Binding molecule specifically binding to Anti-NPR1 antibody and medical use thereof

Abstract

Provided are a binding molecule specifically binding to an anti-NPR1 antibody and the medical use thereof. Specifically provided is the use of the binding molecule specifically binding to the anti-NPR1 antibody in the preparation of a drug for reversing the side effects of the anti-NPR1 antibody.

Description

Specific binding molecules of anti-NPR1 antibodies and their pharmaceutical applications

[0001] This application claims priority to Chinese patent application CN202411820728.9, filed on December 11, 2024. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to binding molecules that specifically bind to anti-NPR1 antibodies and their pharmaceutical uses. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] NPR1 agonists, such as NPR1 agonist antibodies or recombinant NPR1 natural receptor proteins, exert biological functions such as vasodilation, blood pressure reduction, sodium excretion and diuresis, and anti-myocardial hypertrophy and fibrosis by binding to NPR1, and can be used to treat diseases such as hypertension and chronic heart failure. NPR1 agonist antibodies have a longer pharmacokinetic (PK) time and more stable efficacy compared to recombinant natural receptor proteins; however, due to these characteristics, inappropriate dosage or other special circumstances can lead to potential side effects such as persistent hypotension and tachycardia. Therefore, developing specific binding molecules that bind to anti-NPR1 antibodies (e.g., antibodies that bind to anti-NPR1 antibodies) to neutralize anti-NPR1 antibodies and reverse side effects is crucial for ensuring safety. Summary of the Invention

[0005] In one respect, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody.

[0006] On the other hand, this disclosure provides a binding molecule that specifically binds to anti-NPR1 antibodies, reversing the hemodynamic effects of anti-NPR1 antibodies.

[0007] In some implementations, the binding molecule that specifically binds to the anti-NPR1 antibody, as described above, reverses the blood pressure reduction associated with the administration of the anti-NPR1 antibody.

[0008] In some implementations, the binding molecule, as described in the preceding one, neutralizes the activity of the anti-NPR1 antibody.

[0009] <Anti-NPR1 antibody>

[0010] In some embodiments, such as the binding molecule described in any of the preceding embodiments, the anti-NPR1 antibody is an NPR1 agonist antibody.

[0011] In some embodiments, as described in any of the preceding embodiments, the anti-NPR1 antibody 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 the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 99, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 100; or

[0012] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 101, 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: 102; wherein:

[0013] The sequence of SEQ ID NO: 99 is:

[0014] The sequence of SEQ ID NO: 100 is:

[0015] The sequence of SEQ ID NO: 101 is:

[0016] The sequence of SEQ ID NO: 102 is:

[0017] In some embodiments, as described in any of the preceding embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Chothia numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody, 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 of the anti-NPR1 antibody, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the Contact numbering rules.

[0018] In some embodiments, as described in any of the preceding embodiments, the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 93, HCDR2 comprises the amino acid sequence of SEQ ID NO: 94, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 98, wherein:

[0019] HCDR1 is: SYWMH SEQ ID NO: 93,

[0020] HCDR2 is: RIDPNSGVTKYNEKFKS SEQ ID NO: 94,

[0021] HCDR3 is: SGGLRRYFDV SEQ ID NO: 95.

[0022] LCDR1 is: SASSVSYMY SEQ ID NO: 96.

[0023] LCDR2 is: DTYNLAS SEQ ID NO: 97.

[0024] LCDR3 is: QQWSSYPPT SEQ ID NO: 98.

[0025] In some embodiments, such as the binding molecule described in the preceding one, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the anti-NPR1 antibody and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0026] In some embodiments, the binding molecule as described in any of the preceding claims, the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 99, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 100, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, the binding molecule as described in any of the preceding claims, the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 99, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 100.

[0027] In some embodiments, the binding molecule as described in any of the preceding claims, the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 101, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 102, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, the anti-NPR1 antibody, as described in any of the preceding embodiments, comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 101, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 102.

[0028] In some embodiments, as described in any of the preceding embodiments, the anti-NPR1 antibody is a monoclonal antibody. In some embodiments, as described in any of the preceding embodiments, the anti-NPR1 antibody is an IgG1 antibody. In some embodiments, the binding molecule as described in any of the preceding claims, the anti-NPR1 antibody comprises a heavy chain and a light chain, the heavy chain comprising SEQ ID NO: 1, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain comprising SEQ ID NO: 2, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, as described in any of the preceding embodiments, the heavy chain of the anti-NPR1 antibody comprises the amino acid sequence of SEQ ID NO: 1, and the light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, as described in any of the preceding embodiments, the amino acid sequence of the heavy chain of the anti-NPR1 antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 2. In some embodiments, as described in any of the preceding embodiments, the heavy chain of the anti-NPR1 antibody consists of amino acid residues 1-448 of SEQ ID NO: 1, and the light chain consists of amino acid residues 1-213 of SEQ ID NO: 2.

[0029] In some embodiments, as described in any of the preceding embodiments, the heavy chain of the anti-NPR1 antibody comprises SEQ ID NO: 3, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 4, or an amino acid sequence having 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, as described in any of the preceding embodiments, the heavy chain of the anti-NPR1 antibody comprises the amino acid sequence of SEQ ID NO: 3, and the light chain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, as described in any of the preceding embodiments, the amino acid sequence of the heavy chain of the anti-NPR1 antibody is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 4. In some embodiments, as described in any of the preceding embodiments, the heavy chain of the anti-NPR1 antibody consists of amino acid residues 1-448 of SEQ ID NO: 3, and the light chain consists of amino acid residues 1-213 of SEQ ID NO: 4.

[0030] In some embodiments, the anti-NPR1 antibody is Hu140H5L5-11 or SFM-140.

[0031] <Binding molecules>

[0032] On the other hand, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody, the binding molecule 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:

[0033] a. The heavy chain variable region comprises one, two, or three HCDR amino acid sequences from SEQ ID NO: 55, 53, 54, 56, 57, 58, or 59, and the light chain variable region comprises one, two, or three LCDR amino acid sequences from SEQ ID NO: 61, 60, or 62; or

[0034] The heavy chain variable region comprises one, two, or three HCDR amino acid sequences from SEQ ID NO: 32, and the light chain variable region comprises one, two, or three LCDR amino acid sequences from SEQ ID NO: 33; or

[0035] b. The heavy chain variable region comprises one, two, or three HCDR amino acid sequences from SEQ ID NO: 77, 76, or 78, and the light chain variable region comprises one, two, or three LCDR amino acid sequences from SEQ ID NO: 79, 80, 81, 82, or 83; or

[0036] The heavy chain variable region contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 34, and the light chain variable region contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 35.

[0037] On the other hand, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody, said binding molecule comprising a heavy chain variable region, wherein said heavy chain variable region comprises HCDR3, wherein:

[0038] a. The heavy chain variable region comprises the amino acid sequence of HCDR3 in SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59; or

[0039] The heavy chain variable region contains the amino acid sequence of HCDR3 in SEQ ID NO: 32; or

[0040] b. The heavy chain variable region comprises the amino acid sequence of HCDR3 in SEQ ID NO: 77, 76 or 78; or

[0041] The heavy chain variable region contains the HCDR3 amino acid sequence in SEQ ID NO: 34.

[0042] On the other hand, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody, said binding molecule comprising a light chain variable region, wherein said light chain variable region comprises LCDR3, wherein:

[0043] a. The light chain variable region comprises the amino acid sequence of LCDR3 in SEQ ID NO: 61, 60 or 62; or

[0044] The light chain variable region contains the amino acid sequence of LCDR3 in SEQ ID NO: 33; or

[0045] b. The light chain variable region comprises the amino acid sequence of LCDR3 in SEQ ID NO: 79, 80, 81, 82 or 83; or

[0046] The light chain variable region contains the amino acid sequence of LCDR3 in SEQ ID NO: 35.

[0047] On the other hand, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody, the binding molecule comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR3 and the light chain variable region comprises LCDR3, wherein:

[0048] a. The heavy chain variable region comprises the amino acid sequence of HCDR3 in SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of LCDR3 in SEQ ID NO: 61, 60 or 62; or

[0049] The heavy chain variable region comprises the amino acid sequence of HCDR3 in SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of LCDR3 in SEQ ID NO: 33; or

[0050] b. The heavy chain variable region comprises the amino acid sequence of HCDR3 in SEQ ID NO: 77, 76 or 78, and the light chain variable region comprises the amino acid sequence of LCDR3 in SEQ ID NO: 79, 80, 81, 82 or 83; or

[0051] The heavy chain variable region contains the HCDR3 amino acid sequence in SEQ ID NO: 34, and the light chain variable region contains the LCDR3 amino acid sequence in SEQ ID NO: 35.

[0052] On the other hand, this disclosure provides a binding molecule that binds to an anti-NPR1 antibody, the binding molecule 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:

[0053] a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 55, 53, 54, 56, 57, 58, or 59, 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: 61, 60, or 62; or

[0054] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 32, 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: 33; or

[0055] b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 77, 76, or 78, 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: 79, 80, 81, 82, or 83; or

[0056] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 34, 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: 35.

[0057] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0058] a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 55, 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: 61; or

[0059] b. The heavy chain variable regions HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 77, 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: 79.

[0060] In some embodiments, for the binding molecule as described in any preceding item, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the Kabat numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the IMGT numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the Chothia numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the AbM numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the binding molecule are defined according to the Contact numbering rules.

[0061] <CDR defined by Kabat numbering rules>

[0062] In some embodiments, for the binding molecule as described in any preceding item, the binding molecule 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:

[0063] a. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 105, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11, wherein:

[0064] LCDR1 is: KARX2X3VGTYVS SEQ ID NO: 105; wherein X2 is D, E, or Q, preferably D; X3 is E or D, preferably E; or

[0065] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 92, wherein:

[0066] LCDR3 is: QQVX1TLPRT SEQ ID NO: 92;

[0067] X1 is selected from N and Q, preferably N.

[0068] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0069] a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 6, HCDR2 contains the amino acid sequence of SEQ ID NO: 7, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 44, 43, 45, or 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0070] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17 or 73.

[0071] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0072] a. The amino acid sequences of HCDR1 in the heavy chain variable region are shown in SEQ ID NO: 6, HCDR2 in SEQ ID NO: 7, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of LCDR1 in the light chain variable region are shown in SEQ ID NO: 44, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0073] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 6, HCDR2 in SEQ ID NO: 7, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 43, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0074] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 6, HCDR2 in SEQ ID NO: 7, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 45, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0075] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 6, HCDR2 in SEQ ID NO: 7, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0076] b. The amino acid sequences of HCDR1 in the heavy chain variable region are shown in SEQ ID NO: 12, HCDR2 in SEQ ID NO: 13, and HCDR3 in SEQ ID NO: 14; the amino acid sequences of LCDR1 in the light chain variable region are shown in SEQ ID NO: 15, LCDR2 in SEQ ID NO: 16, and LCDR3 in SEQ ID NO: 17; or

[0077] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 12, HCDR2 in SEQ ID NO: 13, and HCDR3 in SEQ ID NO: 14. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 15, LCDR2 in SEQ ID NO: 16, and LCDR3 in SEQ ID NO: 73.

[0078] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0079] a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 6, HCDR2 contains the amino acid sequence of SEQ ID NO: 7, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 44, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0080] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17.

[0081] In some embodiments, the binding molecule as described in any preceding item, the binding molecule 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:

[0082] HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11.

[0083] In some embodiments, the binding molecule as described in any preceding item, the binding molecule 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:

[0084] HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, LCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17.

[0085] In some embodiments, the binding molecule as described in any preceding item, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the binding molecule and LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Kabat numbering rules.

[0086] <CDRs defined by the Chothia numbering rules>

[0087] In some embodiments, the binding molecule as described in any preceding item, the binding molecule 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:

[0088] a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 18, 46, or 47, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 44, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0089] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 18, 46, or 47, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 43, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0090] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 18, 46, or 47, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 45, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0091] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 18, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0092] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 74 or 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17; or

[0093] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 74 or 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 73.

[0094] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0095] a. The amino acid sequence of HCDR1 in the heavy chain variable region is as shown in SEQ ID NO: 18, 46 or 47, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 19, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 8; the amino acid sequence of LCDR1 in the light chain variable region is as shown in SEQ ID NO: 44, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 10, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 11; or

[0096] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 18, 46 or 47, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 19, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 8; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 43, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 10, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 11; or

[0097] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 18, 46 or 47, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 19, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 8; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 45, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 10, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 11; or

[0098] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 18, HCDR2 in SEQ ID NO: 19, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0099] b. The amino acid sequence of HCDR1 in the heavy chain variable region is as shown in SEQ ID NO: 74 or 20, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 21, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 14; the amino acid sequence of LCDR1 in the light chain variable region is as shown in SEQ ID NO: 15, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 17; or

[0100] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 74 or 20, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 21, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 14. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 15, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 16, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 73.

[0101] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0102] a. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18, HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; or

[0103] b. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 74, HCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, LCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17.

[0104] In some embodiments, the binding molecule as described in any of the preceding items, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the binding molecule and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Chothia numbering rules.

[0105] <CDRs defined by the IMGT numbering rules>

[0106] In some embodiments, the binding molecule as described in any of the preceding items, the binding molecule 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:

[0107] a. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, 51 or 52, HCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, LCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; or

[0108] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 22, 51, or 52, HCDR2 contains the amino acid sequence of SEQ ID NO: 23, and HCDR3 contains the amino acid sequence of SEQ ID NO: 24; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 48, LCDR2 contains the amino acid sequence of SEQ ID NO: 26, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0109] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 22, 51, or 52, HCDR2 contains the amino acid sequence of SEQ ID NO: 23, and HCDR3 contains the amino acid sequence of SEQ ID NO: 24; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 50, LCDR2 contains the amino acid sequence of SEQ ID NO: 26, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0110] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 22, HCDR2 contains the amino acid sequence of SEQ ID NO: 23, and HCDR3 contains the amino acid sequence of SEQ ID NO: 24; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 25, LCDR2 contains the amino acid sequence of SEQ ID NO: 26, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0111] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 75 or 27, HCDR2 contains the amino acid sequence of SEQ ID NO: 28, and HCDR3 contains the amino acid sequence of SEQ ID NO: 29; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 30, LCDR2 contains the amino acid sequence of SEQ ID NO: 31, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17; or

[0112] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 75 or 27, HCDR2 contains the amino acid sequence of SEQ ID NO: 28, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 30, LCDR2 contains the amino acid sequence of SEQ ID NO: 31, and LCDR3 contains the amino acid sequence of SEQ ID NO: 73.

[0113] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0114] a. The amino acid sequence of HCDR1 in the heavy chain variable region is as shown in SEQ ID NO: 22, 51 or 52, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 23, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 24; the amino acid sequence of LCDR1 in the light chain variable region is as shown in SEQ ID NO: 49, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 26, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 11; or

[0115] The amino acid sequence of HCDR1 in the heavy chain variable region is shown in SEQ ID NO: 22, 51 or 52; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 23; the amino acid sequence of HCDR3 is shown in SEQ ID NO: 24; the amino acid sequence of LCDR1 in the light chain variable region is shown in SEQ ID NO: 48; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 26; and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11; or

[0116] The amino acid sequence of HCDR1 in the heavy chain variable region is shown in SEQ ID NO: 22, 51 or 52; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 23; the amino acid sequence of HCDR3 is shown in SEQ ID NO: 24; the amino acid sequence of LCDR1 in the light chain variable region is shown in SEQ ID NO: 50; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 26; and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11; or

[0117] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 22, HCDR2 in SEQ ID NO: 23, and HCDR3 in SEQ ID NO: 24; the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 25, LCDR2 in SEQ ID NO: 26, and LCDR3 in SEQ ID NO: 11; or

[0118] b. The amino acid sequence of HCDR1 in the heavy chain variable region is as shown in SEQ ID NO: 75 or 27, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 28, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 29; the amino acid sequence of LCDR1 in the light chain variable region is as shown in SEQ ID NO: 30, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 31, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 17; or

[0119] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 75 or 27, the amino acid sequences of HCDR2 are shown in SEQ ID NO: 28, and the amino acid sequences of HCDR3 are shown in SEQ ID NO: 19. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 30, the amino acid sequences of LCDR2 are shown in SEQ ID NO: 31, and the amino acid sequences of LCDR3 are shown in SEQ ID NO: 73.

[0120] In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 of the binding molecule, as described in any of the preceding embodiments, are defined according to the IMGT numbering rules.

[0121] In some embodiments, the binding molecule as described in any of the preceding claims 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:

[0122] a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 22, HCDR2 contains the amino acid sequence of SEQ ID NO: 23, and HCDR3 contains the amino acid sequence of SEQ ID NO: 24; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 49, LCDR2 contains the amino acid sequence of SEQ ID NO: 26, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or

[0123] b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 75, HCDR2 contains the amino acid sequence of SEQ ID NO: 28, and HCDR3 contains the amino acid sequence of SEQ ID NO: 29, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 30, LCDR2 contains the amino acid sequence of SEQ ID NO: 31, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17.

[0124] In some embodiments, the binding molecule is an antibody. In some embodiments, the binding molecule is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the binding molecule is a chimeric antibody. In some embodiments, the binding molecule is a humanized antibody.

[0125] In some embodiments, the binding molecule as described in any of the preceding embodiments is an antibody fragment. In some embodiments, the binding molecule as described in any of the preceding embodiments is an antibody fragment selected from Fab, Fab′, F(ab′)2, Fd, Fv, scFv, dsFv, and dAb. In some embodiments, the binding molecule as described in any of the preceding embodiments is an antibody fragment selected from Fab, Fab′, F(ab′)2, Fv, scFv, and dsFv.

[0126] In some embodiments, the binding molecule is an antibody, as described in any of the preceding embodiments. In some embodiments, the binding molecule is a multispecific antibody. In some embodiments, the binding molecule is a bispecific antibody. In some embodiments, the binding molecule is a monoclonal antibody. In some embodiments, the binding molecule is a monospecific antibody.

[0127] In some embodiments, the binding molecule as described in any of the preceding embodiments is humanized, reversed mutation, modulated affinity, removed T cell epitopes, reduced antibody deamidation, and / or reduced antibody isomerization.

[0128] In some embodiments, the binding molecule as described in any of the preceding embodiments is obtained by removing / reducing TCE and has one or more variations in one or more CDRs, the variations resulting in a reduction in the immunogenicity of the binding molecule.

[0129] In some implementations, the binding molecule, as described in the preceding one, contains the frame region (FR) of the human antibody.

[0130] In some embodiments, the binding molecule as described in any of the preceding claims, wherein the heavy chain variable region of the binding molecule comprises FR1, FR2, and FR3 derived from IGHV4-30-4*01 or IGHV2-26*01, and FR4 derived from IGHJ6*01, and is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 6Q, 27F, 29L, 30T, 44G, 49G, 71K, 73N, 76S, 78V, and / or 80F; and / or the light chain variable region of the binding molecule comprises FR1, FR2, and FR3 derived from IGKV4-1*01, and FR4 derived from IGKJ4*01, and is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 9K, 43S, 85D, and / or 87H. In some embodiments, the binding molecule as described in any of the preceding embodiments, wherein the heavy chain variable region HCDR1 of the binding molecule comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, 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 1E, 6Q, 27F, 29L, 30T, 44G, 49G, 71K, 73N, 76S, 78V, and / or 80F; and the light chain variable region LCDR1 of the binding molecule comprises the amino acid sequence of SEQ ID NO: 44, 43, 45, or 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11, and the FR of the light chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 9K, 43S, 85D, and / or 87H. In some embodiments, the above-mentioned variable regions and CDRs are defined according to the Kabat numbering rules.

[0131] In some embodiments, the binding molecule as described in any of the preceding embodiments, wherein the heavy chain variable region of the binding molecule comprises FR1, FR2, and FR3 derived from IGHV1-69*02 and FR4 derived from IGHJ6*01, and is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 27Y, 28A, 40R, and / or 94G; and / or the light chain variable region of the binding molecule comprises FR1, FR2, and FR3 derived from IGKV1-39*01 and FR4 derived from IGKJ4*01, and is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 43T, 44V, and / or 71Y. In some embodiments, the binding molecule as described in any of the preceding embodiments, wherein the heavy chain variable region HCDR1 of the binding molecule comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, 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 27Y, 28A, 40R, and / or 94G; and the light chain variable region LCDR1 of the binding molecule comprises the amino acid sequence of SEQ ID NO: 15, LCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17 or 73, and the FR of the light chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 43T, 44V, and / or 71Y. In some embodiments, the above-mentioned variable regions and CDRs are defined according to the Kabat numbering rules.

[0132] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0133] a. The heavy chain variable region comprises SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and the light chain variable region comprises SEQ ID NO: 61, 60 or 62, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; or

[0134] The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 33, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; or

[0135] b. The heavy chain variable region comprises SEQ ID NO: 77, 76, or 78, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the light chain variable region comprises SEQ ID NO: 79, 80, 81, 82, or 83, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0136] The heavy chain variable region comprises SEQ ID NO: 34, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the light chain variable region comprises SEQ ID NO: 35, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0137] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0138] a. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61, 60 or 62; or

[0139] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 33; or

[0140] b. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, 76, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79, 80, 81, 82, or 83; or

[0141] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 34, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 35.

[0142] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0143] a. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0144] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0145] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or

[0146] b. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, 76, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or

[0147] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or

[0148] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or

[0149] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or

[0150] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 76, 77 or 78, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 83.

[0151] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0152] a. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0153] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0154] b. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, 76, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or

[0155] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or

[0156] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or

[0157] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or

[0158] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 76, 77 or 78, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 83.

[0159] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0160] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0161] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0162] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0163] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0164] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0165] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0166] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0167] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0168] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0169] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0170] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0171] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0172] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or

[0173] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 59, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 60.

[0174] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0175] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or

[0176] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or

[0177] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or

[0178] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or

[0179] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or

[0180] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or

[0181] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or

[0182] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or

[0183] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or

[0184] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or

[0185] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or

[0186] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or

[0187] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83; or

[0188] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83; or

[0189] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 78, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 83.

[0190] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0191] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 55, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0192] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0193] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0194] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0195] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 57, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0196] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0197] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 59, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0198] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0199] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0200] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 55, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0201] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0202] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 57, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0203] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60; or

[0204] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 59, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60.

[0205] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0206] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 79; or

[0207] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 79; or

[0208] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 79; or

[0209] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80; or

[0210] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80; or

[0211] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80; or

[0212] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81; or

[0213] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81; or

[0214] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81; or

[0215] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 82; or

[0216] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 82; or

[0217] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 82; or

[0218] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 83; or

[0219] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 83; or

[0220] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 83.

[0221] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0222] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or

[0223] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 77, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 79.

[0224] In some embodiments, the binding molecule as described in any of the preceding embodiments further 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 derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region is a heavy chain constant region derived from human IgG1. In some embodiments, the Fc region of the heavy chain constant region comprises L234A and L235A substitutions. In some embodiments, the light chain constant region is a light chain constant region derived from human κ or λ. In some embodiments, the light chain constant region is a light chain constant region derived from human κ. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63 or 37, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 38.

[0225] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0226] a. The heavy chain comprises SEQ ID NO: 66, 64, 65, 67, 68, 69 or 70, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it, and the light chain comprises SEQ ID NO: 72 or 71, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with it; or

[0227] The heavy chain comprises SEQ ID NO: 40, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 39, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; or

[0228] b. The heavy chain comprises SEQ ID NO: 90, 89, or 91, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the light chain comprises SEQ ID NO: 84, 85, 86, 87, or 88, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0229] The heavy chain comprises SEQ ID NO: 42, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the light chain comprises SEQ ID NO: 41, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0230] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0231] a. The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, 64, 65, 67, 68, 69 or 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 72 or 71; or

[0232] The heavy chain comprises the amino acid sequence of SEQ ID NO: 40, and the light chain comprises the amino acid sequence of SEQ ID NO: 39; or

[0233] b. The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, 89, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 84, 85, 86, 87, or 88; or

[0234] The heavy chain contains the amino acid sequence of SEQ ID NO: 42, and the light chain contains the amino acid sequence of SEQ ID NO: 41.

[0235] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0236] a. The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, 64, 65, 67, 68, 69 or 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0237] The heavy chain comprises the amino acid sequence of SEQ ID NO: 64, 65, 66, 67, 68, 69 or 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0238] b. The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, 89, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 84; or

[0239] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, 90, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0240] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, 90, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 86; or

[0241] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, 90, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 87; or

[0242] The heavy chain contains the amino acid sequence of SEQ ID NO: 89, 90 or 91, and the light chain contains the amino acid sequence of SEQ ID NO: 88.

[0243] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0244] The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0245] The heavy chain comprises the amino acid sequence of SEQ ID NO: 64, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0246] The heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0247] The heavy chain comprises the amino acid sequence of SEQ ID NO: 67, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0248] The heavy chain comprises the amino acid sequence of SEQ ID NO: 68, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0249] The heavy chain comprises the amino acid sequence of SEQ ID NO: 69, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0250] The heavy chain comprises the amino acid sequence of SEQ ID NO: 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0251] The heavy chain comprises the amino acid sequence of SEQ ID NO: 64, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0252] The heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0253] The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0254] The heavy chain comprises the amino acid sequence of SEQ ID NO: 67, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0255] The heavy chain comprises the amino acid sequence of SEQ ID NO: 68, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0256] The heavy chain comprises the amino acid sequence of SEQ ID NO: 69, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or

[0257] The heavy chain contains the amino acid sequence of SEQ ID NO: 70, and the light chain contains the amino acid sequence of SEQ ID NO: 71.

[0258] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0259] The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, and the light chain comprises the amino acid sequence of SEQ ID NO: 84; or

[0260] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 84; or

[0261] The heavy chain comprises the amino acid sequence of SEQ ID NO: 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 84; or

[0262] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0263] The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0264] The heavy chain comprises the amino acid sequence of SEQ ID NO: 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 85; or

[0265] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 86; or

[0266] The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, and the light chain comprises the amino acid sequence of SEQ ID NO: 86; or

[0267] The heavy chain comprises the amino acid sequence of SEQ ID NO: 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 86; or

[0268] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 87; or

[0269] The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, and the light chain comprises the amino acid sequence of SEQ ID NO: 87; or

[0270] The heavy chain comprises the amino acid sequence of SEQ ID NO: 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 87; or

[0271] The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 88; or

[0272] The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, and the light chain comprises the amino acid sequence of SEQ ID NO: 88; or

[0273] The heavy chain contains the amino acid sequence of SEQ ID NO: 91, and the light chain contains the amino acid sequence of SEQ ID NO: 88.

[0274] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0275] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0276] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0277] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0278] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 67, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0279] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0280] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 69, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0281] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 70, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0282] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0283] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0284] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0285] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 67, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0286] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0287] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 69, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71; or

[0288] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 70, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71.

[0289] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0290] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84; or

[0291] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84; or

[0292] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84; or

[0293] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 85; or

[0294] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 85; or

[0295] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 85; or

[0296] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 86; or

[0297] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 86; or

[0298] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 86; or

[0299] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 87; or

[0300] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 87; or

[0301] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 87; or

[0302] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 88; or

[0303] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 88; or

[0304] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 88.

[0305] In some embodiments, the binding molecule as described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0306] The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or

[0307] The heavy chain contains the amino acid sequence of SEQ ID NO: 90, and the light chain contains the amino acid sequence of SEQ ID NO: 84.

[0308] In some embodiments, the binding molecules provided in this disclosure are used at EC concentrations of less than 0.1 ng / mL (e.g., less than 0.05 ng / mL, less than 0.035 ng / mL, less than 0.033 ng / mL, less than 0.020 ng / mL, less than 0.015 ng / mL, less than 0.010 ng / mL, less than 0.005 ng / mL). 50 The value binds to anti-NPR1 antibodies (e.g., SFM-140), the EC 50 The value was measured using ELISA.

[0309] In some embodiments, the binding molecule provided in this disclosure binds to an anti-NPR1 antibody (e.g., SFM-140) with a KD value of less than 1.00E-8M (e.g., less than 1.00E-9M, less than 5.00E-10M, less than 3.00E-10M, less than 9.00E-11M, less than 8.00E-11M), said KD value being measured by Biacore.

[0310] In some embodiments, the binding molecules provided in this disclosure are used at an IC50 concentration of less than 1.0 ng / mL (e.g., less than 0.5 ng / mL, less than 0.04 ng / mL, less than 0.3 ng / mL, less than 0.2 ng / mL). 50 The IC50 value blocks the binding of anti-NPR1 antibodies (e.g., SFM-140) to the NPR1-overexpressing CHO-K1 recombinant cell line hNPR1-CHO-K1. 50 The value was measured using ELISA.

[0311] In some embodiments, the binding molecules provided in this disclosure are used at IC50 concentrations of less than 50 ng / mL (e.g., less than 20 ng / mL, less than 10 ng / mL, less than 5 ng / mL, less than 4.8 ng / mL, less than 4.5 ng / mL, less than 4.3 ng / mL). 50 The IC50 value blocks the binding of anti-NPR1 antibodies (e.g., SFM-140) to the NPR1-overexpressing CHO-K1 recombinant cell line hNPR1-CHO-K1. 50 The value was measured using FACS.

[0312] In some implementations, the binding molecules provided in this disclosure can block the production of cGMP by hNPR1-CHO-K1 cells.

[0313] In some embodiments, this disclosure also provides a separate binding molecule that competitively binds an anti-NPR1 antibody or its epitope to a binding molecule as described in any of the preceding embodiments.

[0314] In some implementations, the anti-NPR1 antibody is an NPR1 agonist antibody.

[0315] In some embodiments, the anti-NPR1 antibody 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:

[0316] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 99, 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: 100; or

[0317] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 101, 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: 102.

[0318] In some embodiments, the anti-NPR1 antibody 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:

[0319] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 93, HCDR2 contains the amino acid sequence of SEQ ID NO: 94, and HCDR3 contains the amino acid sequence of SEQ ID NO: 95, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 96, LCDR2 contains the amino acid sequence of SEQ ID NO: 97, and LCDR3 contains the amino acid sequence of SEQ ID NO: 98.

[0320] In some embodiments, the anti-NPR1 antibody 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:

[0321] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 99, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 100; or

[0322] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 101, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 102.

[0323] In some embodiments, the anti-NPR1 antibody is a monoclonal antibody. In some embodiments, the anti-NPR1 antibody is an IgG1 antibody. In some embodiments, the heavy chain amino acid sequence of the anti-NPR1 antibody is as shown in SEQ ID NO: 1, and the light chain amino acid sequence is as shown in SEQ ID NO: 2. In some embodiments, the heavy chain amino acid sequence of the anti-NPR1 antibody is as shown in SEQ ID NO: 3, and the light chain amino acid sequence is as shown in SEQ ID NO: 4.

[0324] In some embodiments, the anti-NPR1 antibody is Hu140H5L5-11 or SFM-140.

[0325] On the other hand, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the binding molecule as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

[0326] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the binding molecules as described in any of the preceding claims, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the binding molecules as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the binding molecules as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 1%-99% of the binding molecules as described in any of the preceding claims. In some embodiments, the pharmaceutical composition contains 2%-98% of the binding molecules as described in any of the preceding claims.

[0327] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0328] On the other hand, this disclosure provides a nucleic acid (e.g., an isolated nucleic acid) that encodes a binding molecule as described in the previous one.

[0329] On the other hand, this disclosure provides a carrier containing nucleic acids as described in the previous one.

[0330] On the other hand, this disclosure provides a host cell containing nucleic acids as described in the previous one.

[0331] On the other hand, this disclosure provides a method for preparing a binding molecule as described in the preceding claim, the method comprising culturing a host cell as described in the preceding claim in a culture medium to accumulate the binding molecule as described in the preceding claim, and recovering the binding molecule from the culture.

[0332] On the other hand, this disclosure provides a method for reversing side effects associated with the administration of an anti-NPR1 antibody, the method comprising administering to an individual in need a binding molecule as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims. In some embodiments, the side effects are related to the production of cGMP. In some embodiments, the side effects are hemodynamically related. In some embodiments, the side effects are hypotension or tachycardia. In some embodiments, the hypotension is persistent hypotension.

[0333] On the other hand, this disclosure provides the use of the conjugating molecule as described in any of the preceding claims, or the pharmaceutical composition as described in any of the preceding claims, in the preparation of a medicament. In some embodiments, the medicament is used to reverse side effects associated with the administration of the anti-NPR1 antibody in individuals of need. In some embodiments, the side effects are related to the production of cGMP. In some embodiments, the side effects are hemodynamically related. In some embodiments, the side effects are hypotension or tachycardia. In some embodiments, the hypotension is persistent hypotension.

[0334] On the other hand, this disclosure provides a conjugating molecule as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, for use as a medicament. In some embodiments, the medicament is used to reverse side effects associated with the administration of an anti-NPR1 antibody in individuals in need. In some embodiments, the side effects are related to the production of cGMP. In some embodiments, the side effects are hemodynamically related. In some embodiments, the side effects are hypotension or tachycardia. In some embodiments, the hypotension is persistent hypotension.

[0335] On the other hand, this disclosure provides a pharmaceutical composition comprising a binding molecule as described in any of the preceding claims and a second therapeutic agent. In some embodiments, the composition is used for a method of reversing side effects associated with the administration of an anti-NPR1 antibody in an individual in need. In some embodiments, the side effects are related to the production of cGMP. In some embodiments, the side effects are hemodynamically related. In some embodiments, the side effects are hypotension or tachycardia. In some embodiments, the hypotension is persistent hypotension. In some embodiments, the individual suffers from an NPR1-related disease or condition.

[0336] Invention Details

[0337] the term

[0338] 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.

[0339] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0340] 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.

[0341] 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).

[0342] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0343] The term "NPR1" refers to natriuretic peptide receptor 1, also known as atrial natriuretic peptide receptor type A (ANP-A, ANPR-A, or NPR-A) and guanylate cyclase A (GC-A), belonging to the cell surface guanylate cyclase receptor family. It is a homodimeric transmembrane guanylate cyclase that catalyzes cGMP synthesis. The term "NPR1" refers to naturally occurring NPR1 proteins (e.g., but not limited to their precursors, mature, modified, or spliced ​​variants). Human NPR1 (SEQ ID NO: 5) protein is from Uniprot: P16066. Amino acid sequences of NPR1 molecules from non-human species (e.g., mice, rats, monkeys, rabbits, dogs, pigs, etc.) are available from public resources, e.g., cynomolgus monkey NPR1 protein NCBI: XP-005541809.1; rat NPR1 protein, Uniprot: P18910. Although this article provides a specific database accession number, those skilled in the art will understand that NPR1 as referred to in this article also covers the corresponding sequence reported in other databases or literature.

[0344] 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.

[0345] The term "amino acid mutation" encompasses 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 substitutions include substitutions with non-naturally occurring amino acids or with 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 may be represented by the format "position + amino acid residue". For example, 9K indicates that the amino acid residue at the 9th site is K. D9K indicates that the amino acid residue at the 9th site has mutated from D to K. When the residue at a specific site is defined in the claim using the format "position + amino acid residue", the original residue at that site does not limit the scope of protection.

[0346] The terms “binding molecule,” “binding agent,” “reversal agent,” “binding and / or reversal agent,” “activator,” and “antidote” are used interchangeably, and in the context of “antibody that specifically binds to NPR1 (anti-NPR1 antibody) or a binding molecule that binds to an anti-NPR1 antibody,” the term “binding molecule” encompasses a variety of molecules that specifically bind to antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by fusing the two, as well as any molecule comprising the aforementioned antibodies, peptides, and antibody fusion proteins, provided they exhibit the desired antigen-binding activity. Exemplarily, the binding molecule herein is an antibody. In some embodiments, the binding molecule is capable of reversing (e.g., partially reversing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%) the antihypertensive effects of an anti-NPR1 antibody (e.g., any antibody disclosed in patent CN202310675148.4; for example, antibody Hu140H5L5-11 or SFM-140). In some embodiments, the binding molecule is capable of reversing (e.g., partially reversing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%) the effect of anti-NPR1 antibodies (e.g., any antibody disclosed in patent CN202310675148.4; e.g., antibody Hu140H5L5-11 or SFM-140) on blood pressure. In some embodiments, the binding molecule is capable of reversing (e.g., partially reversing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%) the effect of anti-NPR1 antibodies (e.g., any antibody disclosed in patent CN202310675148.4; e.g., antibody Hu140H5L5-11 or SFM-140) on cGMP. In a further specific aspect provided herein, the binding molecule is capable of blocking the binding of anti-NPR1 antibodies to their antigens (e.g., NPR1). As a set of non-limiting examples, the binding molecule is capable of binding to one or more anti-NPR1 antibodies described herein. As a set of non-limiting examples, the binding molecule is capable of binding to an anti-NPR1 antibody that competes with one or more anti-NPR1 antibodies described herein. As a set of non-limiting examples, the binding molecule is capable of binding to an anti-NPR1 antibody that binds to the same epitope as one or more anti-NPR1 antibodies described herein.In one particular aspect, as used herein, the terms “anti-Hu140H5L5-11,” “anti-Hu140H5L5-11 antibody,” “anti-Hu140H5L5-11 humanized antibody,” “binding molecule specifically binding to Hu140H5L5-11,” “Hu140H5L5-11 antagonist,” “Hu140H5L5-11 binder and / or antagonist,” “Hu140H5L5-11 activator,” and “Hu140H5L5-11 antidote,” etc., are used interchangeably and refer to a binding molecule or antagonist that specifically binds to the anti-NPR1 antibody Hu140H5L5-11. Non-limiting examples of anti-Hu140H5L5-11 humanized antibodies are described in this disclosure. In one particular aspect, an anti-NPR1 antibody can be any known antibody, for example, it can be the antibody disclosed in patent CN202310675148.4 (which is incorporated herein by reference in its entirety).

[0347] 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 binding fragments (or binding portions), as long as they exhibit the desired antibody binding activity.

[0348] The term "binding molecule" is used in the broadest sense to encompass molecules that specifically bind to antibodies, including but not limited to antibodies, other peptides with antibody-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 antibody-binding activity. The antibody-binding molecules described herein comprise a variable region (VH) and a variable region (VL), which together constitute the antibody-binding domain. Exemplarily, the antibody-binding molecules described herein are monoclonal antibodies, monospecific antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies).

[0349] 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 known as the 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 known as the variable light domain or light chain variable domain), followed by a constant light domain (light chain constant region, CL).

[0350] 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.

[0351] 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.

[0352] 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 various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0353] Table 1. Relationship between CDR numbering systems

[0354] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0355] 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 an antigen (which may be an antibody in this disclosure) that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, Fd, F(ab′)2, and single-domain antibodies (sdAb, such as VH, VL, VHH, or V). HH ), single-chain Fab (scFab), biantibody, linear antibody, single-chain antibody (e.g., scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0356] The term “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 or VL or VHH), single-chain Fab (scFab), single-chain antibodies (e.g., scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0357] 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 engineered Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. 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 deleting the C-terminal lysine (residue 447 according to the EU numbering system) or by deleting 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.

[0358] 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.

[0359] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0360] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0361] 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.

[0362] 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).

[0363] The terms "human 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 ​​residues 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 generated in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0364] 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.

[0365] 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).

[0366] 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).

[0367] 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.

[0368] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, 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 antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0369] The term "antigen" refers to a molecule or molecular part that can be bound by binding molecules such as antigen-binding proteins (including, for example, antibodies). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0370] 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 discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the antibody loses binding to the conformational epitope. 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, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0371] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a particular antigen or epitope with a higher affinity than to other antigens or epitopes. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0372] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope in the manner described above for specific binding.

[0373] The terms “anti-NPR1 antibody,” “NPR1 antibody,” and “antibody that binds to NPR1” are used interchangeably in this document and refer to antibodies that can bind to NPR1 with sufficient affinity.

[0374] The terms “antibody that binds to NPR1 antibody,” “antibody that binds to anti-NPR1 antibody,” “antibody that specifically binds to anti-NPR1 antibody,” “NPR1 neutralizing antibody,” and “anti-idiotype antibody that binds to NPR1 antibody” are used interchangeably in this document and refer to antibodies that can bind to anti-NPR1 antibody with sufficient affinity.

[0375] The term "anti-idiotype antibody" refers to an antibody or antibody-binding fragment (e.g., a Fab fragment; e.g., a dAb; e.g., a single-arm antibody) that specifically binds to the antigen-binding region of another antibody (e.g., any known anti-NPR1 antibody or its antigen-binding fragment; e.g., any anti-NPR1 antibody or its antigen-binding fragment; e.g., Hu140H5L5-11 or SFM-140). Anti-idiotype antibodies are typically generated against the antigen-binding region or complementarity-determining region (CDR) (idiotype) of the target antibody. Anti-specific antibodies can be generated by the various methods described above, see, for example, Pan et al., 1995, FASEB J.9:43-49. Any reversal agents and / or binding molecules described herein may also be described as anti-idiotype antibodies.

[0376] The term "NPR1 agonist" refers to a molecule that activates, increases, or enhances NPR1 activity or stabilizes the activating conformation of NPR1. In some embodiments, the term "NPR1 agonist" refers to an antibody that specifically binds to NPR1 and activates or increases at least one biological activity of NPR1. In some embodiments, the NPR1 agonist may bind to NPR1 in the presence or absence of a ligand (e.g., ANP or BNP). In some embodiments, the biological activity includes, but is not limited to, reducing or lowering blood pressure in an individual upon administration of the NPR1 agonist antibody. In some embodiments, the biological activity includes hemodynamic changes (e.g., decreased blood pressure) in an individual suffering from a disease or condition such as hypertension or heart failure. The term includes, for example, NPR1 agonist antibodies as defined in CN202310675148.4.

[0377] The term "reversal" includes raising blood pressure in an individual whose blood pressure had been lowered due to administration of an NPR1 agonist. The increase in blood pressure can be measured using any standard blood pressure assessment means known in the art, such as a sphygmomanometer. The increase can be to the level prior to treatment with the agonist antibody or to a level that results in sufficient hemodynamic stability. Hemodynamic effects can include indirect effects, effects associated with the reduction in blood pressure. These effects can also be reversed using the binding molecules disclosed herein. Hemodynamic effects mentioned herein can include physiological parameters such as blood pressure and heart rate, or clinical signs such as dizziness, lightheadedness, blurred vision, nausea, and fatigue. In some embodiments, the binding molecule specifically binds to an NPR1 agonist and reverses the hemodynamic effects induced by the NPR1 agonist. In some embodiments, the binding molecule includes an antibody that specifically binds to an anti-NPR1 antibody as disclosed herein.

[0378] 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.

[0379] 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).

[0380] 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.

[0381] 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-naturally occurring, 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, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise stated, a particular 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.

[0382] 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.

[0383] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions; wherein, when performing optimal alignment of two sequences, gaps are introduced where necessary to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered part of the 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, such as 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.

[0384] 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.

[0385] 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 passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, 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 stipitis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *C. polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium graminearum*, *Fusarium venenatum*, *Physcomitrella patens*, *Neurospora crassa*, and *Yarrowia lipolytica*.

[0386] As used in this disclosure, 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 therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny will have identical DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells.

[0387] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0388] The term "pharmaceutical composition" means a mixture containing one or more of the binding molecules described herein and other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0389] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0390] 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.

[0391] "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.

[0392] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. 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 and 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 cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0393] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired 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 prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0394] The terms "recurrence," "relapse," and "relapsed" refer to the recovery of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0395] The terms "refractory" or "resistant" refer to cancers or diseases that do not respond to treatment.

[0396] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such 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 embodiments, 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, particularly 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 administered 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. A complete therapeutic or preventative effect may not occur after the administration of a single dose, but may occur after the administration of 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.

[0397] This disclosure of the binding molecules

[0398] This disclosure provides a binding molecule that can reverse hypotension or tachycardia caused by administration of anti-NPR1 antibodies, and also has many advantageous properties such as good therapeutic activity, safety, pharmacokinetic properties and drugability (e.g., solubility, viscosity, purity and stability).

[0399] Exemplary binding molecules (e.g., antibodies that specifically bind to anti-NPR1 antibodies).

[0400] For example, the binding molecule disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0401] a. The amino acid sequences of HCDR1 in the heavy chain variable region are shown in SEQ ID NO: 6, HCDR2 in SEQ ID NO: 7, and HCDR3 in SEQ ID NO: 8; the amino acid sequences of LCDR1 in the light chain variable region are shown in SEQ ID NO: 44, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 11; or

[0402] b. The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 12, HCDR2 are shown in SEQ ID NO: 13, and HCDR3 are shown in SEQ ID NO: 14; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 15, LCDR2 are shown in SEQ ID NO: 16, and LCDR3 are shown in SEQ ID NO: 17.

[0403] For example, the binding molecule disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0404] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 55, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 61; or

[0405] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 79.

[0406] For example, the binding molecule disclosed herein comprises a heavy chain and a light chain, wherein:

[0407] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72; or

[0408] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 90, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84.

[0409] antibody structure

[0410] In some implementations, the antibodies provided herein are full-length antibodies.

[0411] In some implementations, the antibodies provided herein are antibody fragments.

[0412] In some embodiments, the antibody 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 antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to 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.

[0413] In some implementations, the antibody 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, which can be the same or different antigens.

[0414] In some embodiments, the antibody 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 antibody 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.

[0415] In some implementations, the antibody fragment is an Fv fragment composed of the VH and VL domains of a single arm of the antibody.

[0416] In some embodiments, the antibody fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody 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 antibody from which it is derived. 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 contain VL-linker-VH or may contain VH-linker-VL.

[0417] In some embodiments, the antibody fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0418] In some implementations, the antibody 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.

[0419] In some embodiments, the antibodies 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.

[0420] In some embodiments, the antibody 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 the non-human antibody (e.g., an antibody providing the CDR sequence).

[0421] Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

[0422] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); mature human (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0423] Variants of the bound molecules

[0424] In some embodiments, amino acid sequence variants of the binding molecules provided herein are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as the ability to bind to anti-NPR1 antibodies.

[0425] Replace, insert, and delete variants

[0426] In some embodiments, antibody 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 2 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 2 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 antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0427] Table 2. Substitution of amino acids

[0428] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0429] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0430] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0431] (3) Acidic: Asp, Glu;

[0432] (4) Alkaline: His, Lys, Arg;

[0433] (5) Residues that affect chain orientation: Gly, Pro;

[0434] (6) Aromatic: Trp, Tyr, Phe.

[0435] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0436] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a 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 immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, 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, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations 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-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody 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.

[0437] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) can be made to 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 either unchanged or contains no more than one, two, or three amino acid substitutions.

[0438] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan 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 if the antibody-antigen interaction 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 antibody and antigen can be identified by studying the crystal structure of the antigen-antibody 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.

[0439] 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 antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

[0440] Recombination method

[0441] The binding molecules can be generated using recombination methods. For these methods, one or more isolated nucleic acids encoding the binding molecule are provided.

[0442] In some embodiments, this disclosure provides isolated nucleic acids encoding the binding molecules as described above. Such nucleic acids can be derived from independently encoding any of the aforementioned polypeptide chains. In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, 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 a host cell containing nucleic acid encoding the polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the binding molecule from the host cell (or host cell culture medium).

[0443] To generate binding molecules through recombination, nucleic acids encoding proteins 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 recombination methods or obtained through chemical synthesis.

[0444] Suitable host cells for cloning or expressing vectors encoding binding molecules 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. After expression, the expression can be separated from the bacterial cell paste in a soluble fraction and further purified.

[0445] 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, US 6040498, US6420548, US 7125978, 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. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0446] Measurement

[0447] The binding molecules provided 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 one aspect, the activity of the binding molecules disclosed herein can be tested, for example, by known methods such as ELISA, Biacore, FACS, etc.

[0448] Treatment methods and routes of administration

[0449] Any of the binding molecules provided in this disclosure may be used for therapeutic purposes. In another aspect, the binding molecules provided in this disclosure may be used in the manufacture or preparation of a medicament. In some embodiments, the disease is a disease or condition associated with NPR1.

[0450] In another aspect, pharmaceutical compositions comprising the said binding molecules are provided, for example, for any of the pharmaceutical uses or treatments described above. In some embodiments, the pharmaceutical composition comprises any binding molecule provided herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0451] The conjugating molecules disclosed herein can be used alone or in combination with other agents for treatment. For example, the antibodies disclosed herein can be administered co-administered with at least one additional therapeutic agent.

[0452] The conjugated molecules (and any other therapeutic agents) 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.

[0453] The conjugating molecules 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 conjugating molecules 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.

[0454] For the prevention or treatment of disease, the appropriate dosage of the combined molecule 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 preventive 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.

[0455] Products

[0456] 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 a binding molecule of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition, wherein the composition comprises a binding molecule of this disclosure; and (b) a second container containing a composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise 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 additional buffers, diluents, filters, needles, and syringes. Detailed Implementation

[0457] Example

[0458] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0459] Example 1. Preparation of anti-human NPR1 antibody

[0460] PCT / CN2024 / 098066 (Application date: 2024-06-07; Priority patent application number: CN202310675148.4) is included herein by reference in its entirety.

[0461] Anti-human NPR1 antibody Hu140H5L5-11 was prepared according to the method disclosed in patent PCT / CN2024 / 098066, wherein:

[0462] Hu140H5L5-11 is used as both an immunogen and a detection reagent. Its specific sequence is as follows:

[0463] Heavy chain of Hu140H5L5-11:

[0464] The light chain of Hu140H5L5-11:

[0465] The anti-human NPR1 murine antibody (SFM-140) was used as a detection reagent, and its specific sequence is as follows:

[0466] Heavy chain of SFM-140:

[0467] SFM-140 light chain:

[0468] Example 2. Preparation of NPR1 recombinant cell line

[0469] Using UniProt or NCBI NPR1 antigen (human NPR1 protein, UniProt number: P16066) as a template for NPR1, a publicly available recombinant cell line for detection was designed, wherein the recombinant cell line for detection is the CHO-K1 cell line that overexpresses the full-length human NPR1 protein.

[0470] Human NPR1 full-length amino acid sequence:

[0471] Note:

[0472] Human NPR1 is a transmembrane protein that exists as a homodimer on the cell membrane surface.

[0473] The double-underlined portion represents the NPR1 extracellular domain (33 to 473).

[0474] The dotted-dash area represents the transmembrane domain (474 ​​to 494).

[0475] The underlined portion represents the intracellular region (Cytoplasmic domain: 495 to 1061);

[0476] The unlabeled portion is the signal peptide.

[0477] CHO-K1 cells were stabilized using the above protein, and after two weeks of pressure selection, subcloning was performed to select single clones. The recombinant cell line with high expression of hNPR1-CHO-K1 was obtained by FACS detection and used for affinity and in vitro function testing.

[0478] Example 3. Preparation and screening of antibodies conjugated to anti-NPR1 antibodies

[0479] Anti-NPR1 antibody Hu140H5L5-11 (SEQ ID NO: 1 and SEQ ID NO: 2) was used as an immunoassay reagent. Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) was used as an adjuvant for cross-immunization of mice. After primary and booster immunization, mice with high or plateauing antibody titers in their serum were selected for spleen cell fusion.

[0480] After fusion, the hybridoma culture supernatant was analyzed and screened based on the hybridoma cell growth density and the results of protein-based binding and blocking assays. High-activity monoclonal hybridoma cell lines were selected, and the cells were promptly expanded, cryopreserved, and subjected to one or two subcloning processes until single-cell clones were obtained. Single cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Ambion, 15596026) (following the manufacturer's instructions) and reverse transcribed (PrimeScript). TM IV cDNA synthesis mix, Takara, cat#6215A). The 1st strand cDNA synthesis reaction solution obtained from reverse transcription was used as a template for PCR reaction (Taq, 10×PCR buffer, dNTP mix, Takara, cat#R001) for PCR amplification followed by sequencing. The amino acid sequences of the CDR and variable region of the screened monoclonal hybridoma cell lines are as follows:

[0481] The heavy and light chain CDR region sequences of the murine antibodies SFM-16-2 and SFM-81-1 are shown in the table below:

[0482] Table 3. CDR sequences of murine antibodies

[0483] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0484] Table 4. CDR sequences of murine antibodies

[0485] Note: The CDRs in the table are CDRs determined according to the Chothia numbering system.

[0486] Table 5. CDR sequences of murine antibodies

[0487] Note: The CDRs in the table are CDRs determined according to the IMGT numbering system. The variable area sequence is as follows:

[0488] SFM-16-2 Heavy Chain Variable Region

[0489] SFM-16-2 Light Chain Variable Zone

[0490] SFM-81-1 Heavy Chain Variable Region

[0491] SFM-81-1 Light Chain Variable Zone

[0492] Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The underlined part is the CDR sequence determined according to the Kabat numbering system, and the ununderlined part is the FR sequence.

[0493] The heavy chain variable region and light chain variable region of murine antibodies SFM-16-2 and SFM-81-1 were recombined with the heavy chain constant region hIgG1-1 and the light chain constant region CL (human κ light chain constant region), respectively, to obtain full-length chimeric antibodies.

[0494] CH1:

[0495] hIgG1-1 (containing L234A and L235A mutations):

[0496] >CL:

[0497] CHI-SFM-16-2 Light Chain:

[0498] CHI-SFM-16-2 Heavy Chain:

[0499] CHI-SFM-81-1 Light Chain:

[0500] CHI-SFM-81-1 Heavy Chain:

[0501] Note: Single underline: CDR; italics: constant region; where CDR is based on Kabat numbering.

[0502] Example 4. Humanization design of mouse antibody SFM-16-2

[0503] By comparing the genetic sequence of the heavy and light chain variable regions with the IMGT human antibody germline gene database using MOE software, a germline gene of the heavy and light chain variable region with high homology to the murine antibody SFM-16-2 was selected as a template. The CDRs of this murine antibody were then 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.

[0504] FR1, FR2, and FR3 of IGKV4-1*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region; FR1, FR2, and FR3 of IGHV4-30-4*01 or IGHV2-26*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region. Optionally, amino acid residues at positions 9, 27, 28, 43, 85, and / or 87 of the light chain variable region were substituted; and / or amino acid residues at positions 1, 6, 27, 29, 30, 44, 49, 71, 73, 76, 78, and / or 80 of the heavy chain variable region were substituted.

[0505] Table 6. Human-centered design of SFM-16-2

[0506] Note: P43S indicates that, according to the Kabat numbering system, the 43rd P is mutated to S, and so on.

[0507] Table 7-1. CDR of SFM-16-2 humanized antibody

[0508] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0509] Table 7-2. CDR of SFM-16-2 humanized antibody

[0510] Note: The CDRs in the table are CDRs determined according to the Chothia numbering system.

[0511] Table 8. CDRs of SFM-16-2 humanized antibody

[0512] Note: The CDRs in the table are CDRs determined according to the IMGT numbering system.

[0513] The light chain variable region and heavy chain variable region sequences of the Hu-SFM-16-2 humanized antibody are as follows:

[0514] >Hu-SFM-16-2-VH1

[0515] >Hu-SFM-16-2-VH2

[0516] >Hu-SFM-16-2-VH3

[0517] >Hu-SFM-16-2-VH4

[0518] >Hu-SFM-16-2-VH5

[0519] >Hu-SFM-16-2-VH6

[0520] >Hu-SFM-16-2-VH7

[0521] >Hu-SFM-16-2-VL1

[0522] >Hu-SFM-16-2-VL2

[0523] >Hu-SFM-16-2-VL3

[0524] Note: In the above sequences, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the non-underlined part is the FR sequence; the double-underlined bold part is the amino acid point mutation.

[0525] The heavy chain variable region and light chain variable region of the SFM-16-2 humanized antibody were recombined with the heavy chain constant region hIgG1-2 (SEQ ID NO: 63) and the light chain constant region CL (human κ light chain constant region) (SEQ ID NO: 38), respectively, to obtain the humanized antibody shown in Table 9 below.

[0526] Table 9. Humanized Antibodies for SFM-16-2

[0527] Note: In the table, “Hu-SFM-16-2-L1H1” indicates that the heavy chain variable region is Hu-SFM-16-2-VH1 (SEQ ID NO: 53), the light chain variable region is Hu-SFM-16-2-VL1 (SEQ ID NO: 60), and the heavy chain constant region is as shown in SEQ ID NO: 63, and the light chain constant region is as shown in SEQ ID NO: 38, which is a humanized antibody. Others are deduced by analogy.

[0528] For example, the modified SFM-16-2 humanized antibody is shown in the table below:

[0529] Table 10. Humanized Antibodies for SFM-16-2

[0530] hIgG1-2- (containing L234A and L235A mutations):

[0531] For example, the full-length sequence of the Hu-SFM-16-2 antibody is as follows:

[0532] Heavy chain sequences of Hu-SFM-16-2-L1H1 and Hu-SFM-16-2-L2H1

[0533] Heavy chain sequences of Hu-SFM-16-2-L1H2 and Hu-SFM-16-2-L2H2

[0534] Heavy chain sequences of Hu-SFM-16-2-L1H3 and Hu-SFM-16-2-L2H3

[0535] Heavy chain sequences of Hu-SFM-16-2-L1H4 and Hu-SFM-16-2-L2H4

[0536] Heavy chain sequences of Hu-SFM-16-2-L1H5 and Hu-SFM-16-2-L2H5

[0537] Heavy chain sequences of Hu-SFM-16-2-L1H6 and Hu-SFM-16-2-L2H6

[0538] Heavy chain sequences of Hu-SFM-16-2-L1H7 and Hu-SFM-16-2-L2H7

[0539] Light chain sequences of Hu-SFM-16-L1H1, Hu-SFM-16-2-L1H2, Hu-SFM-16-2-L1H3, Hu-SFM-16-2-L1H4, Hu-SFM-16-2-L1H5, Hu-SFM-16-2-L1H6, and Hu-SFM-16-2-L1H7

[0540] Light chain sequences of Hu-SFM-16-L2H1, Hu-SFM-16-2-L2H2, Hu-SFM-16-2-L2H3, Hu-SFM-16-2-L2H4, Hu-SFM-16-2-L2H5, Hu-SFM-16-2-L2H6, and Hu-SFM-16-2-L2H7

[0541] Note: In the above sequences, the underlined parts are the CDR sequences determined according to the Kabat numbering system, and the italicized parts are antibody constant regions. The double-underlined and bolded parts are amino acid point mutations.

[0542] Example 5. Humanization design of mouse antibody SFM-81-1

[0543] By comparing the genetic sequence of the heavy and light chain variable regions with the IMGT human antibody germline gene database using MOE software, a germline gene of the heavy and light chain variable regions with high homology to the murine antibody SFM-81-1 was selected as a template. The CDRs of this murine antibody were then 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.

[0544] FR1, FR2, and FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region; FR1, FR2, and FR3 of IGHV1-69*02 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region. Optionally, amino acid residues at positions 43, 44, 71, and / or 92 of the light chain variable region and amino acid residues at positions 27, 28, 40, and / or 94 of the heavy chain variable region were substituted.

[0545] Table 11. Human-centered design of SFM-81-1

[0546] Table 12. CDR of SFM-81-1 humanized antibody

[0547] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0548] Table 13. CDR of SFM-81-1 humanized antibody

[0549] Note: The CDRs in the table are CDRs determined according to the Chothia numbering system.

[0550] Table 14. CDR of SFM-81-1 humanized antibody

[0551] Note: The CDRs in the table are CDRs determined according to the IMGT numbering system.

[0552] The light chain variable region / heavy chain variable region sequence of the Hu-SFM-81-1 humanized antibody is as follows: >Hu-SFM-81-1-VH1

[0553] >Hu-SFM-81-1-VH2

[0554] >Hu-SFM-81-1-VH3

[0555] >Hu-SFM-81-1-VL1

[0556] >Hu-SFM-81-1-VL2

[0557] >Hu-SFM-81-1-VL3

[0558] >Hu-SFM-81-1-VL4

[0559] >Hu-SFM-81-1-VL5

[0560] Note: In the above sequences, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the non-underlined part is the FR sequence; the double-underlined bold part is the amino acid point mutation.

[0561] The heavy chain variable region and light chain variable region of the Hu-SFM-81-1 humanized antibody were recombined with the heavy chain constant region hIgG1-2 (SEQ ID NO: 63) and the light chain constant region CL (human κ light chain constant region) (SEQ ID NO: 38), respectively, to obtain the humanized antibody shown in Table 15 below.

[0562] Table 15. Humanized Antibodies for SFM-81-1

[0563] Note: In the table, “Hu-SFM-81-1-L1H1” indicates that the heavy chain variable region is Hu-SFM-81-1-VL1 (SEQ ID NO: 79), the light chain variable region is Hu-SFM-81-1-VH1 (SEQ ID NO: 76), and the heavy chain constant region is as shown in SEQ ID NO: 63, and the light chain constant region is as shown in SEQ ID NO: 38, which is a humanized antibody. Others are deduced by analogy.

[0564] For example, the modified SFM-81-1 humanized antibody is shown in the table below:

[0565] Table 16. Humanized Antibodies for SFM-81-1

[0566] For example, the full-length sequence of the Hu-SFM-81-1 antibody is as follows:

[0567] Light chains of Hu-SFM-81-1-L1H1, Hu-SFM-81-1-L1H2, and Hu-SFM-81-1-L1H3

[0568] Light chains of Hu-SFM-81-1-L2H1, Hu-SFM-81-1-L2H2, and Hu-SFM-81-1-L2H3

[0569] Light chains of Hu-SFM-81-1-L3H1, Hu-SFM-81-1-L3H2, and Hu-SFM-81-1-L3H3

[0570] Light chains of Hu-SFM-81-1-L4H1, Hu-SFM-81-1-L4H2, and Hu-SFM-81-1-L4H3

[0571] Light chains of Hu-SFM-81-1-L5H1, Hu-SFM-81-1-L5H2, and Hu-SFM-81-1-L5H3

[0572] Heavy chains of Hu-SFM-81-1-L1H1, Hu-SFM-81-1-L2H1, Hu-SFM-81-1-L3H1, Hu-SFM-81-1-L4H1, and Hu-SFM-81-1-L5H1

[0573] Heavy chains of Hu-SFM-81-1-L1H2, Hu-SFM-81-1-L2H2, Hu-SFM-81-1-L3H2, Hu-SFM-81-1-L4H2, and Hu-SFM-81-1-L5H2

[0574] Heavy chains of Hu-SFM-81-1-L1H3, Hu-SFM-81-1-L2H3, Hu-SFM-81-1-L3H3, Hu-SFM-81-1-L4H3, and Hu-SFM-81-1-L5H3

[0575] Note: In the above sequences, the underlined parts are the CDR sequences determined according to the Kabat numbering system, and the italicized parts are antibody constant regions. The double-underlined and bolded parts are amino acid point mutations.

[0576] The negative control antibody used in this disclosure is C25-hIgG1 (where the VH / VL sequence is derived from the C25 antibody of patent US6114143A).

[0577] >C25-HC

[0578] C25-LC

[0579] Note: Italics indicate the antibody constant region.

[0580] Test case

[0581] Test Example 1. ELISA detection of the binding affinity of antibodies to anti-NPR1 antibodies.

[0582] The binding affinity of the antibodies against anti-NPR1 disclosed herein was detected using an ELISA method. SFM-140 antibodies (SEQ ID NO: 3 and SEQ ID NO: 4) were diluted to 1 μg / mL with PBS, 100 μL / well, and coated into 96-well plates (Corning, CLS3590-100EA) and incubated overnight at 4°C. After washing, 5% skim milk powder was added, and the plates were blocked at 37°C for 3 h. After washing, different concentrations of antibodies binding to anti-NPR1 were added, and the plates were incubated at 37°C for 1 h. After washing, HRP-goat anti-human IgG secondary antibody (Jackson, 109-035-003) was added, and the plates were incubated at 37°C for 45 min. After washing the plate, add TMB chromogenic substrate (KPL, 52-00-03), incubate at room temperature for 5-10 min, stop the reaction with 1M H2SO4, and read the absorbance at 450 nm using a VERSAmax microplate reader (Molecular Devices). The results are shown in Table 17.

[0583] Table 17. Binding ability of antibodies binding to anti-NPR1 antibodies to SFM-140 antibodies

[0584] The results showed that all the antibodies disclosed herein that bind to the anti-NPR1 antibody could bind to the SFM-140 antibody with strong affinity.

[0585] Test Example 2. Biacore detects the binding affinity of antibodies to anti-NPR1 antibodies.

[0586] The affinity of the antibody binding to the anti-NPR1 antibody in this disclosure for the SFM-140 antibody was determined using the Biacore method. A CM5 biosensor chip (Cytiva, BR-1005-30) conjugated with an anti-mouse antibody (Cytiva, BR-1008-38) was used to affinity-capture the mouse antimolecule (SFM-140 antibody). The antibody sample was then passed over the chip surface, and the reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl pH 1.5 (Cytiva, BR-1003-54). A 1:1 model was used for data fitting. The binding and dissociation of each antibody are shown in Table 18.

[0587] Table 18. Binding affinity of antibodies to anti-NPR1 antibodies

[0588] The results showed that all the antibodies disclosed herein that bind to the anti-NPR1 antibody could bind to the SFM-140 antibody with strong affinity.

[0589] Test Example 3. ELISA detection of the effect of antibodies binding to anti-NPR1 antibodies on blocking the binding of anti-NPR1 antibodies in cells overexpressing NPR1.

[0590] The blocking effect of the antibody binding to anti-NPR1 in the CHO-K1 recombinant cell line hNPR1-CHO-K1 overexpressing NPR1 against the anti-NPR1 antibody was detected using an ELISA method. 2*10 4 hNPR1-CHO-K1 cells were cultured in 96-well plates (corning, 3599). After 48 hours, the supernatant was discarded, and each well was washed with 50 μL of PBS. Then, 50 μL of 4% paraformaldehyde fixative (Beyotime, P0099-500 mL) was added, and the plates were incubated for 45 minutes. After washing, 5% skim milk powder was added, and the plates were blocked at 37°C for 3 hours. After blocking, the plates were washed again, and antibody solutions were prepared using 20 nM natriuretic peptide (ANP 1-28, GenScript, RP11927) and 1% BSA solution as solvents. 1 μg / mL biotin-SFM-140 (biotinylated SFM-140 antibody) and different concentrations of antibody samples conjugated with anti-NPR1 antibodies were mixed in equal volumes and incubated at 25°C for 1 hour. The mixture was then transferred to 96-well plates and incubated at 37°C for 1 hour. After incubation, the plate was washed, and high-sensitivity streptavidin peroxidase polymeric secondary antibody (Sigma, S2438-250UG) was added. The plate was incubated at 37°C for 45 min. After washing, TMB chromogenic substrate (KPL, 52-00-03) was added, and the plate was incubated at room temperature for 5-10 min. The reaction was terminated by adding 1M H2SO4. The absorbance was read at 450 nm using a VERSAmax microplate reader (Molecular Devices). The results are shown in Table 19.

[0591] Table 19. Blocking effect of antibody-cell binding on anti-NPR1 antibodies

[0592] The results showed that both SFM-16-2 and SFM-81-1 series antibodies that bind to anti-NPR1 antibodies blocked the binding of the CHO-K1 recombinant cell line hNPR1-CHO-K1, which overexpresses NPR1, to anti-NPR1 antibodies.

[0593] Test Example 4. FACS Detection of Cell Binding Blocking Effect of Antibody Binding to Anti-NPR1

[0594] The blocking effect of the antibody against anti-NPR1 in the CHO-K1 recombinant cell line hNPR1-CHO-K1 overexpressing NPR1 on the binding of the disclosed antibody was detected using the FACS method. 1*10 6hNPR1-CHO-K1 cells / mL were centrifuged to precipitate the cells and transferred to 96-well plates (corning, 3795). Antibody solutions were prepared using 20 nM natriuretic peptide (ANP 1-28, GenScript, RP11927) and 1% BSA solution as solvents. Equal volumes of 6 μg / mL biotin-SFM-140 (biotinylated SFM-140 antibody) and antibody samples of different concentrations bound to anti-NPR1 antibodies were mixed and incubated at 25°C for 30 min. The mixture was then transferred to 96-well plates and incubated at 4°C for 1 h. After washing twice with 1% BSA, streptavidin secondary antibody (PE-labeled streptavidin, Biolegend, 405204) was added, and the plates were incubated at 4°C for 45 min. After washing twice with 1% BSA, fluorescence signal values ​​were read using FACS. The results are shown in Table 20.

[0595] Table 20. Blocking effect of antibody-cell binding to anti-NPR1 antibody (FACS)

[0596] The results showed that both Hu-SFM-16-2-L2H3 and Hu-SFM-81-1-L1H2 blocked the binding of the NPR1-overexpressing CHO-K1 recombinant cell line hNPR1-CHO-K1 to the anti-NPR1 antibody, with Hu-SFM-16-2-L2H3 exhibiting stronger binding blocking ability.

[0597] Test Example 5. cGMP Blocking Detection of Antibody Binding to Anti-NPR1

[0598] The blocking effect of anti-NPR1 antibodies on cGMP production was detected using homogeneous time-resolved fluorescence (HTRF). Trypsin-digested hNPR1-CHO-K1 cells were added to complete culture medium to terminate digestion, centrifuged, washed with EBSS, and re-counted. Cells were resuspended in twice the concentration of reaction buffer (EBSS solution containing 10 mM MgCl2, 20 mM HEPES, and 1 mM IBMX) and adjusted to 3E6 cells / mL. 5 μL of each cell was added to each well of a 384-well plate (PE, optiplate-384). Different concentrations of anti-NPR1 antibodies were prepared using EBSS and mixed with an equal volume of Hu140H5L5-11 at a fixed concentration, and incubated at room temperature for 30 min. 5 μL of each antibody was added to each well of the 384-well plate, centrifuged at 300g for 1 min, and incubated at 37°C for 0.5 h. Dissolve d2 (acceptor) and europium cavitation compound (donor) from the cGMP kit (Cisbio, 62GM2PEG). Then, add 5 μL / well of d2 followed by 5 μL / well of europium cavitation compound to a 384-well plate. Centrifuge at 300g for 1 min and incubate at room temperature for 1 h. Use the HTRF module in Envision to read the signal values. The results are shown in Table 21.

[0599] Table 21. Blocking effect of antibodies binding to anti-NPR1 antibodies on cGMP production.

[0600] HTRF test results showed that the antibodies disclosed herein that bind to anti-NPR1 antibodies can block the production of cGMP on hNPR1-CHO-K1.

[0601] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, 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. A binding molecule that binds to an anti-NPR1 antibody, said anti-NPR1 antibody comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and said light chain variable region comprises 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: 99, 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: 100; 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: 101, 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:

102.

2. The binding molecule according to claim 1, wherein the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 93, HCDR2 contains the amino acid sequence of SEQ ID NO: 94, and HCDR3 contains the amino acid sequence of SEQ ID NO: 95; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 96, LCDR2 contains the amino acid sequence of SEQ ID NO: 97, and LCDR3 contains the amino acid sequence of SEQ ID NO:

98. Preferably, The heavy chain variable region comprises SEQ ID NO: 99, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 100, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises SEQ ID NO: 101, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 102, or an amino acid sequence having at least 80% sequence identity with it; More preferably, The anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain comprises SEQ ID NO: 3, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity with it.

3. A binding molecule that binds an anti-NPR1 antibody, said binding molecule 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: a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 55, 53, 54, 56, 57, 58, or 59, 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: 61, 60, or 62; 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: 32, 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: 33; or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 77, 76, or 78, 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: 79, 80, 81, 82, or 83; 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: 34, 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:

35.

4. A binding molecule for binding an anti-NPR1 antibody, said binding molecule 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: a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 6, HCDR2 contains the amino acid sequence of SEQ ID NO: 7, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 105, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO:

92.

5. The binding molecule according to any one of claims 1 to 4, wherein the binding molecule 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: a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 6, HCDR2 contains the amino acid sequence of SEQ ID NO: 7, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 44, 43, 45, or 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17 or 73; Preferably, a. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 6, HCDR2 contains the amino acid sequence of SEQ ID NO: 7, and HCDR3 contains the amino acid sequence of SEQ ID NO: 8; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 44, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 11; or b. The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO:

17.

6. The binding molecule according to any one of claims 1 to 5, wherein the binding molecule is an antibody or an antibody fragment; preferably a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antibody fragment; more preferably a humanized antibody or an antibody fragment selected from Fab, Fab′, F(ab′)2, Fv, scFv and dsFv.

7. The binding molecule according to any one of claims 1 to 6, wherein the binding molecule comprises a heavy chain variable region and a light chain variable region, wherein: a. The heavy chain variable region comprises SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 61, 60 or 62, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 33, or an amino acid sequence having at least 80% sequence identity with it; or b. The heavy chain variable region comprises SEQ ID NO: 77, 76, or 78, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 79, 80, 81, 82, or 83, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises SEQ ID NO: 34, or an amino acid sequence having at least 80% sequence identity with it, and the light chain variable region comprises SEQ ID NO: 35, or an amino acid sequence having at least 80% sequence identity with it; Preferably, a. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, 53, 54, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, 56, 57, 58 or 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or b. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, 76, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, 77, or 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 76, 77 or 78, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 83; More preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61; or The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 77, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

79.

8. The binding molecule according to any one of claims 1 to 7, wherein the binding molecule further comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is a heavy chain constant region derived from human IgG1, and the light chain constant region is a light chain constant region derived from human κ. More preferably, the heavy chain constant region contains the amino acid sequence of SEQ ID NO: 63 or 37, and the light chain constant region contains the amino acid sequence of SEQ ID NO:

38.

9. The binding molecule according to claim 8, wherein the binding molecule comprises a heavy chain and a light chain, wherein: a. The heavy chain comprises SEQ ID NO: 66, 64, 65, 67, 68, 69 or 70, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 72 or 71, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain comprises SEQ ID NO: 40, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity with it; or b. The heavy chain comprises SEQ ID NO: 90, 89, or 91, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 84, 85, 86, 87, or 88, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain comprises SEQ ID NO: 42, or an amino acid sequence having at least 80% sequence identity with it, and the light chain comprises SEQ ID NO: 41, or an amino acid sequence having at least 80% sequence identity with it; Preferably, a. The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, 64, 65, 67, 68, 69 or 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 64, 65, 66, 67, 68, 69 or 70, and the light chain comprises the amino acid sequence of SEQ ID NO: 71; or b. The heavy chain comprises the amino acid sequence of SEQ ID NO: 90, 89, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 84; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, 90, or 91, 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: 89, 90, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 86; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 89, 90, or 91, and the light chain comprises the amino acid sequence of SEQ ID NO: 87; or The heavy chain contains the amino acid sequence of SEQ ID NO: 89, 90 or 91, and the light chain contains the amino acid sequence of SEQ ID NO: 88; More preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, and the light chain comprises the amino acid sequence of SEQ ID NO: 72; or The heavy chain contains the amino acid sequence of SEQ ID NO: 90, and the light chain contains the amino acid sequence of SEQ ID NO:

84.

10. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 9, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

11. A nucleic acid encoding a binding molecule as described in any one of claims 1 to 9.

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

13. A method for preparing the binding molecule of any one of claims 1 to 9, the method comprising culturing a host cell as described in claim 12 in a culture medium to accumulate the binding molecule as described in any one of claims 1 to 9, and recovering the binding molecule from the culture.

14. Use of the combined molecule of any one of claims 1 to 9, or the pharmaceutical composition of claim 10, in the preparation of a medicament for reversing side effects associated with the administration of anti-NPR1 antibody; preferably, the side effects are related to the production of cGMP; more preferably, the side effects are hemodynamic effects; most preferably, the side effects are hypotension or tachycardia.

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