosure provides a method for producing an antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, the method comprising culturing host cells as described in the preceding claims in a culture medium to form and accumulate antigen-binding molecules that specifically bind to A2AP as described in any of the preceding claims, and recovering the antigen-binding molecules from the culture.

[0115] On the other hand, this disclosure provides the use of an antigen-binding molecule that specifically binds to A2AP as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, in the preparation of a medicament for the prevention or treatment of a disease or condition.

[0116] On the other hand, this disclosure provides a method for preventing or treating a disease or condition, the method comprising administering to a subject an antigen-binding molecule that specifically binds to A2AP as described in the preceding claim, or a conjugate as described in the preceding claim, or a pharmaceutical composition as described in the preceding claim.

[0117] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to A2AP as described in the preceding claim, or a conjugate as described in the preceding claim, or a pharmaceutical composition as described in the preceding claim, for use as a medicament. In some embodiments, the medicament is used to prevent or treat a disease or condition.

[0118] In some implementations, the disease or condition as described in any of the preceding embodiments is a condition or disease associated with ischemic events caused by partial or complete vascular occlusion.

[0119] In some implementations, the disease or condition described in any of the preceding embodiments is ischemic stroke, thrombosis, cerebral stroke, coronary syndrome, peripheral artery disease, myocardial infarction, or pulmonary embolism.

[0120] In some implementations, the disease or condition described in any of the preceding claims is acute pulmonary embolism, acute ischemic stroke, acute coronary syndrome, deep vein thrombosis, arterial thrombosis, venous thrombosis, or shunt thrombosis.

[0121] In some embodiments, the antigen-binding molecules that specifically bind to A2AP provided in this disclosure are in EC5 concentrations of less than 10 nM (e.g., less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, less than 0.05 nM, less than 0.01 nM). 50 The value is combined with human A2AP, the EC 50 The value was measured using ELISA. Detailed Implementation

[0122] the term

[0123] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0124] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0125] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0126] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0127] The term "A2AP" or "α2AP" stands for "α2 anti-plasmin," also known as "Serpin F2" (F member 2 of the serpin family), 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 synthesized as a 491-amino acid precursor with a 27-amino acid signal peptide. The secreted form exhibits a short propeptide (residues 28-39) and a mature chain (residues 40-491). The reference sequence for human A2AP is available from the UniProtKB / Swiss-Prot database, accession number P08697-1 (SEQ-ID NO:3). Although a specific database accession number is given, those skilled in the art will understand that the A2AP referred to herein also encompasses the corresponding sequences reported in other databases or literature.

[0128] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0129] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0130] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this document, the amino acid residue at a specific site can be represented by the format "position + amino acid residue". For example, 228P indicates that the amino acid residue at position 228 is P. S228P indicates that the amino acid residue at position 228 has mutated from S to P. It should be understood that when the amino acid sequence is defined by the format "position + residue" in the claims, the amino acid before the mutation at that site does not constitute a limitation on the technical solution. In this document, "the Fc region contains amino acid mutations of 228P, 234A, and 235A" means that the amino acid mutation in the Fc region includes a mutation at position 228 to proline (P), a mutation at position 234 to alanine (A), and a mutation at position 235 to alanine (A).

[0131] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0132] The term "oligonucleotide" refers to oligonucleotide compounds that are typically less than about 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNA interference (RNAi) oligonucleotides (e.g., antisense oligonucleotides (ASO), siRNAs, shRNAs, dsRNAs, etc.), microRNAs (miRNAs), spacer polymers, mixers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, and guide nucleic acids (e.g., Cas9 guide RNAs). Oligonucleotides can be single-stranded or double-stranded. Oligonucleotides can be sense or antisense oligonucleotides.

[0133] The term "antigen-binding molecule" is used in the broadest sense to encompass a variety of molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as well as any molecule containing the aforementioned antibodies, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. Exemplarily, the antigen-binding molecules described herein are monoclonal antibodies, monospecific antibodies, polyclonal antibodies, and multispecific antibodies.

[0134] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, antibody fragments, and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0135] The term "antigen-binding fragment" encompasses full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, e.g., VH, VL, or VHH), single-chain Fab (scFab), single-chain antibodies (e.g., scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabetic, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above. Methods for generating and preparing these antigen-binding fragments are well known in the art.

[0136] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd, single-domain antibodies (sdAb, such as VH, VL, or VHH), single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0137] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0138] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing the Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0139] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0140] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0141] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0142] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between these numbering systems is well known to those skilled in the art and is exemplified as shown in Table A below.

[0143] Table A. Relationships between CDR numbering systems

[0144] Unless otherwise stated, the variable areas and CDRs in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0145] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0146] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0147] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies produced in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The term "human antibody" explicitly excludes humanized antibodies.

[0148] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0149] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0150] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0151] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0152] The term "monoclonal antibody" refers to a group of antibodies that are essentially homogeneous, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibodies typically comprise a variety of different antibodies with varying amino acid sequences in their variable domains, and they generally target different epitopes specifically. "Monoclonal" should not be interpreted as requiring the production of the antibody through any particular method.

[0153] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigenic epitopes of the same antigen. Various structures of bispecific antibodies have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-based bispecific antibodies. Based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies. Based on structural symmetry, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among these, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They exhibit lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively larger molecular weight. The Fc fragment facilitates antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life.

[0154] The term "antigen" refers to a molecule or molecular part that can be selectively bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0155] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain non-continuous amino acids (conformal epitopes), such as those spatially proximal due to antigen folding. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0156] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0157] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0158] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0159] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0160] "Separated nucleic acid" refers to a nucleic acid molecule that has been separated from its components in its natural environment. Separated nucleic acid encoding a polypeptide refers to one or more nucleic acid molecules encoding a polypeptide, including one or more such nucleic acid molecules in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0161] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0162] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned; gaps may be introduced, where necessary, to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0163] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0164] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. Mutant progeny are included herein, which have the same function or biological activity as cells screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The following are listed: *C. polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, *Neurospora crassa*, and *Yarrowia lipolytica*.

[0165] The terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived from them, regardless of the number of passages. It should also be understood that due to intentional or unintentional mutations, not all progeny will have exactly the same DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells.

[0166] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, and the description includes the circumstances under which the event or circumstances may or may not occur.

[0167] The term "pharmaceutical composition" means a mixture containing one or more antibodies, immunoconjugates or antibody-drug conjugates described herein, and other chemical components, such as physiological / pharmaceutical carriers, diluents or excipients.

[0168] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0169] The terms "subject" or "individual" include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein. In some embodiments, the individual or subject is a human being.

[0170] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0171] The term "sample" refers to a collection (such as fluid, cells, or tissue) separated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0172] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention on the individual being treated, and can be implemented for prevention or during a clinicopathological process. The expected effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis.

[0173] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some implementations, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when given to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. Complete treatment or prevention may not occur after a single dose, but may occur after a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0174] This disclosure discloses antigen-binding molecules that specifically bind to A2AP.

[0175] This disclosure provides antigen-binding molecules that specifically bind to A2AP and possess numerous advantageous properties, such as good affinity, therapeutic activity, safety, pharmacokinetic properties, and drugability (e.g., solubility, viscosity, purity, and stability).

[0176] Exemplary antigen-binding molecules that specifically bind to A2AP

[0177] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0178] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 5, HCDR2 in SEQ ID NO: 86, and HCDR3 in SEQ ID NO: 87; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 88, LCDR2 in SEQ ID NO: 89, and LCDR3 in SEQ ID NO: 10, wherein:

[0179] HIX1X2DX3DKX4YNPALKS SEQ ID NO: 86;

[0180] RGAX5YGSSFGWYFDV SEQ ID NO: 87;

[0181] SAX6X7X8IX9X 10 X 11 YLH SEQ ID NO: 88;

[0182] RASX 12 LAS SEQ ID NO: 89;

[0183] X1 is W or H, preferably W; X2 is W or S, preferably W; X3 is N, D or E, preferably N; X4 is S or G, preferably S.

[0184] X5 can be Y or H, with Y being preferred;

[0185] X6 is T or Q, T is preferred; X7 is S or Q, S is preferred; X8 is S or R, S is preferred; X9 is S, R, or T, S is preferred; X 10 S, K, or T, with S being the preferred choice; X 11 It can be N or D, with N being preferred;

[0186] X 12 It can be N or D, with N being preferred.

[0187] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0188] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 5, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 47, 18, 6, 45, 46 or 48, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 7 or 54. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 8, 33, 32, 34, 35, 36, 37, 38 or 39, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 9 or 43, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 10.

[0189] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0190] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 5, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 47, 18 or 6, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 7. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 8 or 33, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 9, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 10.

[0191] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0192] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 5, HCDR2 are shown in SEQ ID NO: 47, and HCDR3 are shown in SEQ ID NO: 7. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 8, LCDR2 are shown in SEQ ID NO: 9, and LCDR3 are shown in SEQ ID NO: 10.

[0193] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0194] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 5, HCDR2 are shown in SEQ ID NO: 47, and HCDR3 are shown in SEQ ID NO: 7. The amino acid sequences of the light chain variable regions LCDR1 are shown in SEQ ID NO: 33, LCDR2 are shown in SEQ ID NO: 9, and LCDR3 are shown in SEQ ID NO: 10.

[0195] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0196] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 5, HCDR2 in SEQ ID NO: 18, and HCDR3 in SEQ ID NO: 7, and the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 8, LCDR2 in SEQ ID NO: 9, and LCDR3 in SEQ ID NO: 10.

[0197] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0198] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 5, HCDR2 in SEQ ID NO: 6, and HCDR3 in SEQ ID NO: 7, and the amino acid sequences of the light chain variable region LCDR1 in SEQ ID NO: 8, LCDR2 in SEQ ID NO: 9, and LCDR3 in SEQ ID NO: 10.

[0199] For example, the antigen-binding molecules that specifically bind to A2AP disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0200] Exemplarily, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein:

[0201] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 are respectively the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 are respectively the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55.

[0202] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact.

[0203] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0204] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0205] The amino acid sequences of the heavy chain variable regions are shown in SEQ ID NO: 81, 73, 22, 19, 20, 21, 22, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80 or 82, and the amino acid sequences of the light chain variable regions are shown in SEQ ID NO: 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63 or 67.

[0206] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0207] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 81 or 22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 55, 68 or 25.

[0208] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0209] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 81, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 55.

[0210] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0211] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 81, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 68.

[0212] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0213] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.

[0214] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0215] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12.

[0216] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0217] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 84, and the amino acid sequence of the light chain is shown in SEQ ID NO: 83 or 85.

[0218] For example, the antigen-binding molecule that specifically binds to A2AP disclosed herein comprises a heavy chain and a light chain, wherein:

[0219] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 84, and the amino acid sequence of the light chain is shown in SEQ ID NO: 83.

[0220] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0221] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 84, and the amino acid sequence of the light chain is shown in SEQ ID NO: 85.

[0222] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0223] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 30.

[0224] In some embodiments, the antigen-binding molecule that specifically binds to A2AP as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0225] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is shown in SEQ ID NO: 16.

[0226] The antigen-binding molecule provided in this disclosure may also contain an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the above-mentioned amino acid sequence, such as a CDR sequence, a variable region sequence, a heavy chain, or a light chain sequence.

[0227] Antigen-binding molecular structure

[0228] In some implementations, the antigen-binding molecule provided herein is a full-length antibody.

[0229] In some implementations, the antigen-binding molecule provided herein is an antigen-binding fragment.

[0230] In some embodiments, the antigen-binding molecule provided herein is an antibody fragment. In some embodiments, the antigen-binding fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antigen-binding molecule via papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains such that interchain disulfide bonds can form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0231] In some implementations, the antigen-binding fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment with two antigen-binding sites, containing linked VH and VL domains within the same polypeptide chain (VH-VL). By using a short linker that prevents pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The two antigens can be the same or different.

[0232] In some embodiments, the antigen-binding fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antigen-binding domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In some embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0233] In some implementations, the antigen-binding fragment is an Fv fragment consisting of the VH and VL domains of a single arm of the antibody.

[0234] In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antigen-binding molecule from which it originates. Unless otherwise specified, the scFv may have VL and VH variable regions in any order herein; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0235] In some embodiments, the antigen-binding fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each of the VH and VL is replaced by a cysteine ​​residue via disulfide bonds between the cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on predictions of the three-dimensional structure of the antigen-binding molecule using known methods.

[0236] In some implementations, the antigen-binding fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain.

[0237] In some embodiments, the antigen-binding molecules provided herein are chimeric antibodies. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0238] In some embodiments, the antigen-binding molecule is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., an antibody providing the CDR sequence).

[0239] Humanized antibodies and their generation methods can refer to existing technologies.

[0240] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using the "best-fit" method; frame regions of common sequences of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions; mature human (somatic mutant) frame regions or human germline frame regions; and frame regions obtained by screening FR libraries.

[0241] Variants of antigen-binding molecules that specifically bind to A2AP

[0242] In some embodiments, amino acid sequence variants of antigen-binding molecules that specifically bind A2AP, as provided herein, are included. For example, improvements in binding affinity and / or other biological properties of the antigen-binding molecule may be desired. Amino acid sequence variants of the antigen-binding molecule can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antigen-binding molecule, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequence of the antigen-binding molecule that specifically binds A2AP. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0243] Replace, insert, and delete variants

[0244] In some embodiments, antigen-binding molecule variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table B under the heading “Preferred Substitutions.” More substantial variations are provided in Table B under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antigen-binding molecule of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0245] Table B. Substitution of Amino Acids

[0246] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0247] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0248] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0249] (3) Acidic: Asp, Glu;

[0250] (4) Alkaline: His, Lys, Arg;

[0251] (5) Residues that affect chain orientation: Gly, Pro;

[0252] (6) Aromatic: Trp, Tyr, Phe.

[0253] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0254] One class of substitution variants involves replacing one or more CDR residues in a parent antigen-binding molecule (e.g., humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased affinity, decreased immunogenicity) relative to the parent antigen-binding molecule, and / or will substantially retain some of the biological properties of the parent antigen-binding molecule. An exemplary substitution variant is an affinity-matured antigen-binding molecule, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, the variant antigen-binding molecule is displayed on a phage, and it is screened for specific biological activities (e.g., binding affinity). Modifications (e.g., substitutions) can be made to the CDRs, for example, to improve the affinity of the antigen-binding molecule. Such alterations can be made to CDR "hotspots"—residues encoded by codons that undergo high-frequency mutations during somatic maturation—and / or residues that come into contact with antigens, while simultaneously testing the binding affinity of the resulting variants VH or VL. In some embodiments of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-guided mutagenesis. A secondary library is then created. The library is then screened to identify any antigen-binding molecule variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0255] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, provided that such changes do not materially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) can be made to the CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0256] One method for identifying residues or regions in an antigen-binding molecule that can serve as mutagenic targets is called "alanine scanning mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the interaction between the antigen-binding molecule and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antigen-binding molecule and the antigen can be identified by studying the crystal structure of the antigen-antigen-binding molecule complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0257] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antigen-binding molecules having an N-terminal methionyl residue. Other insertion variants of antigen-binding molecules include fusions of the N- or C-terminus of an antigen-binding molecule with an enzyme or a peptide that extends the serum half-life of the antigen-binding molecule.

[0258] Recombination method

[0259] Antigen-binding molecules that specifically bind to A2AP can be generated using recombinant methods. For these methods, one or more nucleic acids encoding antigen-binding molecules that specifically bind to A2AP are provided.

[0260] In some embodiments, this disclosure provides nucleic acids encoding antigen-binding molecules that specifically bind A2AP as described above. Such nucleic acids can be derived from independently encoding any of the aforementioned polypeptide chains. In some embodiments, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In some embodiments, this disclosure provides host cells comprising such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method includes culturing host cells comprising nucleic acids encoding said polypeptide or fusion protein, as provided above, under suitable expression conditions, and optionally recovering said antigen-binding molecules that specifically bind A2AP from the host cells (or host cell culture medium).

[0261] To recombinantly generate antigen-binding molecules that specifically bind to A2AP, nucleic acids encoding the protein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, or generated through recombinant methods or obtained through chemical synthesis.

[0262] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules that specifically bind to A2AP include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. Following expression, the vector can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0263] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548, US7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0.

[0264] Measurement

[0265] The antigen-binding molecules that specifically bind to A2AP disclosed herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In some embodiments, the activity of the antigen-binding molecules that specifically bind to A2AP disclosed herein is tested, for example, by known methods such as ELISA, Western blotting, etc.

[0266] Treatment methods and routes of administration

[0267] Any antigen-binding molecule that specifically binds to A2AP provided in this disclosure may be used for therapeutic purposes. In some embodiments, the antigen-binding molecule that specifically binds to A2AP provided in this disclosure is used in the manufacture or preparation of a medicament.

[0268] In some embodiments, a pharmaceutical composition comprising the antigen-binding molecule that specifically binds to A2AP is provided, for example, for any of the pharmaceutical uses or treatment methods described above. In some embodiments, the pharmaceutical composition comprises any of the antigen-binding molecules that specifically bind to A2AP provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0269] The antigen-binding molecule that specifically binds to A2AP disclosed herein can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecule disclosed herein can be administered co-administered with at least one other therapeutic agent.

[0270] The antigen-binding molecules (and any other therapeutic agents) that specifically bind to A2AP disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0271] The antigen-binding molecules that specifically bind to A2AP disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The antigen-binding molecules that specifically bind to A2AP may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at other dosages, and at any route determined empirically / clinically as appropriate.

[0272] For the prevention or treatment of disease, the appropriate dosage of the antigen-binding molecule that specifically binds to A2AP disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either once or after a series of treatments.

[0273] Products

[0274] In another aspect of this disclosure, an article of manufacture is provided comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule that specifically binds to A2AP as disclosed herein. The label or package insert indicates that the use of the composition is for the treatment of the selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition comprising an antigen-binding molecule that specifically binds to A2AP as disclosed herein; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further include a packaging insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including other buffers, diluents, filters, needles, and syringes.

[0275] Example

[0276] The following examples and test cases further describe the invention, and these examples and test cases should not be construed as limiting the scope of the invention. The examples or test cases of the invention do not include detailed descriptions of conventional methods. Experimental methods without specific conditions are generally performed under conventional conditions, as such methods are well known to those skilled in the art and described in many publications, such as *Molecular Cloning* (Green MR, Sambrook J. *A Laboratory Manual*, 4th, 2012) published by Cold Spring Harbor Laboratory, or *Antibody Engineering: Methods and Protocols* (*Antibody Engineering: Methods and Protocols* (Humana Press, 2018) published by Springer Protocols), or according to the conditions recommended by the manufacturer of the experimental materials. Some experimental materials without a specific source are obtained commercially.

[0277] Example 1. Preparation and screening of A2AP (alpha-2-antiplasmin) antibody

[0278] This disclosure describes the preparation of a monoclonal antibody targeting human A2AP using hybridoma technology. Asn was selected. 13 hA2AP-His DNA and Rat A2AP-His proteins serve as immunogens, among which Asn 13 hA2AP-His (SEQ ID NO: 1) is selected based on the functional sequence of A2AP_HUMAN (P08697) (SEQ ID NO: 3). It is formed by the natural cleavage of amino acids P at position 12 and N at position 13 of hA2AP in vivo, accounting for approximately 70% of peripheral A2AP. It is constructed by linking a His tag to the C-terminus of the DNA sequence. Rat A2AP-His (SEQ ID NO: 2) is a fusion protein constructed and expressed based on the Rat A2AP (NP_001011892.1) (SEQ ID NO: 4) sequence by linking a His tag to its C-terminus. Gold Adjuvant (Sigma Cat No. T2684) was used as an adjuvant for cross-immunization of mice. After primary and booster immunizations, mice with high antibody titers in their serum were selected for spleen cell fusion.

[0279] Asn 13 hA2AP-His protein sequence:

[0280] Rat A2AP-His protein sequence:

[0281] The following is the source of the full-length hA2AP sequence (A2AP_HUMAN), accession number: P08697

[0282] The following is the source of the full-length rat A2AP sequence (Rattus norvegicus serpin family F member 2 (Serpinf2)), accession number: NP_001011892.1

[0283] After fusion, the hybridoma culture supernatant was analyzed based on the hybridoma cell growth density, and antibodies specifically binding to human, cynomolgus monkey, rabbit, or rat A2AP antigens were screened. High-activity monoclonal hybridoma cell lines were obtained. Single-cell clones were expanded, and the culture supernatant was collected. Mouse antibodies secreted from the supernatant were extracted using Protein A magnetic beads for in vitro plasmin activity qualitative analysis. Single-cell clones that significantly promoted plasmin activity were selected. Hybridoma cells in their logarithmic growth phase were collected, and RNA was extracted using NucleoZol (MN) (following the kit instructions) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced. The CDR and amino acid sequences of the variable region of the screened monoclonal hybridoma cell line mouse antibody 298-1 are as follows:

[0284] Table 1. CDR sequences of anti-A2AP antibody 298-1 obtained through hybridoma screening Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0285] The variable region sequence is as follows:

[0286] >298-1 Mouse-derived heavy chain variable region

[0287] >298-1 Mouse-derived light chain variable region Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The double underscores are the CDR sequence determined according to the Kabat numbering system, and the non-underscore parts are the FR sequence.

[0288] The heavy chain variable region and light chain variable region of the murine anti-298-1 antibody were recombined with the heavy chain constant region hIgG4 (S228P, F234A, L235A, SEQ ID NO: 14) and the human κ light chain constant region CL (SEQ ID NO: 15), respectively, to obtain the full-length chimeric antibody CHI298-1.

[0289] CH1:

[0290] Heavy chain constant region hIgG4 (S228P, F234A, L235A): CH1-Fc:

[0291] >CL:

[0292] CHI298-1 Light Chain:

[0293] CHI298-1 Heavy Chain: Note: Double underline: CDR; italics: constant region; where CDR is based on Kabat numbering.

[0294] Example 2.298-1 antibody humanization

[0295] Using MOE software, the genetic sequence of the heavy and light chain variable region of the mouse antibody was compared with the IMGT human antibody heavy and light chain variable region germline gene database. A germline gene with high homology to CHI298-1 was selected as a template, and the CDRs of this mouse antibody were transplanted into the corresponding human templates, forming a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific embodiments, the CDR amino acid residues were determined and annotated using the Kabat numbering system.

[0296] The humanized antibody for CHI298-1 uses FR1, FR2, and FR3 of IGKV4-1*01 or IGKV7-3*01, and FR4 of IGKJ2*01, i.e., FGQGTKLEIK (SEQ ID NO:90), as the template for the light chain framework region; and FR1, FR2, and FR3 of IGHV2-70*04, and FR4 of IGHJ6*01, i.e., WGQGTTVTVSS (SEQ ID NO:91), as the template for the heavy chain framework region. Optionally, amino acid residues at positions 4, 22, 43, 60, 71, and / or 81 of the light chain variable region of the humanized antibody are substituted; and / or amino acid residues at positions 1, 30, 82, and / or 55 of the heavy chain variable region of the humanized antibody are substituted (where the positions of the mutation sites are determined according to the Kabat numbering rules). The mutation design of the variable region of the humanized antibody CHI298-1 is shown in Table 2 below:

[0297] Table 2. Humanized antibodies against CHI298-1 Note: P43S indicates that the 43rd P is mutated to S according to the Kabat numbering system, and so on.

[0298] Meanwhile, CHI298-1 was humanized using a computer-aided antibody humanization design method. FR1, FR2, and FR3 of IGKV3D-20*02 and FR4 of IGKJ4*01, i.e., FGGGTKVEIK (SEQ ID NO:92), were used as templates for the light chain framework region. The FR portion of the light chain included mutations at sites 1, 4, 13, and 58 (the positions of the mutation sites were determined according to Kabat numbering rules). FR1, FR2, and FR3 of IGHV2-70*04 and FR4 of IGHJ2*01, i.e., WGRGTLVTVSS (SEQ ID NO:93), were used as templates for the heavy chain framework region. The FR portion of the heavy chain included mutations at sites 30 and 10 (the positions of the mutation sites were determined according to Kabat numbering rules). A D55E mutation was introduced on top of VH5 to reduce the potential risk of isomerization. The mutation design of the variable region of the humanized antibody CHI298-1 based on computer-aided antibody humanization design is shown in Table 3 below.

[0299] Table 3. AI-based humanized antibodies designed for CHI298-1

[0300] The CDR of the humanized antibody CHI298-1 is as follows:

[0301] Table 4. CDR of CHI298-1 humanized antibody

[0302] The light chain variable region / heavy chain variable region sequences of the CHI298-1 humanized antibody are as follows:

[0303] >huCHI298-1VH1

[0304] >huCHI298-1VH2

[0305] >huCHI298-1VH3

[0306] >huCHI298-1VH4

[0307] >huCHI298-1VH5

[0308] >hu CHI298-1VH6

[0309] >hu CHI298-1VL1

[0310] >hu CHI298-1VL2

[0311] >hu CHI298-1VL3

[0312] >hu CHI298-1VL4 Note: In the above sequences, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the double underscores are the CDR sequences determined according to the Kabat numbering system; the un-underlined parts are the FR sequences; the bolded parts are amino acid point mutations.

[0313] The heavy chain variable region and light chain variable region of the CHI298-1 humanized antibody were recombined with the heavy chain constant region hIgG4 (S228P, F234A, L235A) (SEQ ID NO: 14) and the light chain constant region CL (SEQ ID NO: 15), respectively, to obtain the humanized antibody shown in Table 5 below.

[0314] Table 5. Humanized Antibodies for CHI298-1 Note: In the table, “hu298-1L1H4” indicates a humanized antibody with a heavy chain variable region of VH4 (SEQ ID NO: 22), a light chain variable region of VL1 (SEQ ID NO: 25), and a heavy chain constant region as shown in hIgG4 (S228P, F234A, L235A) (SEQ ID NO: 14), and a light chain constant region as shown in CL (SEQ ID NO: 15). Others are deduced by analogy.

[0315] For example, the full-length sequence of the humanized antibody against A2AP hu298-1L1H4 is as follows:

[0316] Heavy chain sequence of hu298-1L1H4 antibody

[0317] The light chain sequence of the hu298-1L1H4 antibody Note: In the above sequences, double underlines represent CDR sequences determined according to the Kabat numbering system, and italicized portions represent antibody constant regions.

[0318] Example 3. Immunogenicity modification of anti-A2AP antibody

[0319] Further point mutations were performed on the CDR or FR regions of the CHI298-1 humanized antibody. Optionally, single-point mutations were combined. The following mutations were performed on hu298-1L1, including single-point and multi-point combination mutations (where the location of the mutation sites was determined according to the Kabat numbering rules), to obtain different variable regions and antibodies.

[0320] Table 6. CDR and FR modification of hu298-1L1 humanized antibody

[0321] The following mutations were performed on hu298-1H4, including single-point and multi-point combination mutations (where the location of the mutation site was determined according to the Kabat numbering rules), to obtain different variable regions and antibodies.

[0322] Table 7. CDR and FR modification of hu298-1H4 humanized antibody

[0323] For example, the variable region sequences of each antibody are obtained as follows:

[0324] >L1-T20Q

[0325] >L1-T26Q

[0326] >L1-S27Q

[0327] >L1-S27aR

[0328] >L1-S29R

[0329] >L1-S29T

[0330] >L1-S30K

[0331] >L1-S30T

[0332] >L1-N31D

[0333] >L1-L47D

[0334] >L1-I48E

[0335] >L1-Y49G

[0336] >L1-N53D

[0337] >L1-T20Q,S27Q

[0338] >L1-T20Q,S27Q,L47D

[0339] >H4-A49T

[0340] >H4-W52H

[0341] >H4-W53S

[0342] >H4-E55N

[0343] >H4-S58G

[0344] >H4-I82V

[0345] >H4-N82bQ

[0346] H4-V85E

[0347] H4-V85T

[0348] H4-T89S

[0349] >H4-Y98H

[0350] >H4-E55N,V85T

[0351] >H4-A49T, V85T Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The double underscores are the CDR sequence determined according to the Kabat numbering system, and the non-underscore parts are the FR sequence.

[0352] The modified anti-A2AP humanized antibody heavy chain variable region (selected from SEQ ID NO: 70-82) and the unmodified huCHI298-1VH4 (SEQ ID NO: 22), and the modified anti-A2AP humanized antibody light chain variable region (selected from SEQ ID NO: 25, 55-69) and the unmodified huCHI298-1VL1 (SEQ ID NO: 25) are recombined with the heavy chain constant region hIgG4 (S228P, F234A, L235A) (SEQ ID NO: 14) and the light chain constant region CL (SEQ ID NO: 15), respectively, to obtain the full-length humanized antibody heavy chain and light chain. The aforementioned heavy chain and light chain are then randomly paired to obtain the full-length humanized antibody.

[0353] For example, the modified anti-A2AP humanized antibody described in Table 8 has the following corresponding sequences for VH (heavy chain variable region), VL (light chain variable region), HCDR (heavy chain CDR region), LCDR (light chain CDR), HC (heavy chain constant region), and LC (light chain constant region):

[0354] Table 8. Modification of A2AP humanized antibody

[0355] The following is an example of the full-length sequence of a humanized anti-A2AP antibody:

[0356] >hu298-1 L1-T20Q;H4-E55N,V85T light chain

[0357] >hu298-1 L1-T20Q;H4-E55N,V85T heavy chain or

[0358] hu298-1 L1-T20Q,S27Q; H4-E55N,V85T heavy chain

[0359] >hu298-1 L1-T20Q,S27Q;H4-E55N,V85T light chain Note: In the above sequences, double underlines represent CDR sequences determined according to the Kabat numbering system, and italicized portions represent antibody constant regions.

[0360] Test case

[0361] Test Example 1: Binding experiment of anti-A2AP antibody to A2AP protein

[0362] Human A2AP protein (SEQ ID NO: 1), monkey A2AP protein (Cyno) (SEQ ID NO: 3 in WO2022058261A2), and rabbit A2AP protein (SEQ ID NO: 2) were diluted to 0.2 μg / mL with PBS and coated into 96-well plates (Corning, CLS3590-100EA) and incubated overnight at 4°C. After washing, 1% BSA was added and the plates were blocked at 37°C for 1 hour. After washing, different concentrations of anti-A2AP antibody were added and the plates were incubated at 37°C for 1 hour. After washing, HRP anti-human IgG (H+L) (Sangon Biotech, B548127) was added and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic substrate (KPL, 52-00-03) was added and the plates were incubated at room temperature for 5-10 minutes. The reaction was terminated by adding 1M H2SO4, and the absorbance was read at 450 nm using a VERSAmax microplate reader (Molecular Devices). The results are shown in Table 9.

[0363] Table 9. Binding ability of anti-A2AP antibodies to A2AP protein

[0364] The results showed that CHI298-1 had nanomolar binding capacity to human, monkey, and rabbit A2AP, and the humanized molecules maintained good binding capacity to human A2AP, while also maintaining good binding to monkey and rabbit A2AP.

[0365] Test Example 2: Anti-A2AP antibody blocks the inactivation of plasmin by A2AP in an in vitro plasma environment.

[0366] This disclosure describes the inhibitory effect of anti-A2AP antibodies on A2AP-mediated plasmin-A2AP covalent binding in an in vitro plasma environment. Standard anticoagulated plasma was used after being diluted 25-fold with phosphate buffer, as follows:

[0367] In a 96-well plate (BeyoGold, FCP968), 20 μL of diluted human plasma was mixed with serially diluted antibody solution (maximum concentration 200 nM, 2-fold serial dilution), and incubated at room temperature for 30 minutes. Then, 20 μL of pre-diluted 8 nM plasmin solution (Haematologic Technologies, HCPM-0140) was added to each well and mixed. Finally, 20 μL of pre-diluted 0.4 mM MeOSuc–Ala–Phe–Lys–AMC substrate (GlpBio, GA23190) was added to each well and mixed. After incubation at room temperature for 1 hour, the fluorescence signal was detected using a microplate reader (excitation wavelength 360 nm, emission wavelength 465 nm). The detection results were used to fit an inhibition curve using GraphPad Prism software, and the IC50 was calculated. 50 Values. The inhibitory effects of each antibody on A2AP activity are shown in Table 10.

[0368] Table 10. Anti-A2AP antibodies block the inactivation of plasmin by A2AP in the in vitro plasma environment.

[0369] The results showed that the humanized antibody disclosed in this study effectively blocked the inactivation of plasmin by A2AP in an in vitro plasma environment.

[0370] Test Example 3: Binding of the modified anti-A2AP antibody to human A2AP protein.

[0371] Human A2AP protein (SEQ ID NO: 1) was diluted to 0.2 μg / mL with PBS and coated into 96-well plates (Corning, CLS3590-100EA), then incubated overnight at 4°C. After washing, 1% BSA was added, and the plates were blocked at 37°C for 1 hour. After washing, different concentrations of anti-A2AP antibody were added, and the plates were incubated at 37°C for 1 hour. After washing, HRP anti-human IgG (H+L) (Sangon Biotech, B548127) was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic substrate (KPL, 52-00-03) was added, and the plates were incubated at room temperature for 5-10 minutes. The reaction was terminated by adding 1M H2SO4, and the absorbance was read at 450 nm using a VERSAmax microplate reader (Molecular Devices). The ELISA results are shown in Table 11.

[0372] Table 11. Binding ability of anti-A2AP antibodies to human A2AP protein

[0373] The results showed that the immunogenicity-optimized modified antibody disclosed herein could maintain its pre-modification activity and effectively bind to human A2AP.

[0374] Test Example 4: The modified anti-A2AP antibody blocked the inactivation of plasmin by A2AP in the in vitro plasma environment.

[0375] This patent describes the inhibitory effect of anti-A2AP antibodies on A2AP-mediated plasmin-A2AP covalent binding in an in vitro plasma environment. Standard anticoagulated plasma was diluted 25-fold with phosphate buffer before use, as detailed below:

[0376] In a 96-well plate (BeyoGold, FCP968), 20 μL of diluted human plasma was mixed with serially diluted antibody solution (maximum concentration 200 nM, 2-fold serial dilution), and incubated at room temperature for 30 minutes. Then, 20 μL of pre-diluted 8 nM plasmin solution (Haematologic Technologies, HCPM-0140) was added to each well and mixed. Finally, 20 μL of pre-diluted 0.4 mM MeOSuc–Ala–Phe–Lys–AMC substrate (GlpBio, GA23190) was added to each well and mixed. After incubation at room temperature for 1 hour, the fluorescence signal was detected using a microplate reader (excitation wavelength 360 nm, emission wavelength 465 nm). The detection results were used to fit an inhibition curve using GraphPad Prism software, and the IC50 was calculated. 50 Values. The inhibitory effects of each antibody on A2AP activity are shown in Table 12.

[0377] Table 12. Anti-A2AP antibodies block the inactivation of plasmin by A2AP in the in vitro plasma environment.

[0378] The results showed that the immunogenicity-optimized modified antibody disclosed herein could maintain its pre-modification activity in an in vitro plasma environment and effectively block the inactivation of plasmin by A2AP.

[0379] Test Example 5: Anti-A2AP antibody promotes in vitro thrombolysis

[0380] This disclosure describes the in vitro thrombolytic effect of anti-A2AP antibodies on tPA in an in vitro plasma environment. The specific method is as follows:

[0381] Pre-diluted tPA (24 μg / mL, Yiqiao Shenzhou, 10157-HNCH2) and thrombin (50 μg / mL, Zedira, T056-200) were mixed in equal volumes, and 2.5 μL was added to each well of a 96-well plate (Corning, 3599). Then, 2.5 μL of 500 mM calcium chloride solution (Sinopharm, 10043-52-4) was added to each well. Simultaneously, in another 96-well plate (JET, TCP002096), 216 μL of standard anticoagulated plasma was mixed with 12 μL of serially diluted antibody solution (maximum concentration 20 μM, 2-fold serial dilution), and incubated at 37°C for 5 minutes. Subsequently, 95 μL of plasma / antibody mixture was taken from each well and transferred to the first 96-well plate. After thorough mixing and sealing with a transparent optical seal, the absorbance at 405 nm was immediately measured using a microplate reader at 37°C, with a measurement frequency of once per minute for 11 consecutive hours. Peak curves were plotted using Visual Basic software. The thrombolysis time of each OD405 curve was used as the 50% clot lysis time index. Finally, inhibition curves were generated by combining the antibody concentrations and their corresponding thrombolysis times using GraphPad Prism software, and the IC50 was calculated. 50 value.

[0382] Table 13. Effects of anti-A2AP antibodies on promoting in vitro thrombolysis

[0383] The results showed that the anti-A2AP antibody exhibited superior thrombolytic activity in clot lysis.

[0384] Test Example 6: Single and Multiple Dose Efficacy of Anti-A2AP Antibody in a Rabbit Deep Vein Thrombosis (DVT) Model

[0385] After anesthetizing the male Japanese white rabbits (Tianjin Yuda Experimental Animal Breeding Co., Ltd.), they were fixed in a supine position. The abdomen was prepared and disinfected. The abdomen was opened through the midline and linea alba. The intestines were protected with saline gauze and pushed to one side. The retroperitoneum was cut open, and the inferior vena cava was freed (from below the left renal vein to above the opening of the iliopsoas vein). At a 2cm interval between the proximal and distal ends of the blood vessel, a 2mm diameter plastic tube was placed parallel to the blood vessel. The proximal and distal blood vessels were ligated respectively. After ligation, the plastic tube was slowly withdrawn. At the same time, the branch veins at this interval were ligated.

[0386] A needle electrode was placed on the endothelium of the ligated inferior vena cava, and a 2mA current was applied for 15 minutes of stimulation. During the stimulation, an arterial clamp was used to close the narrowed area and block the vein. After the stimulation, the needle hole was sutured closed and the arterial clamp was released, followed by layer-by-layer suturing.

[0387] Rabbits were randomly divided into a model control group (administered with PBS) and two groups (hu298-1 L1_T20Q; H4_E55N, V85T3, 10, and 30 mg / kg), with eight rabbits in each group. Administered the medication 30 minutes after the electrical stimulation ended, via slow intravenous injection over 1 minute in the marginal ear vein. The endpoint of the experiment was 3 hours after administration.

[0388] The detection indicators were thrombus wet weight and plasma coagulation parameters TT, PT, APTT and FIB measured 3 hours after administration.

[0389] Experimental results showed that the wet weight of thrombi decreased by 20.8% (P>0.05), 35.3% (P<0.05), and 46.4% (P<0.01) in the hu298-1 L1_T20Q;H4_E55N,V85T groups at 3, 10, and 30 mg / kg, respectively.

[0390] hu298-1 L1_T20Q; H4_E55N, V85T had no significant effect on the four coagulation parameters.

[0391] Table 14. Effect on wet weight of DVT thrombi in rabbits (mean ± SD, n = 8) Note: Compared with the model control group: * P<0.05, ** P<0.01.

[0392] Table 15. Effects of in vivo administration of anti-A2AP antibody on plasma coagulation parameters TT, PT, APTT, and FIB. Note: Compared with the model control group: * P<0.05, ** P<0.01.

[0393] Test Example 7: Multidose efficacy of anti-A2AP antibody in a rat model of cerebral stroke (MCAO)

[0394] Rats (provided by Vital Rivers, Beijing) were anesthetized with isoflurane gas and fixed in a supine position on the operating table. The skin was incised along the midline of the neck, and the right common carotid artery and external carotid artery were dissected. The pterygopalatine artery, a branch of the internal carotid artery, was isolated and clamped. A small incision was made at the external carotid artery, and a thrombus prepared from whole blood was injected into the cranium with 0.4 mL of physiological saline. The catheter was carefully withdrawn, and the skin was sutured. In the sham surgery group, only the skin was incised along the midline of the neck, and the right common carotid artery and external carotid artery were dissected; no thrombus was injected.

[0395] 55 minutes after thromboembolism, students were eligible for enrollment based on a neurological function score ≥8 (9-11 points for those with relatively uniform damage, excluding those with excessive or mild damage) and a decrease in cerebral blood flow ≥50% compared to the baseline value on the same side as detected by laser speckle imaging. Administering medication began 60 minutes after thromboembolism.

[0396] Rats that successfully established the model were randomly divided into groups of 10 animals each: a model control group, and groups treated with CHI298-1 at 3 mg / kg, 10 mg / kg, and 30 mg / kg. A sham-operated group of 10 rats was also included. The test drug groups received a single intravenous injection of the drug via the tail vein, while the sham-operated and model control groups received a single intravenous injection of PBS (propagated PBS). The endpoint was 24 hours after drug administration.

[0397] The results showed that, compared with the sham-operated group, rats in the model control group exhibited significant behavioral impairments 1 hour after embolization and 24 hours after modeling (P < 0.001). Compared with the model control group, all drug-treated groups showed significant improvement in neurological function scores 24 hours after administration. The improvement rates of CHI298-1 at 3, 10, and 30 mg / kg were 12.0% (P > 0.05), 28.0% (P < 0.01), and 34.7% (P < 0.001), respectively, showing a dose-dependent effect.

[0398] Table 16. Effects on neurological function in rats with thromboembolic stroke (mean ± SD, n = 10) Note 1. Compared with the sham surgery group: ### P<0.001; 2. Compared with the model control group: ** P<0.01, *** P<0.001.

[0399] Compared with the sham-operated group, the cerebral blood flow in the model control group rats was significantly reduced 1 hour after thromboembolism (before drug administration), and no significant change was observed 3 hours after drug administration. Compared with the model control group, each drug administration group showed varying degrees of increase in cerebral blood flow 3 hours after drug administration. The improvement rates of CHI298-1 at 3, 10, and 30 mg / kg were 14.67% (P<0.05), 18.92% (P<0.001), and 21.88% (P<0.001), respectively, showing a dose-dependent effect.

[0400] Table 17. Effects of drugs on the rate of change (%) of cerebral blood flow in rats with thromboembolic stroke (mean ± SD, n = 10) Note 1. Compared with the sham surgery group: ### P<0.001; 2. Compared with the model control group: * P<0.05, ** P<0.01, *** P<0.001.

[0401] Compared with the sham-operated group, rats in the model control group showed significant cerebral infarction (P < 0.001). Compared with the model control group, the proportion of cerebral infarction area was significantly reduced in each treatment group 24 hours after administration. The improvement rates in the CHI298-1 3, 10, and 30 mg / kg groups were 22.6% (P > 0.05), 50.3% (P < 0.01), and 58.2% (P < 0.001), respectively.

[0402] Table 18. Effects of drugs on the ratio of cerebral infarction area in rats with thromboembolic stroke (mean ± SD, n = 10) Note 1. Compared with the sham surgery group: ### P<0.001; 2. Compared with the model control group: *** P<0.001.

[0403] Compared with the model control group, the fraction of cerebral hemorrhages 24 hours after administration of CHI298-1 at 3, 10, and 30 mg / kg was not significantly increased, and the number of cases of cerebral hemorrhage was not significantly increased (1 / 10).

[0404] Table 19. Effects of drugs on thromboembolic stroke and cerebral hemorrhage in rats (mean ± SD, n = 10) Note: 1. Compared with the sham surgery group: P>0.05; 2. Compared with the model control group: P>0.05.

[0405] Test Example 8: Pharmacokinetic Analysis of Anti-A2AP Antibody in Cynomolgus Monkeys

[0406] Crab-eating macaques weighing 3-5 kg ​​(Suzhou Tuowei Biotechnology Co., Ltd.) were selected, and three animals (3 males or 2 males and 1 female) were housed in a standard stainless steel cage in a general-grade enclosure, individually. The temperature was set at 20-26℃, and the relative humidity at 40-70%, with a controlled 12-hour light / dark cycle. Anti-A2AP antibody was administered intravenously at a dose of 10 mpk. Food and water were allowed before and during administration. All animals were given the test sample / control sample via a 10 mL syringe via the hind limb vein. Blood samples were collected from the cephalic vein of the forelimb after the single administration, with the day of administration designated as day 0 (D0). Blood samples were collected at the corresponding detection time points. The collected whole blood was placed in an inert separating gel coagulation tube and allowed to stand at room temperature for serum precipitation (no more than 30 minutes). Then, the samples were centrifuged at 2600g for 10 minutes at 4℃, and the serum (at least 360 μL) was collected after centrifugation.

[0407] Blood drug concentration assays were performed by coating DELFIA plates (PerkinElmer #AAAND-0001) with 100 μL of 1 μg / mL Goat Anti-Human IgG, Monkey ads-UNLB (Southern Biotech #2049-01) and incubating overnight at 4°C. After washing with washing buffer, the plates were blocked with blocking buffer at 37°C for 1–2 hours. Subsequently, serum samples containing STD, QC, and the sample to be tested were added separately and incubated at 25°C and 350 rpm for 1.5–2 hours with shaking. After washing the plate 5 times with wash buffer, Goat Anti-Human IgG and Monkey Ads-BIOT (1:5000) were added and incubated at 25°C and 350 rpm for 1–1.5 hours with shaking. After washing the plate 5 times with wash buffer, 100 μL of SA-EU (1:1000) was added and incubated at 25°C and 350 rpm for 1–1.5 hours. After washing the plate 5 times with wash buffer, 100 μL of DELFIA Enhancement Solution was added and incubated in the dark for 30 minutes. The plate was then read, and the pharmaceutical concentrations in serum were calculated and data processed according to the STD standard curve. Pharmacokinetic parameters were calculated using a Phoenix WinNonlin 7.0 non-compartmental model.

[0408] The results showed that the t1 / 2 of hu298-1 L1_T20Q; H4_E55N, V85T was 14.5±1.0 days, the AUC 0-672h was 57577±5791ug / ml*h, and the CL was 3.5±0.4ml / day / kg.

[0409] Table 20. Pharmacokinetics of anti-A2AP antibody in cynomolgus monkeys

[0410] Although the invention has been described in detail with the aid of examples for clarity, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

1. An antigen binding molecule that specifically binds A2AP, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 86, and HCDR3 contains the amino acid sequence of SEQ ID NO:

87. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 88, LCDR2 contains the amino acid sequence of SEQ ID NO: 89, and LCDR3 contains the amino acid sequence of SEQ ID NO:

10.

2. An antigen binding molecule that specifically binds A2AP, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein: The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, 22, 19, 20, 21, 23, 24, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, 68, 26, 27, 28, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 69; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO:

12. Preferably, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 73, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 73, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 68, 69, or 57; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79, or 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25, 26, 27, 28, 55, 64, 65, or 66; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, 19, 20, 21, or 23, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12, 25, 26, 27, or 28; More preferably, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81 or 22, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55, 25, or 68; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55 or 68; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO:

12. Most preferably, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 55; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 81, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 68; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO:

25.

3. The antigen-binding molecule that specifically binds to A2AP according to claim 1 or 2, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, 18, 6, 45, 46 or 48, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7 or 54, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, 33, 32, 34, 35, 36, 37, 38 or 39, LCDR2 contains the amino acid sequence of SEQ ID NO: 9 or 43, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10; Preferably, The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, 18, or 6, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8 or 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10; or The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47 or 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8 or 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO:

10. More preferably, The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10; or The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 47, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10; or The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO: 10; or The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 5, HCDR2 contains the amino acid sequence of SEQ ID NO: 6, and HCDR3 contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 8, LCDR2 contains the amino acid sequence of SEQ ID NO: 9, and LCDR3 contains the amino acid sequence of SEQ ID NO:

10.

4. The antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 3, wherein the antigen-binding molecule that specifically binds to A2AP is an antibody; Preferably, the antigen-binding molecule that specifically binds to A2AP is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; More preferably, the antigen-binding molecule that specifically binds to A2AP is a humanized antibody.

5. The antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 4, wherein: The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63, or 67, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 81, 73, 22, 19, 20, 21, 23, 24, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, or 82, and ... NO: Amino acid sequences with at least 80% sequence identity, such as 55, 64, 65, 66, 25, 26, 27, 28, 57, 68, 69, 56, 58, 59, 60, 61, 62, 63, or 67; or The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 11, 19, 20, 21 or 23 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, 19, 20, 21 or 23, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66; Preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 25, 26, 27, 28, 64, 65 or 66 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, 25, 26, 27, 28, 64, 65 or 66; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, 69 or 57 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 68, 69 or 57; or The heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79 or 82 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79 or 82, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 25, 26, 27, 28, 55, 64, 65 or 66 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 25, 26, 27, 28, 55, 64, 65 or 66; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

12. More preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 25, 26, 27, 28, 64, 65, or 66; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, 69 or 57; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, 24, 70, 75, 76, 77, 78, 79 or 82, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25, 26, 27, 28, 55, 64, 65 or 66; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, 19, 20, 21 or 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, 25, 26, 27, 28, 55, 64, 65 or 66; Most preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

12.

6. The antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 5, wherein the antigen-binding molecule that specifically binds to A2AP is an antigen-binding fragment; Preferably, the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, Fv, scFv and dsFv.

7. The antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 5, wherein the antigen-binding molecule that specifically binds to A2AP comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is the human IgG4 heavy chain constant region or a variant thereof, and the light chain constant region is the human κ light chain constant region or a variant thereof; More preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

15.

8. The antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 5, 7, wherein the antigen-binding molecule that specifically binds to A2AP comprises a heavy chain and a light chain, wherein: The heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 84 or 29, and the light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 83, 85, or 30; or The heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 17, and the light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16; Preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 84 or 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 83, 85, or 30; or The heavy chain contains the amino acid sequence of SEQ ID NO: 17, and the light chain contains the amino acid sequence of SEQ ID NO: 16; More preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 83 or 85; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 83 or 30; or The heavy chain contains the amino acid sequence of SEQ ID NO: 17, and the light chain contains the amino acid sequence of SEQ ID NO: 16; Most preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 83; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 84, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 30; or The heavy chain contains the amino acid sequence of SEQ ID NO: 17, and the light chain contains the amino acid sequence of SEQ ID NO:

16.

9. A pharmaceutical composition comprising an antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

10. A nucleic acid encoding an antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 8.

11. A host cell comprising the nucleic acid as described in claim 10.

12. A method for preventing or treating a disease or condition, the method comprising administering to a subject an antigen-binding molecule that specifically binds to A2AP according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9; Preferably, the disease or condition is a condition or illness associated with ischemic events caused by partial or complete vascular occlusion; More preferably, the disease or condition is ischemic stroke, thrombosis, thromboembolism, stroke, coronary syndrome, peripheral artery disease, myocardial infarction, or pulmonary embolism; Most preferably, the disease or condition is acute pulmonary embolism, acute ischemic stroke, acute coronary syndrome, deep vein thrombosis, arterial thrombosis, venous thrombosis, or shunt thrombosis.

Images