Antibody conjugate and application thereof

Abstract

The invention provides an antibody conjugate (smacAb) and application of the antibody conjugate (smacAb). The invention also provides a construction method of the smacAb library and a screening method of the smacAb. The smacAb prepared by the invention has higher affinity, inhibitory activity and specificity, and the constructed smacAb library has diversity and a library capacity of about 109.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an antibody conjugate and application thereof in smacAb library construction and antibody or smacAb screening. Background Art

[0002] Phage display technology is a biotechnology that inserts the DNA sequence of foreign proteins or polypeptides into the appropriate position of the structural gene of the phage coat protein, so that the foreign gene is expressed along with the expression of the coat protein. At the same time, the foreign protein is displayed on the surface of the phage as the phage is reassembled.

[0003] Phage display technology can be used to screen fully human antibodies for the treatment of various diseases. For example, patent CN112851814A screened out BCMA-specific antibodies with strong binding ability and high affinity through multiple rounds of panning and ELISA.

[0004] However, it is difficult to screen out antibodies with weak binding ability using phage display technology. Therefore, the present application covalently binds small molecule compounds to specific sites of the antibody to further enhance the binding ability of the antibody to the target antigen, thereby screening out functional antibodies. Summary of the invention

[0005] In a first aspect, the present invention provides an antibody conjugate (smacAb), which comprises a covalent conjugate formed by site-specific conjugation of an antibody or an antigen-binding fragment thereof with a small molecule compound, wherein the small molecule compound and the antibody or its antigen-binding fragment jointly participate in binding to a target antigen.

[0006] The small molecule compound can bind to the target antigen.

[0007] The antibody or antigen-binding fragment thereof and the small molecule compound can bind to the same type or class of target antigens.

[0008] The antibody or antigen-binding fragment thereof can be coupled to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) small molecule compounds, wherein when two small molecule compounds are coupled, the small molecule compounds may be the same or different, and when three or more small molecule compounds are coupled, the small molecule compounds may be all the same, or completely different, or partially the same.

[0009] The small molecule compound is coupled to the complementarity determining region (CDR) of the antibody or its antigen-binding fragment; preferably, the small molecule compound is coupled to the heavy chain CDR or light chain CDR of the antibody or its antigen-binding fragment, such as heavy chain CDR1, CDR2 or CDR3 or light chain CDR1, CDR2 or CDR3.

[0010] Preferably, the small molecule compound is coupled to the heavy chain CDR3 or light chain CDR3 of the antibody or its antigen-binding fragment. Further preferably, the small molecule compound is coupled to the light chain CDR3 of the antibody or its antigen-binding fragment.

[0011] The CDR may be 3-20 aa in length, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0012] The coupling position can be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th amino acid on the CDR.

[0013] In a specific embodiment of the present invention, the small molecule compound is coupled to the light chain CDR3 of the antibody or its antigen-binding fragment.

[0014] The light chain CDR3 length is 3-20 aa, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0015] The small molecule compound is coupled to any amino acid residue of the light chain CDR3 of the antibody or its antigen-binding fragment, preferably at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0016] The light chain CDR3 includes QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTFG (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 23) NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0017] The coupling position may or may not be mutated.

[0018] The antibody or antigen-binding fragment thereof is coupled to the small molecule compound via a bioorthogonal reaction. Preferably, the bioorthogonal reaction comprises a Michael addition reaction or a Huisgen reaction.

[0019] Preferably, the Michael addition reaction comprises: introducing cysteine ​​Cys at the site of the antibody or its antigen-binding fragment, modifying the maleimide terminal group on the small molecule compound, and performing the Michael addition reaction. For example, the amino acid residue at the coupling position is mutated to cysteine ​​Cys, and then the maleimide terminal group is modified on the small molecule compound to perform the Michael addition reaction. Of course, it can also include that the CDR region itself contains cysteine, and then directly reacts with the small molecule compound modified with the maleimide terminal group to perform the Michael addition reaction.

[0020] Preferably, the Huisgen reaction comprises: introducing UAA-N3 (introducing an azide group through a non-natural amino acid) at a site of an antibody or an antigen-binding fragment thereof, modifying a cyclooctyne terminal group or a derivative thereof on a small molecule compound, and performing a Huisgen reaction; the cyclooctyne terminal group or a derivative thereof is selected from any one of DIFO, BCN, DIBAC, DIBO, ADIBO or DBCO.

[0021] The small molecule compound and the antibody or its antigen-binding fragment further include a linker group. Preferably, the linker group can be a connecting peptide, a PEG group,

[0022]

[0023]

[0024]

[0025] Preferably, the connecting peptide may include LLTPPPPPLFPPPFF (SEQ ID NO: 240, J Mol Biol. 2013; 425: 2100–32), (EAAAK)n (n=2–5, wherein n=2, 3, 4, 5 are SEQ ID NOs: 241, 242, 243, 244, Proteins. 2004; 57: 829–38), E4K4 (SEQ ID NO: 245, J Biol Chem. 2014; 289: 25460–7), (E4R4)2 (SEQ ID NO: 246, J Biol Chem. 2014; 289: 25460–7), (E4K4)2 (SEQ ID NO: 148, J Biol Chem. 2014; 289: 25460–7), KLYPYDVPDYA (SEQ ID NO: 139, J Mol Biol.2005;354:546–55), PKPSTPPGSS (SEQ ID NO:130, J Biotechnol.2015;199:90–7), GSPAG (SEQ IDNO:121, Nucleic Acids Res.2020;48(4):e24), GGASPAGG (SEQ ID NO:113, Nucleic Acids AcidsRes.2020;48(4):e24), GGASPAAPAPAG (SEQ ID NO:106, Nucleic Acids Res.2020;48(4):e24), AMGPSSGAPGGGGS (SEQ ID NO:98, Enzyme Microb Technol.2016;82:105-109), or with SEQ ID The sequences shown in any of NO:98, 106, 113, 121, 130, 139, 148 or 240-246 have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity.

[0026] Preferably, 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) PEG groups are further included between the small molecule compound and the antibody or its antigen-binding fragment. Preferably, 3-5 PEG groups are further included between the small molecule compound and the antibody or its antigen-binding fragment.

[0027] For example, the structure of PEG3-DBCO is as follows:

[0028]

[0029] For example, the structure of PEG3-Mal is as follows:

[0030]

[0031] Preferably, the small molecule compound is an inhibitor of the target antigen, such as PSB12379 and ASF.

[0032] The target antigen can be a target protein, peptide, lipid, polysaccharide or polynucleotide. Preferably, the target protein is selected from membrane proteins, membrane transport proteins, receptor proteins, complexes formed by membrane proteins and membrane proteins, and complexes formed by membrane proteins and other proteins.

[0033] Further preferably, the membrane protein may be a peripheral membrane protein, an intrinsic membrane protein, or a lipid-anchored protein;

[0034] Further preferably, the membrane transport protein may be a carrier protein or a channel protein; preferably, the channel protein may be a sodium ion channel protein, a calcium ion channel protein, a chloride ion channel protein, a potassium ion channel protein, an Ach receptor channel, or an amino acid receptor channel;

[0035] Further preferably, the receptor protein may be an internal receptor, a cell surface receptor, an ion channel receptor, a G-protein coupled receptor (GPCRs) and an enzyme-linked receptor.

[0036] In a specific embodiment of the present invention, the target antigen may be Na v 1.1、Na v 1.2 Na v 1.3 Na v 1.4 Na v 1.5 Na v 1.6 Na v 1.7 Na v 1.8 Na v1.9, CD73, VEGFA, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integr in receptor, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12 R, IL15Rα, IL17R, IL23R, PCSK9, TNF-α, TNFR, RANKL, complement protein C3, complement protein C5, GPCR, GLP1R, CD3, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, B7-1, B7-2, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, F AP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3 or 5T4.

[0037] The antibody conjugate may be:

[0038]

[0039]

[0040]

[0041] Wherein, R is a small molecule compound, R1 is R2 is R3 is

[0042] The antibody or antigen-binding fragment thereof may be Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0043] The antibody or antigen-binding fragment thereof is a CD73 antibody or an antigen-binding fragment thereof, and the CD73 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0044] The VLCDR1 comprises any one of SEQ ID NOs: 30-39, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 30-39;

[0045] The VLCDR2 comprises any one of SEQ ID NOs: 40-45, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 40-45;

[0046] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0047] The VHCDR1 comprises any one of SEQ ID NOs: 46-55, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 46-55;

[0048] The VHCDR2 comprises any one of SEQ ID NOs: 56-64, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 56-64;

[0049] The VHCDR3 comprises any one of SEQ ID NOs: 65-72, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 65-72.

[0050] VLCDR1 and VLCDR2 include:

[0051] Antibody Number VL CDR1 VL CDR2 10D8 RASQSISSWLA (SEQ ID NO: 30) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 10A7 RASQGISRWLA (SEQ ID NO: 31) NLLIYGASTLQSGVPP (SEQ ID NO: 41) 4C10 RATQAISNYLA (SEQ ID NO: 32) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 5E12 RTSQGITDPLN (SEQ ID NO: 33) KLLIYKTSSLESGVPS (SEQ ID NO: 42) 5H12 RASQGLSSWLA (SEQ ID NO: 34) KLLIYDASNLETGVPS (SEQ ID NO: 43) 6D11 RASQNIGTYLA (SEQ ID NO: 35) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2B RASQGINNYLA (SEQ ID NO: 36) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2G QASHDISKYLN (SEQ ID NO: 37) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2A RASETIGSWLA (SEQ ID NO: 38) NLLIYAASTLQSGVSS (SEQ ID NO: 44) 10F8 QASQDISNYLN (SEQ ID NO: 39) NLLIYAASTLQSGVPS (SEQ ID NO: 45)

[0052] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0053]

[0054]

[0055] The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 73-82, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 73-82.

[0056] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 83-92, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 83-92.

[0057] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof is a CD73 antibody or an antigen-binding fragment thereof, the small molecule compound is PSB12379, and the structure of PSB12379 is:

[0058]

[0059] In a specific embodiment of the present invention, the antibody conjugate is a light chain CDR3 of a CD73 antibody or an antigen-binding fragment thereof and (PEG) 2-10 -PSB12379 was coupled via Huisgen reaction.

[0060] The antibody or antigen-binding fragment thereof is Na v 1.7 Antibodies or antigen-binding fragments thereof, wherein the Nav 1.7 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0061] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100;

[0062] The VLCDR2 comprises any one of SEQ ID NOs: 101-105 or 107-108, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101-105 or 107-108;

[0063] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0064] The VHCDR1 comprises any one of SEQ ID NOs: 109-112 or 114, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109-112 or 114;

[0065] The VHCDR2 comprises any one of SEQ ID NOs: 115-120 or 122-123, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115-120 or 122-123;

[0066] The VHCDR3 comprises the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132.

[0067] VLCDR1 and VLCDR2 include:

[0068]

[0069] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0070]

[0071] The heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 142-147 or 149-150, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 142-147 or 149-150.

[0072] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141.

[0073] The antibody or antigen-binding fragment thereof is Na v 1.2 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.2 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0074] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156;

[0075] The VLCDR2 comprises any one of SEQ ID NOs: 102, 103, 157-163, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 102, 103, 157-163;

[0076] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0077] The VHCDR1 comprises any one of SEQ ID NOs: 110, 111, 164-170, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 110, 111, 164-170;

[0078] The VHCDR2 comprises any one of SEQ ID NOs: 116, 117, 171-177, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 116, 117, 171-177;

[0079] The VHCDR3 comprises any one of SEQ ID NOs: 125, 126, 178-183, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 125, 126, 178-183.

[0080] VLCDR1 and VLCDR2 include:

[0081]

[0082]

[0083] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0084]

[0085]

[0086] The heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 143, 144, 191-197, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 143, 144, 191-197.

[0087] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190.

[0088] The antibody or antigen-binding fragment thereof is Na v 1.6 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.6 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0089] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203;

[0090] The VLCDR2 comprises any one of SEQ ID NOs: 101, 103, 104, 204-209, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101, 103, 104, 204-209;

[0091] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0092] The VHCDR1 comprises any one of SEQ ID NOs: 109, 111, 210-215, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109, 111, 210-215;

[0093] The VHCDR2 comprises any one of SEQ ID NOs: 115, 117, 118, 216-221, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115, 117, 118, 216-221;

[0094] The VHCDR3 comprises any one of SEQ ID NOs: 124, 126, 127, 222-227, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 124, 126, 127, 222-227.

[0095] VLCDR1 and VLCDR2 include:

[0096]

[0097]

[0098] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0099]

[0100]

[0101] The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 142, 144, 145, 234-239, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 142, 144, 145, 234-239.

[0102] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233.

[0103] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof is Na v 1.2 Antibodies or antigen-binding fragments thereof, Na v 1.6 Antibodies or antigen-binding fragments thereof or Na v1.7 Antibodies or antigen-binding fragments thereof, wherein the small molecule compound is an aromatic sulfonamide, preferably 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine (ASF), having the structure:

[0104]

[0105] In a specific embodiment of the present invention, the antibody conjugate is Na v 1.2 Antibodies or antigen-binding fragments thereof, Na v 1.6 Antibodies or antigen-binding fragments thereof or Na v 1.7 Light chain CDR3 of an antibody or antigen-binding fragment thereof and (PEG) 2-10 -ASF was coupled via Huisgen reaction.

[0106] The second aspect of the present invention provides a method for preparing the antibody conjugate described in the first aspect, the preparation method comprising forming a covalent conjugate by site-specific conjugation of an antibody or an antigen-binding fragment thereof with a small molecule compound, wherein the small molecule compound and the antibody or its antigen-binding fragment jointly participate in binding to the target antigen.

[0107] The small molecule compound can bind to the target antigen.

[0108] In a third aspect of the present invention, a CD73 antibody or an antigen-binding fragment thereof is provided, wherein the CD73 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0109] The VLCDR1 comprises any one of SEQ ID NOs: 30-39, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 30-39;

[0110] The VLCDR2 comprises any one of SEQ ID NOs: 40-45, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 40-45;

[0111] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0112] The VHCDR1 comprises any one of SEQ ID NOs: 46-55, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 46-55;

[0113] The VHCDR2 comprises any one of SEQ ID NOs: 56-64, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 56-64;

[0114] The VHCDR3 comprises any one of SEQ ID NOs: 65-72, or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 65-72.

[0115] Among them, VLCDR1 and VLCDR2 respectively include:

[0116] Antibody Number VL CDR1 VL CDR2 10D8 RASQSISSWLA (SEQ ID NO: 30) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 10A7 RASQGISRWLA (SEQ ID NO: 31) NLLIYGASTLQSGVPP (SEQ ID NO: 41) 4C10 RATQAISNYLA (SEQ ID NO: 32) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 5E12 RTSQGITDPLN (SEQ ID NO: 33) KLLIYKTSSLESGVPS (SEQ ID NO: 42) 5H12 RASQGLSSWLA (SEQ ID NO: 34) KLLIYDASNLETGVPS (SEQ ID NO: 43) 6D11 RASQNIGTYLA (SEQ ID NO: 35) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2B RASQGINNYLA (SEQ ID NO: 36) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2G QASHDISKYLN (SEQ ID NO: 37) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2A RASETIGSWLA (SEQ ID NO: 38) NLLIYAASTLQSGVSS(SEQ ID NO:44) 10F8 QASQDISNYLN (SEQ ID NO: 39) NLLIYAASTLQSGVPS(SEQ ID NO: 45)

[0117] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0118]

[0119]

[0120] The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 73-82, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 73-82.

[0121] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 83-92, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 83-92.

[0122] The antibody or antigen-binding fragment thereof is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0123] A fourth aspect of the present invention provides a Na v 1.2 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.2 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0124] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156;

[0125] The VLCDR2 comprises any one of SEQ ID NOs: 102, 103, 157-163, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 102, 103, 157-163;

[0126] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0127] The VHCDR1 comprises any one of SEQ ID NOs: 110, 111, 164-170, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 110, 111, 164-170;

[0128] The VHCDR2 comprises any one of SEQ ID NOs: 116, 117, 171-177, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 116, 117, 171-177;

[0129] The VHCDR3 comprises any one of SEQ ID NOs: 125, 126, 178-183, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 125, 126, 178-183.

[0130] Among them, VLCDR1 and VLCDR2 respectively include:

[0131]

[0132] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0133]

[0134]

[0135] The heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 143, 144, 191-197, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 143, 144, 191-197.

[0136] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190.

[0137] The antibody or antigen-binding fragment thereof is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0138] A fifth aspect of the present invention provides a Na v 1.6 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.6 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0139] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203;

[0140] The VLCDR2 comprises any one of SEQ ID NOs: 101, 103, 104, 204-209, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101, 103, 104, 204-209;

[0141] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0142] The VHCDR1 comprises any one of SEQ ID NOs: 109, 111, 210-215, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109, 111, 210-215;

[0143] The VHCDR2 comprises any one of SEQ ID NOs: 115, 117, 118, 216-221, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115, 117, 118, 216-221;

[0144] The VHCDR3 comprises any one of SEQ ID NOs: 124, 126, 127, 222-227, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 124, 126, 127, 222-227.

[0145] Among them, VLCDR1 and VLCDR2 respectively include:

[0146]

[0147] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0148]

[0149]

[0150] The heavy chain variable region comprises any one of SEQ ID NOs: 142, 144, 145, 234-239, or is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs: 142, 144, 145, 234-239;

[0151] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233.

[0152] The antibody or antigen-binding fragment thereof is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0153] A sixth aspect of the present invention provides a Na v 1.7 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.7 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0154] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100;

[0155] The VLCDR2 comprises any one of SEQ ID NOs: 101-105 or 107-108, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101-105 or 107-108;

[0156] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0157] The VHCDR1 comprises any one of SEQ ID NOs: 109-112 or 114, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109-112 or 114;

[0158] The VHCDR2 comprises any one of SEQ ID NOs: 115-120 or 122-123, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115-120 or 122-123;

[0159] The VHCDR3 comprises the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132.

[0160] Among them, VLCDR1 and VLCDR2 respectively include:

[0161]

[0162]

[0163] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0164]

[0165]

[0166] The heavy chain variable region comprises any one of SEQ ID NOs: 142-147 or 149-150, or is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs: 142-147 or 149-150;

[0167] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141.

[0168] The antibody or antigen-binding fragment thereof is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0169] In a seventh aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the above-mentioned CD73 antibody or antigen-binding fragment thereof, the above-mentioned Na v 1.2 Antibodies or antigen-binding fragments thereof, the above-mentioned Na v 1.6 Antibodies or antigen-binding fragments thereof or the above-mentioned Na v 1.7 Antibodies or antigen-binding fragments thereof.

[0170] The eighth aspect of the present invention provides a vector, wherein the vector expresses the above-mentioned antibody or antigen-binding fragment thereof or comprises the above-mentioned nucleic acid molecule.

[0171] The ninth aspect of the present invention provides a host cell or immune cell, wherein the host cell or immune cell comprises the above-mentioned antibody or antigen-binding fragment thereof, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector.

[0172] The host cell or immune cell expresses the above-mentioned antibody or antigen-binding fragment thereof.

[0173] Preferably, the immune cells include but are not limited to lymphocytes (eg, T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes, and mast cells.

[0174] The tenth aspect of the present invention provides a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0175] The eleventh aspect of the present invention provides a cell, wherein the cell expresses the chimeric antigen receptor mentioned above.

[0176] The twelfth aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, the preparation method comprising introducing the above-mentioned nucleic acid molecule into cells and inducing cell expression.

[0177] The thirteenth aspect of the present invention provides the application of small molecule compounds, wherein the application includes:

[0178] A) Construction of smacAb library;

[0179] Preferably, the small molecule compound is site-specifically coupled to the antibody or antigen-binding fragment thereof in the antibody or antigen-binding fragment thereof library to form a covalent conjugate, namely, the smacAb library;

[0180] B) smacAb screening;

[0181] Preferably, the small molecule compound is site-specifically coupled to an antibody or an antigen-binding fragment thereof in a library of antibodies or antigen-binding fragments thereof to form a covalent conjugate, namely, a smacAb library, and smacAb is obtained by target antigen screening;

[0182] C) Increase the binding strength or affinity between the antibody and the target antigen;

[0183] Preferably, the small molecule compound and the antibody are site-specifically coupled to form a covalent conjugate, wherein the small molecule compound and the antibody jointly participate in the binding with the target antigen;

[0184] D) Application in diagnosis and / or treatment of diseases;

[0185] E) Use in the preparation of drugs for diagnosing and / or treating diseases.

[0186] Preferably, the disease may be a tumor, an infectious disease or inflammation.

[0187] Preferably, the drug comprises the antibody conjugate described in the first aspect.

[0188] The small molecule compound is selected from PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine.

[0189] The target antigen may be a target protein, peptide, lipid, polysaccharide or polynucleotide. Preferably, the target protein is selected from membrane proteins, membrane transport proteins, receptor proteins, complexes formed by membrane proteins and membrane proteins, and complexes formed by membrane proteins and other proteins.

[0190] Further preferably, the membrane protein may be a peripheral membrane protein, an intrinsic membrane protein, or a lipid-anchored protein;

[0191] Further preferably, the membrane transport protein may be a carrier protein or a channel protein; preferably, the channel protein may be a sodium ion channel protein, a calcium ion channel protein, a chloride ion channel protein, a potassium ion channel protein, an Ach receptor channel, or an amino acid receptor channel;

[0192] Further preferably, the receptor protein may be an internal receptor, a cell surface receptor, an ion channel receptor, a G-protein coupled receptor (GPCRs) and an enzyme-linked receptor.

[0193] The antibody or antigen-binding fragment thereof is selected from the above-mentioned antibodies or antigen-binding fragments thereof.

[0194] The connection mode between the small molecule compound and the antibody or its antigen-binding fragment and the linker group therebetween are described in the first aspect.

[0195] In a fourteenth aspect, the present invention provides a smacAb library, wherein the smacAb library comprises forming covalent conjugates by site-specifically coupling small molecule compounds with antibodies or antigen-binding fragments thereof in an antibody or antigen-binding fragment library.

[0196] The small molecule compound is coupled to the complementarity determining region (CDR) of the antibody or its antigen-binding fragment; preferably, the small molecule compound is coupled to the heavy chain CDR3 or light chain CDR3 of the antibody or its antigen-binding fragment, and further preferably, the small molecule compound is coupled to the light chain CDR3 of the antibody or its antigen-binding fragment.

[0197] The light chain CDR3 includes QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTFG (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 23) NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0198] The small molecule compound is selected from PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amino

[0199] The antibody or antigen-binding fragment thereof is coupled with a small molecule compound via a bioorthogonal reaction; preferably, the bioorthogonal reaction comprises a Michael addition reaction or a Huisgen reaction.

[0200] The small molecule compound and the antibody or its antigen-binding fragment further include a linker group, preferably, the linker group can be a connecting peptide, a PEG group,

[0201]

[0202]

[0203]

[0204] Preferably, the connecting peptide may include LLTPPPPPLFPPPFF (SEQ ID NO: 240), (EAAAK)n (n=2-5, wherein n=2, 3, 4, 5 are SEQ ID NOs: 241, 242, 243, 244, respectively), E4K4 (SEQ ID NO: 245), (E4R4)2 (SEQ ID NO: 246), (E4K4)2 (SEQ ID NO: 148), KLYPYDVPDYA (SEQ ID NO: 139), PKPSTPPGSS (SEQ ID NO: 130), GSPAG (SEQ ID NO: 121), GGASPAGG (SEQ ID NO: 113), GGASPAAPAPAG (SEQ ID NO: 106), AMGPSSGAPGGGGS (SEQ ID NO: 98), or SEQ ID NO: 1 The sequences shown in any of NO:98, 106, 113, 121, 130, 139, 148 or 240-246 have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity.

[0205] The small molecule compound further comprises 2-10 PEG groups between the small molecule compound and the antibody or its antigen-binding fragment. Preferably, the small molecule compound further comprises 3-5 PEG groups between the small molecule compound and the antibody or its antigen-binding fragment.

[0206] The smacAb library comprises 10 9 Capacity of smacAb.

[0207] In a fifteenth aspect of the present invention, a method for constructing a smacAb library is provided, the method comprising forming a covalent conjugate by site-specific coupling of the displayed antibody or antigen-binding fragment thereof with a small molecule compound.

[0208] The display can be performed using a single-stranded filamentous phage display system, a lambda phage display system or a T4 phage display system.

[0209] The antibody or antigen-binding fragment thereof can be coupled to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) small molecule compounds, wherein when two small molecule compounds are coupled, the small molecule compounds may be the same or different, and when three or more small molecule compounds are coupled, the small molecule compounds may be all the same, or completely different, or partially the same.

[0210] The small molecule compound is covalently coupled to the complementarity determining region (CDR) of the antibody or its antigen-binding fragment; preferably, the small molecule compound is covalently coupled to the heavy chain CDR or light chain CDR of the antibody or its antigen-binding fragment, such as heavy chain CDR1, CDR2 or CDR3 or light chain CDR1, CDR2 or CDR3.

[0211] Preferably, the small molecule compound is covalently coupled to the heavy chain CDR3 or light chain CDR3 of the antibody or its antigen-binding fragment. Further preferably, the small molecule compound is coupled to the light chain CDR3 of the antibody or its antigen-binding fragment.

[0212] The CDR may be 3-20 aa in length, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0213] The coupling position can be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th amino acid on the CDR.

[0214] The light chain CDR3 length is 3-20 aa.

[0215] The small molecule compound is coupled to any amino acid residue of the light chain CDR3 of the antibody or its antigen-binding fragment, preferably at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0216] The light chain CDR3 includes QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTFG (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 23) NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0217] The coupling position may or may not be mutated.

[0218] The antibody or antigen-binding fragment thereof is coupled with the small molecule compound via a bioorthogonal reaction; preferably a Michael addition reaction or a Huisgen reaction.

[0219] Preferably, the Michael addition reaction comprises: introducing cysteine ​​Cys at the site of the antibody or its antigen-binding fragment, modifying the maleimide terminal group on the small molecule compound, and performing the Michael addition reaction. For example, the amino acid residue at the coupling position is mutated to cysteine ​​Cys, and then the maleimide terminal group is modified on the small molecule compound to perform the Michael addition reaction. Of course, it can also include that the CDR region itself contains cysteine, and then directly reacts with the small molecule compound modified with the maleimide terminal group to perform the Michael addition reaction.

[0220] Preferably, the Huisgen reaction comprises: introducing UAA-N3 at the site of the antibody or its antigen-binding fragment, modifying the cyclooctyne terminal group or its derivative on the small molecule compound, and performing the Huisgen reaction; the cyclooctyne terminal group or its derivative is selected from any one of DIFO, BCN, DIBAC, DIBO, ADIBO or DBCO.

[0221] The small molecule compound and the antibody or its antigen-binding fragment further include a linker group, preferably, the linker group can be a connecting peptide, a PEG group,

[0222]

[0223]

[0224]

[0225] Preferably, the connecting peptide may include LLTPPPPPLFPPPFF (SEQ ID NO: 240), (EAAAK)n (n=2-5, wherein n=2, 3, 4, 5 are SEQ ID NOs: 241, 242, 243, 244, respectively), E4K4 (SEQ ID NO: 245), (E4R4)2 (SEQ ID NO: 246), (E4K4)2 (SEQ ID NO: 148), KLYPYDVPDYA (SEQ ID NO: 139), PKPSTPPGSS (SEQ ID NO: 130), GSPAG (SEQ ID NO: 121), GGASPAGG (SEQ ID NO: 113), GGASPAAPAPAG (SEQ ID NO: 106), AMGPSSGAPGGGGS (SEQ ID NO: 98), or SEQ ID NO: 1 The sequences shown in any of NO:98, 106, 113, 121, 130, 139, 148 or 240-246 have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity.

[0226] The small molecule compound further comprises 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) PEG groups between the small molecule compound and the antibody or its antigen-binding fragment. Preferably, the small molecule compound further comprises 3-5 PEG groups between the small molecule compound and the antibody or its antigen-binding fragment.

[0227] Preferably, the small molecule compound is an inhibitor of the target antigen, such as PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine.

[0228] The target antigen can be a target protein, peptide, lipid, polysaccharide or polynucleotide. Preferably, the target protein is selected from membrane proteins, membrane transport proteins, receptor proteins, complexes formed by membrane proteins and membrane proteins, and complexes formed by membrane proteins and other proteins.

[0229] Further preferably, the membrane protein may be a peripheral membrane protein, an intrinsic membrane protein, or a lipid-anchored protein;

[0230] Further preferably, the membrane transport protein may be a carrier protein or a channel protein; preferably, the channel protein may be a sodium ion channel protein, a calcium ion channel protein, a chloride ion channel protein, a potassium ion channel protein, an Ach receptor channel, or an amino acid receptor channel;

[0231] Further preferably, the receptor protein may be an internal receptor, a cell surface receptor, an ion channel receptor, a G-protein coupled receptor (GPCRs) and an enzyme-linked receptor.

[0232] For example: v 1.1、Na v 1.2 Na v 1.3 Na v 1.4 Na v 1.5 Na v 1.6 Na v 1.7 Na v 1.8 Na v 1.9, CD73, VEGFA, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integr in receptor, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12 R, IL15Rα, IL17R, IL23R, PCSK9, TNF-α, TNFR, RANKL, complement protein C3, complement protein C5, GPCR, GLP1R, CD3, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, B7-1, B7-2, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, F AP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3 or 5T4.

[0233] The antibody or antigen-binding fragment thereof may be Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0234] In a specific embodiment of the present invention, the construction method comprises:

[0235] A) obtaining CDR library fragments and assembling them into corresponding positions of the framework antibody to obtain library phagemids;

[0236] B) introducing the library phagemid into E. coli, and displaying the antibody or antigen-binding fragment thereof on the phage surface under the action of a helper phage;

[0237] C) The displayed antibodies or antigen-binding fragments thereof (preferably CDR regions) are coupled with small molecule compounds through a bioorthogonal reaction (preferably Michael addition reaction or Huisgen reaction) to obtain a smacAb library.

[0238] The CDR library fragments are derived from B lymphocytes, and preferably, the B lymphocytes are derived from humans.

[0239] The method of obtaining the CDR library fragments includes separating mononuclear lymphocytes from peripheral blood, extracting RNA, reverse transcribing it into cDNA as a template, and obtaining the CDR library fragments by PCR.

[0240] The backbone antibody is derived from Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody, single chain antibody, IgG, IgA, IgM, IgD or IgE.

[0241] The backbone antibody comprises a light chain and / or a heavy chain.

[0242] The light chain is derived from kappa (κ) or lambda (λ).

[0243] The heavy chain is derived from μ chain, γ chain, α chain, δ chain or ε chain.

[0244] The library phagemid also contains a cleavage sequence inserted at the site where the backbone antibody and the p3 protein are fused. Preferably, the cleavage sequence is selected from any one or a combination of two or more of SEQ ID NOs: 1-4.

[0245] The sixteenth aspect of the present invention provides a smacAb library obtained by the above construction method or the use of the above smacAb library in antibody or smacAb screening.

[0246] In the seventeenth aspect of the present invention, a method for screening antibodies or antigen-binding fragments thereof or smacAb is provided, the screening method comprising adding a target antigen to the smacAb library obtained by the above-mentioned construction method or the above-mentioned smacAb library, and collecting antibodies or antigen-binding fragments thereof that bind to the target antigen.

[0247] The target antigen may be a target antigen expressed on the surface of an overexpressing cell, that is, cells overexpressing the target antigen may be added for screening.

[0248] The collection of antibodies or antigen-binding fragments thereof bound to the target antigen includes an elution step, and the elution can be performed by an acidic solution such as a glycine-HCl solution (pH 2-3) or an alkaline solution such as a 0.1 M triethylamine solution or by enzyme cleavage.

[0249] Preferably, the elution can be an enzyme cleavage elution, for example, recognizing and cleaving the cleavage site on the cleavage sequence.

[0250] The cleavage is thrombin or sequence-specific nickel-assisted cleavage.

[0251] Preferably, the screening method comprises one or more steps of binding, elution and amplification.

[0252] Preferably, the screening method can be a specific screening of antibodies that diagnose and bind to a specific protein subtype, for example, removing the specific protein subtype as a negative screening.

[0253] Preferably, the screening method can be competitive screening or non-competitive screening.

[0254] The eighteenth aspect of the present invention provides an antibody or an antigen-binding fragment thereof, or smacAb obtained by the above-mentioned screening method.

[0255] In a nineteenth aspect, the present invention provides a method for detecting a target antigen, which comprises contacting the target antigen with the above-mentioned antibody conjugate or the above-mentioned antibody or antigen-binding fragment thereof.

[0256] The twentieth aspect of the present invention provides a use of the above-mentioned antibody conjugate, the above-mentioned antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell or immune cell or the above-mentioned smacAb library in the preparation of products for diagnosing and / or treating diseases.

[0257] The disease is selected from tumors, inflammation, and infectious diseases.

[0258] The twenty-first aspect of the present invention provides a drug or a diagnostic kit, wherein the drug or the diagnostic kit comprises the above-mentioned antibody conjugate, the above-mentioned antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell or immune cell.

[0259] The twenty-second aspect of the present invention provides a method for treating a disease, which comprises applying the above-mentioned antibody conjugate, the above-mentioned antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned chimeric antigen receptor, the above-mentioned host cell, the above-mentioned immune cell or smacAb.

[0260] Preferably, the method comprises applying an effective amount of the above-mentioned antibody conjugate, the above-mentioned antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned chimeric antigen receptor, the above-mentioned host cell, the above-mentioned immune cell or smacAb to a subject in need.

[0261] The disease may be a tumor, an infectious disease or an inflammation.

[0262] The "antibody or antigen-binding fragment thereof" described in the present invention represents a structure having conventional similarity to Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody, single chain antibody or full-length antibody, and does not represent whether its binding strength to the target antigen is high or low. The antibody or antigen-binding fragment thereof may bind to the target antigen with high, extremely high, low, extremely low, or even no binding. For example, the smacAb library constructed by the present invention must have conventional similarity to Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody, single chain antibody or full-length antibody structure with high, low or no binding strength in the smacAb library. For another example, the antibody conjugate obtained by screening from the smacAb library must have a very strong ability to bind to the target antigen, but this may be the effect of small molecule compounds, or the effect of antibodies or their antigen-binding fragments, or more often the synergistic effect of coupling small molecule compounds with antibodies or their antigen-binding fragments, etc.

[0263] The "antigen-binding fragment" of the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody can specifically bind to an epitope on the target molecule of the antibody. It includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2 and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or more than two CDRs from the heavy chain or light chain of an antibody. Among them, Fab, that is, a monovalent fragment consisting of VL, VH, CL and CH1 domains. F(ab')2, that is, a divalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region. Fd, an Fd fragment consisting of VH and CH1 domains. Fv, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' in which the cysteine ​​residue of the constant domain carries at least one free thiol group. Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain, and CH1 is CH1 that constitutes the heavy chain constant region.

[0264] The "non-natural amino acid" of the present invention is a compound containing an amino group and a carboxyl group that is not naturally found in proteins. Preferably, the non-natural amino acid is any non-natural amino acid known in the art. Further preferably, the non-natural amino acid includes but is not limited to N-ethylaspartyl, hydroxylysine, 3-hydroxyproline, 2-aminobutyric acid, β-alanine, β-aminopropionic acid, 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, allo-isoleucine, isodesmosine, 4-hydroxyproline, allo-hydroxylysine, 2-aminoisobutyric acid, N-methylglycine, N-methylisoleucine, 3-aminoisobutyric acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, ornithine, norleucine, norvaline, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine or 2-aminopimelic acid, etc. Of course, the "non-natural amino acids" described in this application may also be modified derivatives of natural amino acids.

[0265] The "method" described in the present invention can be used for the purpose of diagnosis and treatment of diseases, or for the purpose of diagnosis and treatment of non-diseases.

[0266] The "infectious disease" described in the present invention is selected from bacterial infection, viral infection or fungal infection; further preferably, viral infection, pneumonia with septic shock, peritonitis, bacteremia, sepsis or septicemia; the viral infection is selected from acute viral infection or chronic viral infection; preferably, influenza virus, parainfluenza virus, herpes virus (such as HSV-1, EBV), measles virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, lymphocytic choriomeningitis virus or human papillomavirus.

[0267] The "inflammation" described in the present invention can be inflammation occurring in any tissue, and the tissues include but are not limited to the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicle, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and leukocytes. Further preferably, the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (e.g., allergic rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity reactions, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis or interstitial cystitis.

[0268] The "tumor" of the present invention can be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased tendency or risk of undesirable cell proliferation, neoplasm or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). Neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblastic cells, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0269] The "subject" described in the present invention can be a human or non-human mammal, or a cell, organ or tissue of a human or non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0270] The "effective amount" of the present invention refers to the amount or dosage of the drug of the present invention that provides the desired treatment or prevention after being administered to an individual or an organ in a single or multiple doses.

[0271] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0272] The term "diagnosis" as used herein refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression of a disease or its possible future progression.

[0273] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0274] The "homology" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39 %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

[0275] Beneficial effects:

[0276] First: connecting small molecule compounds to the CDR3 region, which is easily accessible to the antigen and fully exposed, does not affect the expression level of the antibody, nor does it affect the affinity, and can form a specific connection, which also ensures the diversity of the smacAb library, as well as 10 9 The storage capacity is about 2000 square meters.

[0277] Second: By inserting a cutting sequence into the fusion site of herFab and p3 protein, specific and efficient elution can be achieved without affecting the activity of the phage.

[0278] Third: Compared with single small molecule compounds or antibodies, the prepared smacAb has higher affinity, inhibitory activity and specificity, and the binding of small molecule compounds to the binding pocket assists the affinity of the antibody and compensates for the lack of epitopes.

[0279] Fourth: The library designed in this application can increase the probability of obtaining functional antibodies.

[0280] Fifth: Through competitive inhibition screening, specific binding screening of target protein subtypes is performed to reverse the preference of ASF small molecules for NaV1.7 and screen out smacAbs that specifically bind to NaV1.7, NaV1.2, and NaV1.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0281] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0282] Figure 1 :Selection of site-directed mutation sites of Herceptin, among which, Figure 1 A is the amino acid sites on the surface of Herceptin, which are colored according to the degree of embedding in the Herceptin-HER2 complex (red, >75%; orange, >50-75%; yellow, >25-50%). The upper part of the dotted line is the heavy chain site, and the lower part is the light chain amino acid site; Figure 1 B is a reducing SDS-PAGE gel electrophoresis of the Herceptin antibody with site-directed mutations at positions 91, 92, and 94 of the light chain to Cys, followed by Coomassie Brilliant Blue staining to observe the protein bands.

[0283] Figure 2 : Phage ELISA was used to detect the expression of herFab.

[0284] Figure 3 : Schematic diagram of the reaction between herFab phage and Cy5 detection molecule, wherein the upper figure is Michael addition reaction (Michael addition) and the lower figure is click reaction (Huisigen Cycloaddition).

[0285] Figure 4 : The coupling reaction of site-directed mutant herFab phage and the corresponding Cy5 detection molecule, wherein Figure A is the chemical structure of UAA; Figure B is the specific detection of the coupling reaction of herFab-UAA and Cy5-DBCO, and Ctrl is the wild-type herFab phage.

[0286] Figure 5 :4 different cleavage sequences were inserted into the fusion site of herFab and p3 protein, and the flow chart of the elution efficiency detection of herFab phage.

[0287] Figure 6 : After the ELISA plate was coated with HER2 antigen, herFab phages with four cleavage sequences were added for binding. After cleavage and elution under different conditions, the phage elution effect was detected. A is the addition of HRP-labeled anti-M13 antibody to the ELISA well after elution, and the remaining phages in the ELISA well plate were detected. The data points are expressed as mean±SD, n=3; B is the phage in the elution solution infecting TG1 bacterial solution, coating the plate and culturing overnight, counting the clones and calculating the titer of the eluted phage.

[0288] Figure 7 : PCR amplifies the CDRs fragment library and prepares it into ssDNA, wherein A is a schematic diagram of PCR primer design, where the primers are located at the corresponding positions of the herFab phagemid; B is agarose gel electrophoresis to identify the CDRs fragment library obtained by PCR reaction; C is the principle of ssDNA preparation, where the dsDNA product labeled with single-side biotin is combined with streptavidin magnetic beads, and the DNA double helix is ​​unwound with NaOH solution to release the ssDNA without biotin labeling into the solution.

[0289] Figure 8 : Overlap PCR product detection of Fab library fragments, where HC is the heavy chain, LC is the light chain, and DNA ladder is the DNA fragment.

[0290] Fig. 9 : Cloning and sequencing results of Fab-UAA library phagemid.

[0291] Fig.10 : The coupling effect of Fab-UAA library phage and Cy5-DBCO, among which the Ctrl group is the wild-type herFab phage.

[0292] Fig.11 : Flowchart of Fab-BN3 library screening for smacAb targeting CD73.

[0293] Fig.12 : Identification of CD73 protein, wherein A is the identification of CD73 purity by Coomassie Brilliant Blue staining after reducing SDS-PAGE gel electrophoresis; B is the identification of CD73 protein purity by SEC.

[0294] Fig.13 :Inhibition curves of PSB12379 (left) and BN3 (right) small molecules on CD73.

[0295] Fig.14: Clonal sequence analysis and clone selection of CD73.

[0296] Fig.15 : The inhibitory activity of 12.5 nM Ab-BN3 on CD73, A is a schematic diagram of the Ab-BN3 structure; B is the incubation of CD73 with 12.5 nM Ab-BN3 and then the addition of AMP substrate for reaction to detect the phosphate concentration in the product.

[0297] Fig.16 : Dose-effect curve of Ab-BN3 inhibiting CD73.

[0298] Fig.17 : Detection of the inhibitory activity of Fab-BN3 on CD73, A is a schematic diagram of the structure of Fab-BN3; B is the phosphate concentration in the product detected by the malachite green phosphorus determination method after pre-incubation of Fab-BN3 with CD73 at different concentrations. Data points are shown as mean±SD, n=2, and Graphpad was used for fitting and plotting.

[0299] Fig.18 : Flowchart of Fab-ASF library screening based on overexpression cell lines.

[0300] Fig.19 : Chemical structures of ASF and ASF-PEG-DBCO.

[0301] Fig. 20 : ASF-PEG-DBCO with different PEG chain lengths inhibits Na V 1.7 Activity.

[0302] Fig.21 :Na V 1.7 Sequencing analysis of clones screened from the library.

[0303] Fig. 22 :Ab-PEG3-ASF versus Na V The inhibition curve is 1.7.

[0304] Fig.23 :Na V 1.7 Selective Ab-ASF for Na V 1.6 Inhibitory activity test.

[0305] Fig.24 :Na V 1.7 Inhibitory Ab-ASF on Na V The inhibitory activity of 1.2.

[0306] Fig.25 :Na V 1.7 Clonal enrichment statistics for competitive library screening.

[0307] Fig.26 :Na V 1.7 Selective Ab-ASF inhibitory activity test, where A is 10nM Ab-PEG3-ASF inhibits Na V 1.7 activity test; B is Ab-PEG3-ASF inhibiting Na V The dose-effect curve of 1.7. Data points are expressed as mean±SD, n=2-4, and Graphpad was used for fitting and plotting.

[0308] Fig. 27 :Na V 1.7 Selectivity test of Ab-ASF.

[0309] Fig.28 :ASF-PEG-DBCO to Na V 1.2 and Na V 1.6 inhibitory activity, specifically: HEK293-Na V 1.2 or HEK293-Na V 1.6 Different concentrations of ASF-PEG-DBCO molecules were added to the cells, and the corresponding inhibitory activity was tested using the whole-cell patch clamp technique. The data are shown as mean±SD, n=2-3.

[0310] Fig.29 :Na V 1.2 Statistics of clone enrichment in non-competitive library screening.

[0311] Fig.30 :Na V 1.2 Clonal enrichment statistics for competitive library screening.

[0312] Fig.31 :Na V 1.2 Selective Ab-ASF inhibitory activity test, where A is 10nM and 100nM Ab-ASF inhibits Na V 1.2 activity test; B is the most active of the two Ab-PEG3-ASF inhibition of Na V The dose-effect curve of 1.2.

[0313] Fig.32 :Na V 1.2 Selectivity testing of Ab-ASF.

[0314] Fig.33 :Na V 1.6 Statistics of clone enrichment in library screening.

[0315] Fig.34 :Na V 1.6 Clonal enrichment statistics for competitive library screening.

[0316] Fig.35 :HEK293-Na V 1.6 The cells were incubated with different concentrations of Ab-ASF at room temperature for 15 min and then the inhibitory activity was tested. A was 10 nM and 100 nM Ab-ASF inhibited Na V 1.6 activity test; B is the most active of the two Ab-PEG3-ASF inhibition of Na V The dose-effect curve of 1.2. Data points are expressed as mean±SD, n=2-4, and Graphpad was used for fitting and plotting.

[0317] Fig.36 : Selectivity test of 5A5-PEG3-ASF.

[0318] Fig.37 :H3-PEG3-ASF Mal Activity test results. DETAILED DESCRIPTION

[0319] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0320] The following is a specific implementation plan:

[0321] 1. An antibody conjugate, comprising a covalent conjugate formed by site-specific conjugation of an antibody or an antigen-binding fragment thereof with a small molecule compound, wherein the small molecule compound and the antibody or its antigen-binding fragment jointly participate in binding to a target antigen.

[0322] 2. According to the antibody conjugate described in embodiment 1, the small molecule compound is conjugated to the complementarity determining region (CDR) of the antibody or its antigen binding fragment; preferably, the small molecule compound is conjugated to the heavy chain CDR3 or light chain CDR3 of the antibody or its antigen binding fragment, and further preferably, the small molecule compound is conjugated to the light chain CDR3 of the antibody or its antigen binding fragment.

[0323] 3. According to the antibody conjugate described in Embodiment 2, the light chain CDR3 length is 3-20 aa.

[0324] 4. According to any one of embodiments 1-3, the small molecule compound is conjugated to any amino acid residue of the light chain CDR3 of the antibody or its antigen-binding fragment, preferably at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0325] 5. According to any one of embodiments 2-4, the light chain CDR3 comprises QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTF (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0326] 6. The antibody conjugate according to any one of embodiments 1-5, wherein the antibody or antigen-binding fragment thereof is coupled to the small molecule compound via a bioorthogonal reaction;

[0327] Preferably, the bioorthogonal reaction comprises a Michael addition reaction or a Huisgen reaction.

[0328] 7. According to any one of embodiments 1-6, the antibody conjugate further comprises a linker group between the small molecule compound and the antibody or its antigen-binding fragment. Preferably, the linker group can be a connecting peptide, a PEG group,

[0329]

[0330]

[0331]

[0332]

[0333] Preferably, the connecting peptide may include a sequence as shown in any one of SEQ ID NOs: 98, 106, 113, 121, 130, 139, 148 or 240-246 or have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 98, 106, 113, 121, 130, 139, 148 or 240-246.

[0334] 8. According to any one of embodiments 1-7, the target antigen may be a target protein, peptide, lipid, polysaccharide or polynucleotide. Preferably, the target protein is selected from membrane proteins, membrane transport proteins, receptor proteins, complexes formed by membrane proteins and membrane proteins, and complexes formed by membrane proteins and other proteins.

[0335] Further preferably, the membrane protein may be a peripheral membrane protein, an intrinsic membrane protein, or a lipid-anchored protein;

[0336] Further preferably, the membrane transport protein may be a carrier protein or a channel protein; preferably, the channel protein may be a sodium ion channel protein, a calcium ion channel protein, a chloride ion channel protein, a potassium ion channel protein, an Ach receptor channel, or an amino acid receptor channel;

[0337] Further preferably, the receptor protein may be an internal receptor, a cell surface receptor, an ion channel receptor, a G-protein coupled receptor (GPCRs) and an enzyme-linked receptor.

[0338] 9. According to the antibody conjugate according to any one of embodiments 1-8, the antibody or its antigen-binding fragment is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0339] 10. The antibody conjugate according to any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof is a CD73 antibody or an antigen-binding fragment thereof, and the CD73 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0340] The VLCDR1 comprises any one of SEQ ID NOs: 30-39, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 30-39;

[0341] The VLCDR2 comprises any one of SEQ ID NOs: 40-45, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 40-45;

[0342] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0343] The VHCDR1 comprises any one of SEQ ID NOs: 46-55, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 46-55;

[0344] The VHCDR2 comprises any one of SEQ ID NOs: 56-64, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 56-64;

[0345] The VHCDR3 comprises any one of SEQ ID NOs: 65-72, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 65-72.

[0346] 11. The antibody conjugate according to embodiment 10, wherein VLCDR1 and VLCDR2 respectively comprise:

[0347] Antibody ID VL CDR1 VL CDR2 10D8 RASQSISSWLA (SEQ ID NO: 30) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 10A7 RASQGISRWLA (SEQ ID NO: 31) NLLIYGASTLQSGVPP (SEQ ID NO: 41) 4C10 RATQAISNYLA (SEQ ID NO: 32) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 5E12 RTSQGITDPLN (SEQ ID NO: 33) KLLIYKTSSLESGVPS(SEQ ID NO: 42) 5H12 RASQGLSSWLA (SEQ ID NO: 34) KLLIYDASNLETGVPS (SEQ ID NO: 43) 6D11 RASQNIGTYLA (SEQ ID NO: 35) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2B RASQGINNYLA (SEQ ID NO: 36) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2G QASHDISKYLN (SEQ ID NO: 37) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2A RASETIGSWLA (SEQ ID NO: 38) NLLIYAASTLQSGVSS(SEQ ID NO:44) 10F8 QASQDISNYLN (SEQ ID NO: 39) NLLIYAASTLQSGVPS(SEQ ID NO: 45)

[0348] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0349]

[0350]

[0351] 12. The antibody conjugate of embodiment 10 or 11, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 73-82, or is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs: 73-82;

[0352] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 83-92, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 83-92.

[0353] 13. According to any one of embodiments 1-12, the antibody or antigen-binding fragment thereof is a CD73 antibody or an antigen-binding fragment thereof, the small molecule compound is PSB12379, and the structure of PSB12379 is:

[0354]

[0355] 14. The antibody conjugate according to any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof is Na v 1.7 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.7 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0356] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100;

[0357] The VLCDR2 comprises any one of SEQ ID NOs: 101-105 or 107-108, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101-105 or 107-108;

[0358] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0359] The VHCDR1 comprises any one of SEQ ID NOs: 109-112 or 114, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109-112 or 114;

[0360] The VHCDR2 comprises any one of SEQ ID NOs: 115-120 or 122-123, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115-120 or 122-123;

[0361] The VHCDR3 comprises the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132.

[0362] 15. The antibody conjugate according to embodiment 14, wherein VLCDR1 and VLCDR2 respectively comprise:

[0363]

[0364] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0365]

[0366]

[0367] 16. The antibody conjugate according to embodiment 14 or 15, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 142-147 or 149-150, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 142-147 or 149-150;

[0368] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141.

[0369] 17. The antibody conjugate according to any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof is Na v 1.2 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.2 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0370] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156;

[0371] The VLCDR2 comprises any one of SEQ ID NOs: 102, 103, 157-163, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 102, 103, 157-163;

[0372] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0373] The VHCDR1 comprises any one of SEQ ID NOs: 110, 111, 164-170, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 110, 111, 164-170;

[0374] The VHCDR2 comprises any one of SEQ ID NOs: 116, 117, 171-177, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 116, 117, 171-177;

[0375] The VHCDR3 comprises any one of SEQ ID NOs: 125, 126, 178-183, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 125, 126, 178-183.

[0376] 18. The antibody conjugate according to embodiment 17, wherein VLCDR1 and VLCDR2 respectively comprise:

[0377]

[0378]

[0379] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0380]

[0381]

[0382] 19. The antibody conjugate according to embodiment 17 or 18, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 143, 144, 191-197, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 143, 144, 191-197;

[0383] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190.

[0384] 20. The antibody conjugate according to any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof is Na v 1.6 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.6 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0385] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203;

[0386] The VLCDR2 comprises any one of SEQ ID NOs: 101, 103, 104, 204-209, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101, 103, 104, 204-209;

[0387] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29;

[0388] The VHCDR1 comprises any one of SEQ ID NOs: 109, 111, 210-215, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109, 111, 210-215;

[0389] The VHCDR2 comprises any one of SEQ ID NOs: 115, 117, 118, 216-221, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115, 117, 118, 216-221;

[0390] The VHCDR3 comprises any one of SEQ ID NOs: 124, 126, 127, 222-227, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 124, 126, 127, 222-227.

[0391] 21. The antibody conjugate according to embodiment 20, wherein VLCDR1 and VLCDR2 respectively comprise:

[0392]

[0393]

[0394] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0395]

[0396]

[0397] 22. The antibody conjugate according to embodiment 20 or 21, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 142, 144, 145, 234-239, or is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs: 142, 144, 145, 234-239;

[0398] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233.

[0399] 23. The antibody conjugate according to any one of embodiments 1-9 and 14-22, wherein the antibody or antigen-binding fragment thereof is Na v 1.2 Antibodies or antigen-binding fragments thereof, Na v 1.6 Antibodies or antigen-binding fragments thereof or Na v 1.7 Antibodies or antigen-binding fragments thereof, wherein the small molecule compound is an aromatic sulfonamide, preferably 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine, and the structure thereof is:

[0400]

[0401] 24. A CD73 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0402] The VLCDR1 comprises any one of SEQ ID NOs: 30-39, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 30-39;

[0403] The VLCDR2 comprises any one of SEQ ID NOs: 40-45, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 40-45;

[0404] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0405] The VHCDR1 comprises any one of SEQ ID NOs: 46-55, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 46-55;

[0406] The VHCDR2 comprises any one of SEQ ID NOs: 56-64, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 56-64;

[0407] The VHCDR3 comprises any one of SEQ ID NOs: 65-72, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 65-72.

[0408] 25. The CD73 antibody or antigen-binding fragment thereof according to embodiment 24, wherein VLCDR1 and VLCDR2 respectively comprise:

[0409] Antibody ID VL CDR1 VL CDR2 10D8 RASQSISSWLA (SEQ ID NO: 30) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 10A7 RASQGISRWLA (SEQ ID NO: 31) NLLIYGASTLQSGVPP (SEQ ID NO: 41) 4C10 RATQAISNYLA (SEQ ID NO: 32) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 5E12 RTSQGITDPLN (SEQ ID NO: 33) KLLIYKTSSLESGVPS(SEQ ID NO: 42) 5H12 RASQGLSSWLA (SEQ ID NO: 34) KLLIYDASNLETGVPS (SEQ ID NO: 43) 6D11 RASQNIGTYLA (SEQ ID NO: 35) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2B RASQGINNYLA (SEQ ID NO: 36) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2G QASHDISKYLN (SEQ ID NO: 37) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2A RASETIGSWLA (SEQ ID NO: 38) NLLIYAASTLQSGVSS(SEQ ID NO:44) 10F8 QASQDISNYLN (SEQ ID NO: 39) NLLIYAASTLQSGVPS(SEQ ID NO: 45)

[0410] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0411]

[0412]

[0413] 26. The CD73 antibody or antigen-binding fragment thereof according to embodiment 24 or 25, wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 73-82, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 73-82;

[0414] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 83-92, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 83-92.

[0415] 27. A kind of Na v 1.2 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.2 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0416] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 94, 95, 151-156;

[0417] The VLCDR2 comprises any one of SEQ ID NOs: 102, 103, 157-163, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 102, 103, 157-163;

[0418] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0419] The VHCDR1 comprises any one of SEQ ID NOs: 110, 111, 164-170, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 110, 111, 164-170;

[0420] The VHCDR2 comprises any one of SEQ ID NOs: 116, 117, 171-177, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 116, 117, 171-177;

[0421] The VHCDR3 comprises any one of SEQ ID NOs: 125, 126, 178-183, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 125, 126, 178-183.

[0422] 28. Na according to embodiment 27 v 1.2 An antibody or antigen-binding fragment thereof, wherein VLCDR1 and VLCDR2 respectively comprise:

[0423]

[0424]

[0425] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0426]

[0427]

[0428] 29. Na according to embodiment 27 or 28 v 1.2 An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 143, 144, 191-197, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 143, 144, 191-197;

[0429] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 134, 135, 184-190.

[0430] 30. A Na v 1.6 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.6 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0431] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 93, 95, 96, 198-203;

[0432] The VLCDR2 comprises any one of SEQ ID NOs: 101, 103, 104, 204-209, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101, 103, 104, 204-209;

[0433] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0434] The VHCDR1 comprises any one of SEQ ID NOs: 109, 111, 210-215, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109, 111, 210-215;

[0435] The VHCDR2 comprises any one of SEQ ID NOs: 115, 117, 118, 216-221, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115, 117, 118, 216-221;

[0436] The VHCDR3 comprises any one of SEQ ID NOs: 124, 126, 127, 222-227, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 124, 126, 127, 222-227.

[0437] 31. Na according to embodiment 30 v 1.6 An antibody or antigen-binding fragment thereof, wherein VLCDR1 and VLCDR2 respectively comprise:

[0438]

[0439]

[0440] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0441]

[0442]

[0443] 32. Na according to embodiment 30 or 31 v 1.6 An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises any one of SEQ ID NOs: 142, 144, 145, 234-239, or is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs: 142, 144, 145, 234-239;

[0444] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 133, 135, 136, 228-233.

[0445] 33. A Na v 1.7 Antibodies or antigen-binding fragments thereof, wherein the Na v 1.7 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3;

[0446] The VLCDR1 comprises a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 93-97 or 99-100;

[0447] The VLCDR2 comprises any one of SEQ ID NOs: 101-105 or 107-108, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101-105 or 107-108;

[0448] The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid;

[0449] The VHCDR1 comprises any one of SEQ ID NOs: 109-112 or 114, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109-112 or 114;

[0450] The VHCDR2 comprises any one of SEQ ID NOs: 115-120 or 122-123, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115-120 or 122-123;

[0451] The VHCDR3 comprises the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NOs: 124-129 or 131-132.

[0452] 34. Na according to embodiment 33 v 1.7 An antibody or antigen-binding fragment thereof, wherein VLCDR1 and VLCDR2 respectively comprise:

[0453]

[0454]

[0455] VHCDR1, VHCDR2 and VHCDR3 respectively include:

[0456]

[0457]

[0458] 35. Na according to embodiment 33 or 34 v 1.7 An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 142-147 or 149-150, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 142-147 or 149-150;

[0459] The light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to a sequence as shown in any one of SEQ ID NOs: 133-138 or 140-141.

[0460] 36. According to any one of embodiments 24-35, the antibody or antigen-binding fragment thereof is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0461] 37. A nucleic acid molecule encoding the CD73 antibody or antigen-binding fragment thereof according to any one of embodiments 24-26 and 36, the Na according to any one of embodiments 27-29 and 36 v 1.2 Antibodies or antigen-binding fragments thereof, Na according to any one of embodiments 30-32 and 36 v 1.6 Antibody or antigen-binding fragment thereof or Na according to any one of embodiments 33-36 v 1.7 Antibodies or antigen-binding fragments thereof.

[0462] 38. A vector, wherein the vector expresses the antibody or antigen-binding fragment thereof described in any one of embodiments 24-36 or comprises the nucleic acid molecule described in embodiment 37.

[0463] 39. A host cell or immune cell, comprising the antibody or antigen-binding fragment thereof described in any one of embodiments 24-36, or the nucleic acid molecule described in embodiment 37, or the vector described in embodiment 38.

[0464] 40. Application of small molecule compounds, the application comprising:

[0465] A) Construction of smacAb library;

[0466] Preferably, the small molecule compound is site-specifically coupled to the antibody or antigen-binding fragment thereof in the antibody or antigen-binding fragment thereof library to form a covalent conjugate, namely, the smacAb library;

[0467] B) smacAb screening;

[0468] Preferably, the small molecule compound is site-specifically coupled to an antibody or an antigen-binding fragment thereof in a library of antibodies or antigen-binding fragments thereof to form a covalent conjugate, namely, a smacAb library, and smacAb is obtained by target antigen screening;

[0469] C) Increase the binding strength or affinity between the antibody and the target antigen;

[0470] Preferably, the small molecule compound and the antibody are site-specifically coupled to form a covalent conjugate, wherein the small molecule compound and the antibody jointly participate in the binding with the target antigen.

[0471] 41. According to the application described in embodiment 40, the small molecule compound is selected from PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine.

[0472] 42. According to the use described in embodiment 40, the target antigen can be a target protein, peptide, lipid, polysaccharide or polynucleotide. Preferably, the target protein is selected from membrane proteins, membrane transport proteins, receptor proteins, complexes formed by membrane proteins and membrane proteins, and complexes formed by membrane proteins and other proteins;

[0473] Further preferably, the membrane protein may be a peripheral membrane protein, an intrinsic membrane protein, or a lipid-anchored protein;

[0474] Further preferably, the membrane transport protein may be a carrier protein or a channel protein; preferably, the channel protein may be a sodium ion channel protein, a calcium ion channel protein, a chloride ion channel protein, a potassium ion channel protein, an Ach receptor channel, or an amino acid receptor channel;

[0475] Further preferably, the receptor protein may be an internal receptor, a cell surface receptor, an ion channel receptor, a G-protein coupled receptor (GPCRs) and an enzyme-linked receptor.

[0476] 43. According to the use described in embodiment 40, the antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment described in any one of embodiments 24-36.

[0477] 44. A smacAb library, comprising forming covalent conjugates by site-specifically coupling small molecule compounds with antibodies or antigen-binding fragments thereof in an antibody or antigen-binding fragment library.

[0478] 45. According to the smacAb library described in embodiment 44, the small molecule compound is coupled to the complementarity determining region (CDR) of the antibody or its antigen binding fragment; preferably, the small molecule compound is coupled to the heavy chain CDR3 or light chain CDR3 of the antibody or its antigen binding fragment, and further preferably, the small molecule compound is coupled to the light chain CDR3 of the antibody or its antigen binding fragment.

[0479] 46. ​​According to the smacAb library described in Embodiment 45, the light chain CDR3 length is 3-20 aa.

[0480] 47. According to any one of embodiments 44-46 of the smacAb library, the small molecule compound is coupled to any amino acid residue of the light chain CDR3 of the antibody or its antigen-binding fragment, preferably position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0481] 48. According to any one of embodiments 45-47 of the smacAb library, the light chain CDR3 comprises QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTF (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0482] 49. According to any one of embodiments 44-48, the smacAb library, wherein the antibody or antigen-binding fragment thereof is coupled to the small molecule compound via a bioorthogonal reaction;

[0483] Preferably, the bioorthogonal reaction comprises a Michael addition reaction or a Huisgen reaction.

[0484] 50. According to any one of the smacAb libraries described in embodiments 44-49, a linker group is further included between the small molecule compound and the antibody or its antigen-binding fragment. Preferably, the linker group can be a connecting peptide, a PEG group,

[0485]

[0486]

[0487] Preferably, the connecting peptide may include a sequence as shown in any one of SEQ ID NOs: 98, 106, 113, 121, 130, 139, 148 or 240-246 or have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with a sequence as shown in any one of SEQ ID NOs: 98, 106, 113, 121, 130, 139, 148 or 240-246.

[0488] 51. According to the smacAb library described in embodiments 44-50, the small molecule compound is selected from PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine.

[0489] 52. According to the smacAb library described in embodiments 44-50, the antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment described in any one of embodiments 24-36.

[0490] 53. A method for constructing a smacAb library, the method comprising forming a covalent conjugate by site-specific coupling of the displayed antibodies or antigen-binding fragments thereof with small molecule compounds.

[0491] 54. According to the construction method of embodiment 53, the construction method comprises:

[0492] A) obtaining CDR library fragments and assembling them into corresponding positions of the framework antibody to obtain library phagemids;

[0493] B) introducing the library phagemid into E. coli, and displaying the antibody or antigen-binding fragment thereof on the phage surface under the action of a helper phage;

[0494] C) The displayed antibodies or antigen-binding fragments thereof are coupled with small molecule compounds through bioorthogonal reactions to obtain a smacAb library.

[0495] 55. According to the construction method described in embodiment 54, the backbone antibody is derived from Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanoantibody, single domain antibody, single chain antibody, IgG, IgA, IgM, IgD or IgE.

[0496] 56. According to the construction method described in embodiment 53 or 54, the antibody or its antigen-binding fragment is Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, nanobody, single domain antibody or single chain antibody.

[0497] 57. According to the construction method described in any one of embodiments 53-56, the antibody or its antigen-binding fragment comprises a light chain CDR3, and the light chain CDR3 comprises QQ*YTTPPT (SEQ ID NO: 5), CQQ*YTTPPT (SEQ ID NO: 6), YCQQ*YTTPPT (SEQ ID NO: 7), YYCQQ*YTTPPT (SEQ ID NO: 8), TYYCQQ*YTTPPT (SEQ ID NO: 9), QQ*YTTPPTF (SEQ ID NO: 10), QQ*YTTPPTFG (SEQ ID NO: 11), QQ*YTTPPTFGQ (SEQ ID NO: 12), QQ*YTTPPTFGQG (SEQ ID NO: 13), CQQ*YTTPPTF (SEQ ID NO: 14), CQQ*YTTPPTFG (SEQ ID NO: 15), CQQ*YTTPPTFGQ (SEQ ID NO: 16), CQQ*YTTPPTFGQG (SEQ ID NO: 17). NO: 17), YCQQ*YTTPPTF (SEQ ID NO: 18), YCQQ*YTTPPTFG (SEQ ID NO: 19), YCQQ*YTTPPTFGQ (SEQ ID NO: 20), YCQQ*YTTPPTFGQG (SEQ ID NO: 21), YYCQQ*YTTPPTF (SEQ ID NO: 22), YYCQQ*YTTPPTFG (SEQ ID NO: 23), YYCQQ*YTTPPTFGQ (SEQ ID NO: 24), YYCQQ*YTTPPTFGQG (SEQ ID NO: 25), TYYCQQ*YTTPPTF (SEQ ID NO: 26), TYYCQQ*YTTPPTFG (SEQ ID NO: 27), TYYCQQ*YTTPPTFGQ (SEQ ID NO: 28) or TYYCQQ*YTTPPTFGQG (SEQ ID NO: 29), or with SEQ ID Any of the sequences shown in NO:5-29 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity, wherein * is a natural amino acid or a non-natural amino acid and is a site at which a small molecule compound is coupled to an antibody or its antigen-binding fragment.

[0498] 58. According to the construction method described in any one of embodiments 53-57, the small molecule compound is selected from PSB12379 or 5-(2-(4-(N-(1,2,4-thiadiazole-5-sulfonamido)-2,5-difluorophenoxy)-5-chlorophenyl)benzisoxazole)-3-amine.

[0499] 59. Use of a smacAb library obtained by the construction method described in any one of embodiments 53-58 or a smacAb library described in any one of embodiments 44-52 in antibody or smacAb screening.

[0500] 60. A method for screening antibodies or antigen-binding fragments thereof or smacAb, the screening method comprising adding a target antigen to a smacAb library obtained by the construction method described in any one of Embodiments 53-58 or a smacAb library described in any one of Embodiments 44-52, and collecting antibodies or antigen-binding fragments thereof that bind to the target antigen.

[0501] 61. Use of the antibody conjugate described in any one of embodiments 1-23, the antibody or antigen-binding fragment thereof described in any one of embodiments 24-36, the nucleic acid molecule described in embodiment 37, the vector described in embodiment 38, the host cell or immune cell described in embodiment 39, or the smacAb library described in any one of embodiments 44-52 in the preparation of a product for diagnosing and / or treating a disease.

[0502] 62. According to the use described in embodiment 61, the disease is selected from tumors, inflammation, and infectious diseases.

[0503] 63. A drug or a diagnostic kit, wherein the drug or the diagnostic kit comprises the antibody conjugate described in any one of embodiments 1-23, the antibody or its antigen-binding fragment described in any one of embodiments 24-36, the nucleic acid molecule described in embodiment 37, the vector described in embodiment 38, or the host cell or immune cell described in embodiment 39.

[0504] 64. A method for treating a disease, the method comprising applying the antibody conjugate described in any one of embodiments 1-23, the antibody or its antigen-binding fragment described in any one of embodiments 24-36, the nucleic acid molecule described in embodiment 37, the vector described in embodiment 38, or the host cell or immune cell described in embodiment 39.

[0505] 65. According to the method described in embodiment 64, the disease is selected from tumors, infectious diseases or inflammation.

[0506] Example 1: Preparation of antibody conjugate (smacAb)

[0507] 1. Select the coupling site of small molecule compounds

[0508] The active small molecule compound is site-specifically coupled to the CDR3 of the light chain of the antibody, so that it binds to the antigen as part of the antibody and exerts its activity. Figure 1 A) as an example, this example selects amino acid 91 of the Herceptin light chain with the highest surface embedding degree of CDR3, followed by amino acid 92 and amino acid 94. First, the amino acids at the above three sites of the Herceptin light chain are mutated to Cys to observe whether the site-directed mutation will affect protein expression.

[0509] The results are as follows Figure 1 As shown in B, the three site-directed mutant antibodies were all successfully expressed, and the expression levels were not much different. The small molecule coupling site was randomly selected to be set to the 91st amino acid of the light chain CDR3 for subsequent experiments.

[0510] 2. Preparation of herFab phage

[0511] Herceptin, also known as trastuzumab, was approved by the FDA in 1998 for the treatment of HER2-overexpressing breast cancer. It is a humanized monoclonal antibody that is widely used in clinical treatment and scientific research. The light chain variable region framework of Herceptin is derived from human kappa light chain variable region subgroup I (Human VL kappa subgroup I), which is the most abundant subgroup in kappa light chains, and the heavy chain variable region framework is derived from human heavy chain variable region subgroup III (Human VH subgroup III), which is the most abundant subgroup in heavy chains.

[0512] The herFab phagemid was introduced into TG1 Escherichia coli and prepared into phage-displayed herFab with the assistance of M13KO7 phage. The display of herFab on the phage surface was tested by phage ELISA experiment. Specifically, 0.5 μg / mL Herceptin antibody and each phage (diluted to OD 268 The ELISA plate was coated with HRP-labeled anti-human kappa LC antibody for binding, and finally HRP substrate TMB was added for color development and the OD was measured with an enzyme reader. 450 The data points are shown as mean ± SD, n = 3. Figure 2 As shown, herFab was successfully expressed on the surface of phage.

[0513] 3. Coupling method

[0514] According to the design of smacAb, it is ensured that the small molecule compound is specifically coupled to the antibody mutation site without reacting with the rest of the antibody and the phage capsid protein, so as to reduce the adverse effects of non-specific coupling of small molecules on library screening. In this example, two specific coupling methods are selected, one of which is the Michael addition reaction between the thiol group of cysteine ​​and the maleimide group (see Figure 3 The other is the click reaction between the azide group of UAA and the DBCO group (Huisigen Cycloaddition, see Figure 3 To confirm the specificity of the coupling reaction, the amino acids at the mutation sites of the phage-displayed herFab were mutated to Cys and UAA (see the structure). Figure 4 A), herFab phages were prepared with site-directed mutations, and then the specificity of the two coupling reactions with the corresponding herFab phages was tested by Cy5-Mal and Cy5-DBCO, respectively.

[0515] The coupling reaction conditions were room temperature for 3 h, the products were subjected to reducing SDS-PAGE gel electrophoresis, and the Cy5 band was detected by Typhoon.

[0516] The results of the coupling product detection of herFab-UAA phage and Cy5-DBCO in different molar ratios are shown in Figure 2. Figure 4 As shown in B, the Ctrl group is a wild-type herFab phage, and no obvious bands appear after the reaction. The herFab-UAA phage has a single band at the light chain position, indicating that specific coupling has occurred. Within a 150-fold ratio, as the added Cy5-DBCO ratio increases, the Cy5 band gradually increases, indicating that the coupling efficiency gradually increases, while there is no obvious difference between the 150-fold and 200-fold bands, indicating that the reaction has been basically saturated.

[0517] Example 2: Optimization of elution method

[0518] In the screening process of phage display antibody library, acidic solution such as glycine-HCl solution (pH 2-3) or alkaline solution such as 0.1M triethylamine solution is usually used for elution, and this elution mode makes the antibody-phage bound to the antigen free to the eluent by dissociating the antigen-antibody complex. Although acidic and alkaline elution modes are widely used in the elution process of phage display antibody library, the stronger the binding force, the more difficult it is to dissociate the antigen-antibody complex, so it is not conducive to the elution of high-affinity antibodies, and this elution mode is also easy to have an adverse effect on phage activity. Therefore, the present embodiment designs to insert a cleavage sequence at the herFab and p3 protein fusion site, and the specific cutting of the cleavage sequence is used to realize the elution of phage.

[0519] Two cleavage methods were selected as phage elution methods and their respective elution effects were observed. The first is thrombin, which can specifically recognize the sequence LVPRGSG (SEQ ID NO: 1) and cut between R and G. The second is sequence-specific nickel-assisted cleavage (SNAC), which is a chemical cleavage assisted by Ni ions (Dang et al., 2019). Three SNAC sequences were specifically selected and labeled as SNAC1, SNAC2 and SNAC3, respectively. Their cleavage sequences are NSLGSQHQAQ (SEQ ID NO: 2), GGSHHTDLPGGS (SEQ ID NO: 3) and PGSHHWG (SEQ ID NO: 4), respectively. The cleavage site of SNAC is between the first GS in the sequence.

[0520] Test whether these four cleavage sequences can achieve efficient elution of phage. The overall process design is as follows Figure 5 As shown, in the experiment, HER2 antigen was coated on the ELISA plate, and after blocking, 4 kinds of herFab phages were added for binding and fully washed to remove unbound phages. Then, different cutting times were set for elution at 37°C, and the eluted phages were collected to infect TG1 bacterial liquid to determine the phage titer. At the same time, the phage ELISA experiment was used to detect the remaining phages in the ELISA plate.

[0521] Phage ELISA results Figure 6 As shown in A, the four herFab phages containing different cleavage sequences can all bind to HER2, and the OD value of the positive control glycine elution group was significantly reduced, indicating that some phages have been dissociated into the eluent. The elution of phages with three SNAC sequences showed similar results. There was no significant difference in their OD values ​​at the three time points of 0.5-2h, and they were equivalent to the OD value of the negative control PBS group, indicating that the cutting efficiency of the three SNAC sequences was poor. The Thrombin cleavage sequence phage showed a better elution effect. The OD value of the 0.5h cleavage group was slightly higher than that of the positive control glycine group. When the cleavage time was extended to 1h, its OD value was equivalent to that of the glycine group, and the OD values ​​of the 2h and 1h cleavage groups were similar, indicating that the cleavage process was basically completed at 1h. The activity test results of the phages in the eluent are shown in Figure 6 As shown in B, the clone numbers of the three cutting time groups of the thrombin cutting sequence phage were all higher than those of the glycine group. It is possible that a better cutting effect can be achieved if the cutting time is controlled within 1 h.

[0522] After comprehensively examining the phage elution efficiency of the four cleavage sequences, the thrombin cleavage sequence was selected as the cleavage sequence for subsequent use. This cleavage sequence can achieve the separation of herFab from phage under mild conditions, and the cleavage efficiency is similar to that of glycine elution. In addition, thrombin cleavage has less effect on phage activity than glycine elution, which is beneficial to maintaining phage activity.

[0523] Example 3: Fab-UAA library construction

[0524] The construction of the Fab-UAA library mainly includes three steps: (1) Obtaining the fragment library of CDRs. During the library construction process, the sequence of the CDR containing the mutation site, namely the light chain CDR3, remains unchanged, and the diversity of the antibody library is achieved through the fragment library of the remaining 5 CDRs. (2) The CDRs fragment library is assembled to the corresponding position of the backbone antibody to prepare the Fab-UAA library phagemid. (3) The Fab-UAA library is displayed on the phage surface to verify the specific coupling of Fab-UAA and Cy5-DBCO again.

[0525] 1. Herceptin was used as the backbone antibody for the construction of the Fab library (see "2. Preparation of herFab phage" in Example 1).

[0526] 2. Acquisition of CDRs library fragments

[0527] Human B lymphocyte genes were used as the source of CDRs library fragments. In the experiment, mononuclear lymphocytes were isolated from the peripheral blood of 5 healthy people and RNA was extracted. The RNA was reverse transcribed into cDNA as a template to obtain CDRs library fragments through PCR reaction. PCR primers were designed as follows: Figure 7 As shown in A, using herFab-UAA phagemid as a vector, a single-side biotin-labeled primer was designed in the FR region of the antibody, and a PCR reaction was performed to obtain the corresponding DNA fragment. A homologous sequence was designed at the junction of adjacent fragments to facilitate the subsequent assembly of the Fab sequence using an overlap PCR reaction. The five fragments containing CDRs were amplified by PCR using cDNA as a template, and the products were identified by agarose gel electrophoresis as shown in Figure 7 As shown in Figure 2, the light chain CDR1 and CDR2 are located on products L2 and L3, respectively, and the heavy chain CDR1, CDR2 and CDR3 are located on products H2, H3 and H5, respectively. The length of each product fragment is between 100 bp and 250 bp, which is consistent with the estimated length. The remaining fragments were amplified by PCR reaction using herFab-UAA phagemid as a template.

[0528] In order to assemble the CDRs library fragments into Fab-UAA library fragments, a multi-fragment overlap PCR reaction is proposed to assemble the heavy chain and light chain separately. In order to successfully carry out the multi-fragment overlap PCR reaction, each PCR fragment needs to be prepared as single-stranded DNA (ssDNA). The preparation principle of ssDNA is as follows Figure 7 As shown in Figure C, since the primers were designed with a single-side biotin label, each PCR product was bound to streptavidin magnetic beads, and then the DNA double strands were dissociated using a NaOH solution. The ssDNA in the double strands that was not labeled with biotin was released into the solution, and the ssDNA of each fragment was obtained by purification and recovery, which was used in the subsequent multi-fragment overlap PCR assembly experiment.

[0529] 3. Assembly of Fab-UAA library phagemid

[0530] In the experiment, a two-step overlap PCR method was used to assemble the ssDNA into Fab library fragments. First, the light chain and heavy chain were assembled into complete chains by overlap PCR. The ssDNA fragments of the light chain included L1, L2, L3 and L4, and the size of their overlap PCR products should be about 800 bp. The ssDNA fragments of the heavy chain included H1, H2, H3, H5 and the artificially synthesized backbone region ssDNA fragment VH-H4, and the size of their overlap PCR products was about 500 bp. The PCR products were identified by agarose gel electrophoresis. Figure 8 As shown, the nucleic acid bands in the product were observed under ultraviolet light. The positions of the light chain and heavy chain were consistent with the prediction, so the PCR product was cut and recovered. The light chain and heavy chain were then assembled into Fab-UAA library fragments, and the gel electrophoresis identification results were shown as follows: Figure 8 As shown, the estimated length of the PCR product is about 1300 bp, and the position of the band is consistent with the expected length, indicating that the CDRs library fragments have been successfully assembled into Fab library fragments.

[0531] Finally, the Fab-UAA library fragments were loaded onto the phagemid by enzyme cutting and ligation. In the experiment, the Fab library fragments were loaded onto the herFab-UAA-thrombin phagemid to construct the Fab-UAA library phagemid. In order to detect the assembly of the Fab-UAA library phagemid, the library phagemid was transformed into Escherichia coli TG1, coated on a plate and cultured into monoclonal colonies, and then 10 monoclonal colonies were randomly selected for Sanger sequencing to observe the Fab sequences they contained. Multiple sequence alignment was performed using the ClustalW Multiple Alignment method. The sequencing results are shown in Figure 2. Fig. 9As shown, the sequencing of clone 5 failed, the heavy chain of clone 6 was missing, and the remaining 8 clones were assembled correctly without duplicate clones, indicating that the CDRs of most clones in the library were assembled correctly and diverse, and the Fab-UAA library phagemid was successfully constructed.

[0532] 4. Preparation and coupling test of Fab-UAA library phage

[0533] In order to obtain a high-capacity phage display Fab-UAA library, the Fab-UAA phagemid library was introduced into Escherichia coli by electroporation. Since electroporation requires the purity of the library phagemid to be as high as possible and the salt concentration to be as low as possible, the Fab-UAA library phagemid was first purified by column and eluted in ultrapure water. At the same time, the UAA expression auxiliary plasmid 2104 was transfected into the TG1 strain and TG1-2104 electroporation competent cells were prepared. Finally, the purified Fab-UAA library phagemid was electroporated into TG1-2104 electroporation competent cells, and the plate was coated for clone counting. The library capacity of the Fab-UAA library was calculated to be 10 9 Left and right, indicating that the high-capacity phage-displayed Fab-UAA was successfully constructed.

[0534] In order to confirm that the Fab-UAA library phage can undergo specific coupling reaction with DBCO-modified small molecules, Cy5-DBCO was used to characterize the coupling effect of Fab-UAA phage. Specifically, Fab-UAA phage was coupled with 200 times molar amount of Cy5-DBCO, the product was subjected to reducing SDS-PAGE gel electrophoresis, and then the Cy5 coupling band was detected by Typhoon. The detection results of Cy5 coupling product are as follows Fig.10 As shown, after the Fab-UAA phage was coupled with Cy5-DBCO, a Cy5 band was observed at the light chain position, which was similar to the coupling result of herFab-UAA, proving that the Fab-UAA library phage can achieve specific coupling of DBCO-modified small molecules.

[0535] So far, a library with a capacity of 10 9 The phage display Fab-UAA library can be specifically coupled with the target protein active small molecule compound with DBCO group through the N3 group carried by UAA, so as to construct a smacAb library suitable for smacAb screening of target protein.

[0536] Example 4: Screening and identification of smacAb with CD73 as target protein

[0537] The flowchart of Fab-BN3 library screening for smacAb targeting CD73 is shown in Fig.11 The specific steps are as follows:

[0538] 1. Expression, purification and identification of CD73 protein

[0539] First, the purified CD73 protein antigen was obtained. The pcDNA3.4-Chis CD73 plasmid was transfected into 293 cells for expression, and the cell culture supernatant containing CD73 protein was collected and purified with a nickel column. The obtained CD73 was stored at -80°C. The purity of CD73 protein was identified by reducing SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. The theoretical molecular weight of CD73 protein is 58.8kD. The electrophoresis results are as follows Fig.12 A, which shows a single band, preliminarily judged that the protein purity is high. And the molecular weight of the band is between 55-70kD, which is in line with expectations. Then SEC was used to verify the purity of CD73. The results are as follows Fig.12 As shown in B, a single peak can be seen at 9.573 min, and the protein purity is over 98%, so the next step of the experiment can be continued.

[0540] 2. Synthesis and activity testing of CD73 small molecule inhibitor BN3

[0541] The CD73 competitive inhibitory small molecule PSB12379 was selected as the core structure, and a PEG3 flexible linker was added between PSB12379 and the coupling group DBCO to synthesize the small molecule inhibitor BN3.

[0542] It is necessary to determine whether the modified small molecule BN3 still has the activity to inhibit CD73. In the experiment, the activity of small molecule inhibitors was tested with recombinantly expressed CD73 protein. CD73 protein hydrolyzes AMP to obtain adenosine and PO4 3- ,PO4 3- It can be quantified by the malachite green phosphorus determination method. Therefore, CD73 is pre-incubated with a small molecule inhibitor, and then the AMP substrate is added for reaction to test the PO4 in the reaction product. 3- Specifically, small molecule inhibitors of different concentrations were pre-incubated with CD73 protein at 37°C for 30 min, AMP substrate was added and reacted at room temperature for 15 min, and malachite green phosphorus determination detection reagent was added and incubated for 30 min before measuring OD 620 The data points are shown as mean±SD, n=2, and the inhibition curves were fitted by Graphpad software.

[0543] The inhibitory activity of the original structure small molecule PSB12379 and the modified small molecule BN3 were tested in the experiment. The small molecule inhibition curves are shown in Figure 2. Fig.13 As shown, IC 50 The concentration statistics are shown in Table 1, where the IC 50 The IC of the modified small molecule BN3 is 1040nM.50 It is 70.46nM. These results show that BN3 retains the inhibitory activity well and can be used for the next step of coupling and screening.

[0544] Table 1: Statistics of inhibitory activity of PSB12379 and BN3

[0545] Small molecules <![CDATA[IC 50 ±SEM(nM)]]> PSB12379 1040±120.4 BN3 70.46±4.778

[0546] 2. Phage display Fab-BN3 library screening

[0547] 1) Fab-BN3 screening of CD73

[0548] according to Fig.11 The library screening process design shown in the figure uses a freshly prepared Fab-BN3 library for CD73 screening. Negative screening with streptavidin magnetic beads is the first choice. The Fab-BN3 library is incubated with streptavidin magnetic beads to remove clones bound to streptavidin magnetic beads in the Fab-BN3 library. Collect the Fab-BN3 library after negative screening, add CD73-biotin protein for binding, and then capture CD73-biotin with new streptavidin magnetic beads. After multiple washes, remove the phages nonspecifically adsorbed on the surface, and then add thrombin for enzyme cleavage and elution. The eluted phages are collected to infect TG1-2104 and amplified with the participation of M13KO7 helper phages to prepare the Fab-BN3 sublibrary for the next round of screening. The screening process is generally repeated for 3-4 rounds.

[0549] 2) Fab-BN3 sequence analysis and clone selection

[0550] After 3-4 rounds of screening, duplicate clones have been detected, so clones need to be selected for subsequent activity testing. First, duplicate clones 10A7 (10A7 / 10F9 group) and 10D8 (10D8 / 10G9 group) were selected. In addition, the VH sequences of the remaining clones were analyzed, with CDR3 sequences given priority, and the enrichment of CDR3 was observed through multiple sequence alignment. The results are as follows Fig.14 As shown, the sequences were grouped into one cluster based on CDR3 similarity, and the sequenced clones were classified into 5 clusters in total. Then, the sequence distribution of CDR2 was compared and analyzed within each cluster, and the sequences were grouped into different subclasses based on sequence similarity, and clones were selected in each subclass for the next step of the experiment.

[0551] In this experiment, a total of 10 clones, including clones 10A7, 10D8, ABN3-2A, ABN3-2B, ABN3-2G, 4C10, 5E12, 5H12, 6D11, and 10F8 (wherein the VL CDR3 sequence is as shown in SEQ ID NO: 5-29, the VL CDR1-2 sequence is shown in Table 2, and the VH CDR1-3 sequence is shown in Table 3), were selected for subsequent expression and activity testing. Among them, 6D11 and 10D8 are from the same cluster of CDRH3 sequences but belong to different CDR2H subclasses, 5H12 and 4C10 are also from the same cluster of CDRH3 sequences but belong to different CDR2H subclasses, ABN3-2B and 10A7 are from unique CDRH3 clusters and CDR2H subclasses, ABN3-2A and ABN3-2G belong to the same CDRH3 cluster and the same CDR2H subclass, and the remaining clones 5E12 and 10F8 are clones that are not similar to the above sequences among the measured sequences and are randomly selected clones.

[0552] Table 2: VL CDR1-2

[0553] Antibody ID VL CDR1 VL CDR2 10D8 RASQSISSWLA (SEQ ID NO: 30) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 10A7 RASQGISRWLA (SEQ ID NO: 31) NLLIYGASTLQSGVPP (SEQ ID NO: 41) 4C10 RATQAISNYLA (SEQ ID NO: 32) KLLIYDVSNVQTGVPS (SEQ ID NO: 40) 5E12 RTSQGITDPLN (SEQ ID NO: 33) KLLIYKTSSLESGVPS(SEQ ID NO: 42) 5H12 RASQGLSSWLA (SEQ ID NO: 34) KLLIYDASNLETGVPS (SEQ ID NO: 43) 6D11 RASQNIGTYLA (SEQ ID NO: 35) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2B RASQGINNYLA (SEQ ID NO: 36) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2G QASHDISKYLN (SEQ ID NO: 37) KLLIYDASNLETGVPS (SEQ ID NO: 43) ABN3-2A RASETIGSWLA (SEQ ID NO: 38) NLLIYAASTLQSGVSS(SEQ ID NO:44) 10F8 QASQDISNYLN (SEQ ID NO: 39) NLLIYAASTLQSGVPS(SEQ ID NO: 45)

[0554] Table 3: VH CDR1-3

[0555]

[0556]

[0557] The amino acid sequences of the heavy chain variable region and light chain variable region of each antibody are as follows:

[0558]

[0559] >10D8.VH

[0560]

[0561] >ABN3-2A.VH

[0562]

[0563] >ABN3-2B.VH

[0564]

[0565] >ABN3-2G.VH

[0566]

[0567] >4C10.VH

[0568]

[0569] >5E12.VH

[0570]

[0571] >5H12.VH

[0572]

[0573] >6D11.VH

[0574]

[0575] >10F8.VH

[0576]

[0577] >10A7.VL

[0578]

[0579] >10D8.VL

[0580]

[0581] >ABN3-2A VL

[0582]

[0583] >ABN3-2B.VL

[0584]

[0585] >ABN3-2G.VL

[0586]

[0587] >4C10.VL

[0588]

[0589] >5E12.VL

[0590]

[0591] >5H12.VL

[0592]

[0593] >6D11.VL

[0594]

[0595] >10F8.VL

[0596]

[0597] 3) Antibody expression and BN3 conjugation

[0598] In order to prepare the selected clones as smacAb for functional testing, the antibodies were first expressed as full-length IgG1. The antibodies were then conjugated to BN3 molecules and functionally tested.

[0599] 3. Functional testing

[0600] 1) Ab-BN3 activity test

[0601] In order to observe the activity of the screened Ab-BN3 and study the mode of action of Ab-BN3 and CD73, the inhibitory activity of Ab-BN3 and the corresponding unconjugated antibody was tested. Fig.15 A) Inhibitory activity against CD73 at a concentration of 12.5 nM (about 2 μg / mL). Fig.15 As shown in B, all 10 Ab-BN3s have certain inhibitory activity.

[0602] In the experiment, five Ab-BN3 with good activity were selected to measure the inhibition curve. Specifically, CD73 was incubated with different concentrations of Ab-BN3, and then AMP substrate was added for reaction. The phosphate concentration in the product was detected by malachite green phosphorus determination method. The data points are shown as mean±SD, n=2, and Graphpad was used for fitting and plotting. The results are shown in Figure 2. Fig.16 As shown, the IC values ​​of 5 Ab-BN3 50 The IC values ​​of each Ab-BN3 were in the range of 0.2-0.8 nM (Table 4). 50 However, the IC of Ab-BN3 calculated from the inhibition curve 90 The concentrations of 10A7-BN3 showed great differences, and the best inhibitory effect was observed with an IC 90 The inhibitory effect of BN3 was 2.9nM, followed by 6D11-BN3 at 8.2nM, and the worst inhibitory effect was 10D8-BN3. 50 and IC 90 The activity of the five Ab-BN3s was significantly improved compared with BN3, which was 70.5nM and 760.4nM respectively. In addition, it was observed in the experiment that most of the antibodies not coupled with BN3 did not have inhibitory activity, and only 6D11 showed a weak inhibitory trend. Therefore, it can be considered that the inhibitory activity of Ab-BN3 is the result of the complementary effect of BN3 and antibodies.

[0603] Table 4: IC of Ab-BN3 inhibiting CD73 50 and IC 90

[0604]

[0605] 2) Fab-BN3 activity test

[0606] Monovalent antibody fragments such as Fab and scFv have a smaller size and are easier to penetrate into the tumor microenvironment, thereby better exerting tumor immunotherapy functions. According to the design of the smacAb library, the Fab library is used for the screening of smacAb, so it is speculated that the smacAb obtained by the screening can function in a monovalent form. In order to prove that the monovalent smacAb still has CD73 inhibitory activity, the IgG1 antibodies of the most active clones 6D11 and 10A7 were cut with papain and the Fab fragments were recovered. Then, the Fab was coupled with BN3 to prepare a monovalent Fab-BN3 ( Fig.17 A) and tested its inhibitory activity against CD73. The inhibition curve is shown in Fig.17 As shown in B, compared with the inhibitory activity of Ab-BN3, the inhibitory activity of the two Fab-BN3s showed opposite trends. 10A7-Fab-BN3 retained the inhibitory activity well, and its IC 50 The IC50 of 6D11-Fab-BN3 is 7.315nM, which is 1 / 5 of that of 10A7-BN3. 50 It increased by more than 10 times, indicating that the activity of monovalent smacAb was significantly reduced.

[0607] Overall, the monovalent Fab-BN3 prepared from the two Ab-BN3s retained the inhibitory activity against CD73, proving that Ab-BN3 interacts with CD73 using Fab-BN3 as the recognition unit, which is consistent with the design of smacAb. The inhibitory activity of the two Fab-BN3s showed different trends compared with the corresponding Ab-BN3, indicating that the two antibodies have different modes of action. 10A7-Fab-BN3 still has strong inhibitory activity and has the potential to be developed into a monovalent antibody.

[0608] 3) Ab-BN3 affinity test

[0609] In order to further verify that the binding of Ab-BN3 is the result of the combined action of the Ab part and the BN3 part, the affinity of Ab-BN3 and the corresponding unconjugated antibody was tested. In the experiment, Octet-BLI was first used to analyze the affinity of Ab-BN3 and CD73. Ab-BN3 was immobilized by proteinAsensor, and then incubated with different concentrations of CD73 solution to obtain the binding signal. The data was collected by the software and fitted and analyzed. The affinity data statistics of each Ab-BN3 are shown in Table 5, where Herceptin-BN3 was used as the control group, and its KD was 1.07×10 -8 M. A total of 8 Ab-BN3s were tested in the experimental group, including the 5 Ab-BN3 groups with the best inhibitory activity and the 3 Ab-BN3 groups with the worst activity among the 10 Ab-BN3s, corresponding to antibodies including ABN-2B, 4C10 and 5E12. The KD of the experimental group was 2.97×10 -10 -4.37×10 -9 Within the M range, the overall KD value is smaller than that of Herceptin-BN3, indicating that the affinity of each Ab-BN3 to CD73 is significantly improved after screening, and the Ab-BN3 in the high-activity group has a smaller KD value than that in the low-activity group, indicating that high affinity is correlated with high inhibitory activity.

[0610] At the same time, the affinity of Ab corresponding to the five most active Ab-BN3 molecules to CD73 was also tested. The results showed that when Ab was in contact with 20μM CD73 solution, only very weak binding signals could be observed, indicating that the affinity of Ab to CD73 was weak.

[0611] Table 5: Affinity data of Ab-BN3 and CD73

[0612]

[0613]

[0614] In summary, the BN3 part in Ab-BN3 is crucial for the generation of affinity, and the appropriate Ab part helps to improve the affinity, so both parts play a role in the binding of Ab-BN3 and CD73, which is consistent with the design of smacAb.

[0615] Example 5: Screening and identification of smacAb with NaV1.7 as target protein

[0616] The Fab-ASF library was incubated with biotinylated target cells and negative cells, and the target cells were obtained by magnetic bead sorting. The bound phages were eluted by thrombin enzyme digestion, and the Fab-ASF library was prepared again after amplification for the next round of screening.

[0617] 1. ASF small molecule synthesis and activity identification

[0618] In order to expand the scope of action of the antibody, a flexible PEG linker was inserted between ASF and DBCO. In the experiment, ASF was coupled with PEG3-DBCO and PEG8-DBCO to prepare ASF-PEG-DBCO molecules with PEG linkers of different lengths (see the structural formula Fig.19 ).

[0619] On this basis, we first verified whether the ASF-PEG-DBCO small molecule still has Na V 1.7 Inhibitory activity. Specifically: HEK293-Na V 1.7 Cells were incubated with different concentrations of ASF-PEG-DBCO for 15 min and then the inhibitory activity was tested by whole-cell patch clamp technique. Data points are shown as mean±SD, n=2-4, and graphpad was used for fitting and plotting. V The inhibition curve of 1.7 is as follows Fig. 20 As shown, the two ASF-PEG-DBCO molecules have V 1.7 all showed inhibitory activity, but ASF-PEG3-DBCO had stronger activity. Its IC 50 It was 2.237 nM, indicating that the long PEG chain had a certain effect on the activity of ASF, so ASF-PEG3-DBCO was selected for subsequent screening.

[0620] 2. Preparation and screening of Fab-ASF library

[0621] Since ASF-PEG3-DBCO has a higher Na V 1.7 Inhibitory activity, the experiment used ASF-PEG3-DBCO to couple with the newly prepared Fab-UAA library to prepare the Fab-PEG3-ASF library for Na V 1.7 Screening. The screening experiment was carried out according to the designed screening process ( Fig.18 ), only Na was used in the first round of screening V 1.7 Overexpressing cells were screened, and 6 times the number of HEK293 cells were added as negative cells for subsequent screening.

[0622] 1) Fab-ASF library targeting Na V 1.7 Screening

[0623] Na V 1.7 Protein library screening was carried out according to the designed screening process ( Fig.18 HEK293-Na V1.7 Overexpression cell lines were used as target cells, and HEK293 was used as negative cells for library screening. A total of 4 rounds of screening were performed in this experiment. After screening, 24 clones of the third round products and 48 clones of the fourth round products were randomly selected for Sanger sequencing analysis, and 22 and 37 valid sequences were obtained, respectively. No duplicate clones appeared in the third round products. In the fourth round of screening, the eluted phage infected TG1 bacterial solution was spread on the plate to grow monoclonal colonies. 48 monoclonal clones were randomly selected for Sanger sequencing, the VH amino acid sequence of Fab was analyzed, and the clone enrichment was statistically analyzed. The results are shown in Fig.21 As shown in Figure 2, four repetitive sequences were enriched in the fourth round of products, including F3, G3, H3 and A4 (wherein, the VL CDR3 sequence is shown in SEQ ID NO: 5-29, the VL CDR1-2 sequence is shown in Table 6, and the VHCDR1-3 sequence is shown in Table 7), among which F3 and A4 sequences were also detected in the third round of products. The clonal enrichment of the fourth round of screening products was significantly improved and repetitive sequences were detected, indicating that for Na V 1.7 For this library screening strategy, four rounds of screening may be necessary. Considering the screening results, four clones, F3, G3, H3 and A4, were selected for antibody expression and conjugation and tested for their effects on Na V 1.7 inhibitory activity and selectivity.

[0624] Table 6: VL CDR1-2

[0625]

[0626]

[0627] Table 7: VH CDR1-3

[0628]

[0629]

[0630] The amino acid sequences of the heavy chain variable region and light chain variable region of each antibody are as follows:

[0631]

[0632] >G3.VL

[0633]

[0634] >H3.VL

[0635]

[0636] >A4.VL

[0637]

[0638] >15F7.VL

[0639]

[0640] >15F9.VL

[0641]

[0642] >15A10.VL

[0643]

[0644]

[0645] >15C12.VL

[0646]

[0647] >F3.VH

[0648]

[0649] >G3.VH

[0650]

[0651] >H3.VH

[0652]

[0653] >A4.VH

[0654]

[0655] >15F7.VH

[0656]

[0657] >15F9.VH

[0658]

[0659] >15A10.VH

[0660]

[0661] >15C12.VH

[0662]

[0663] 2) Antibody expression and conjugation with ASF

[0664] In order to detect the effect of the screened smacAb on Na V To determine the inhibitory activity of 1.7, each clone was first expressed as a full-length IgG1 antibody. Ab-UAA was then coupled with ASF-PEG3-DBCO to prepare Ab-PEG3-ASF, and its inhibitory activity and selectivity were tested.

[0665] 3. Ab-PEG3-ASF inhibits Na V 1.7 Activity Test

[0666] In order to detect the effect of the screened smacAb on Na V The inhibitory activity of Ab-PEG3-ASF on Na V 1.7 inhibitory effect. Specifically: HEK293-Na V 1.7 Cells were incubated with different concentrations of Ab-PEG3-ASF for 15 min and the cellular sodium current was measured by whole-cell patch clamp technique. Data points are shown as mean±SD, n=2-3, and Graphpad was used for fitting and plotting. Ab-PEG3-ASF for Na V The inhibition curve of 1.7 is as follows Fig. 22 As shown in Table 8, the IC50 values ​​of Ab-PEG3-ASF inhibited Na V 1.7 IC 50 The inhibitory activity of H3-PEG3-ASF was the best among the four smacAbs, with an IC 50 About 1nM.

[0667] Table 8: Ab-PEG3-ASF inhibits Na V 1.7 IC 50

[0668] Ab-PEG3-ASF <![CDATA[IC 50 ±SEM(nM)]]> F3-PEG3-ASF 2.692±0.6870 G3-PEG3-ASF 1.880±0.3943 H3-PEG3-ASF 1.129±0.1913 A4-PEG3-ASF 1.218±0.5341

[0669] The experimental results show that the four Ab-PEG3-ASFs selected by the principle of repeated cloning priority through the screening of the Fab-PEG3-ASF library all have Na V 1.7 inhibitory activity. Therefore, the screening of smacAb library can obtain V 1.7 Ab-PEG3-ASF with good inhibitory activity preliminarily verified the applicability of smacAb design and library screening strategy for screening functional antibodies against complex membrane proteins.

[0670] 4. Specificity test of Ab-PEG3-ASF inhibitory activity

[0671] According to literature reports, ASF small molecules have a V 1.2 and Na V 1.6 also has a certain inhibitory activity. In order to detect the ASFsmacAb obtained by screening, V 1.7 inhibitory activity and whether it can achieve selective inhibition of subtype proteins. V 1.2 and Na V 1.6 inhibitory activity, 3-4 concentration points were tested, and the highest concentration was 500nM or 1μM. V 1.6 Cells were incubated with different concentrations of Ab-PEG3-ASF at room temperature for 15 min before testing. Data points are shown as mean±SD, n=2-3, and Graphpad was used for fitting and plotting. V 1.6 Inhibitory activity test results Fig.23 As shown, none of the four Ab-PEG3-ASFs showed any effect on Na V 1.6 of inhibition.

[0672] HEK293-Na V 1.2 The cells were incubated with different concentrations of Ab-PEG3-ASF at room temperature for 15 min and then tested. The data points are shown as mean±SD, n=2-3, and Graphpad was used for fitting and plotting. V The results of the inhibitory activity test of 1.2 are as follows Fig.24 As shown, G3-PEG3-ASF has a certain Na V 1.2 Inhibitory activity: The inhibition rate was about 60% at the highest concentration tested, 500 nM. Except for G3-PEG3-ASF, the other three Ab-PEG3-ASFs showed no inhibition of Na at concentrations of 500 nM or below. V 1.2 inhibition phenomenon.

[0673] In summary, through Na V 1.7 phage display Fab-PEG3-ASF library screening with target, the selected 4 Ab-PEG3-ASF have Na2O3 equivalent to ASF small molecule V 1.7 inhibitory activity, and 3 of them Ab-PEG3-ASF does not have Na V 1.2 and Na V 1.6, indicating that Ab-PEG3-ASF has a selective Na V 1.7 Inhibitory activity: smacAb design can achieve selective inhibition of ion channel subtype proteins. However, the screening products were also enriched for Na V1.2 G3-ASF-PEG3 has inhibitory activity, so the next step is to optimize the library screening strategy to increase the screening product's effect on Na V Selectivity of 1.7.

[0674] 5. Screening, optimization and identification of smacAb library

[0675] In order to further improve the library screening products to Na V The selectivity of 1.7 was optimized. In the library screening process, HEK293-Na V 1.2 Cells and HEK293-Na V 1.6 cells were used as negative cells to replace HEK293 cells for subtype competitive screening of the library (competitive screening for short), hoping to fully remove the Na V 1.2 and Na V 1.6 The combined clones. Similarly, the library screening products were subjected to Sanger sequencing of monoclonal colonies and the Fab genes of the obtained clones were analyzed. The clones were selected based on the principle of repeat clone priority to express Ab-UAA and coupled with ASF-PEG3-DBCO to prepare Ab-PEG3-ASF. The Na V 1.7 inhibitory activity and specificity.

[0676] Targeting Na V 1.7 Competitive Fab-ASF Library Screening: Targeting Na with Fab-PEG3-ASF Library V 1.7 competitive library screening. In the first round of screening, only target cells, namely Na V 1.7 Overexpression cells were screened. Starting from the second round of screening, HEK293-Na V 1.2 Cells and HEK293-Na V 1.6 cells were used as negative cells for competitive screening of the library, and a total of 4 rounds of screening were performed. Monoclonal colonies were randomly selected from the screening products to detect the Fab variable region genes and observe the sequence enrichment of the screening products. V Randomly select single clones from the competitive library screening products of 1.7 for Sanger sequencing, analyze the variable region sequence of Fab and count the sequence enrichment. The sequence enrichment statistics of the competitive library screening products are as follows: Fig.25 As shown, five repeat sequences were detected, two of which were A4 and H3, which had appeared in the non-competitive library screening products and had been verified to have selective Na V 1.7 inhibitory activity, and the other two repeat sequences were newly appeared clones 15F7 and 15A10. In addition, clone 15C12 was found, which was very similar to A4.

[0677] Considering the results of clone sequencing, 15F7, 15A10 and 15C12 (VL CDR3 sequences are shown in SEQ ID NO: 5-29, VL CDR1-2 sequences are shown in Table 6, and VH CDR1-3 sequences are shown in Table 7) were selected as four clones with high sequence enrichment, and another clone 15F9 was randomly selected for subsequent experiments. In subsequent experiments, these five clones were expressed and prepared as Ab-PEG3-ASF and the inhibitory activity and selectivity were tested.

[0678] 6. Ab-PEG3-ASF versus Na V 1.7 Inhibitory activity test

[0679] Given that the above experiments have successfully prepared Na V 1.7 The smacAb obtained by competitive library screening now needs to be tested for the effect of each Ab-PEG3-ASF on the target protein Na V The inhibitory activity of Ab-PEG3-ASF was still tested using Na V 1.7 The overexpression cell lines were tested by whole-cell patch clamp technique. First, the effect of 10 nM Ab-PEG3-ASF on Na V 1.7 inhibitory activity of HEK293-Na V 1.7 After incubating cells with different concentrations of Ab-PEG3-ASF at room temperature for 15 minutes, the inhibitory activity test results were as follows: Fig.26 As shown in A, 15F9-PEG3-ASF had almost no inhibitory activity, 15F7-PEG3-ASF had an inhibition rate of about 60%, and the remaining three Ab-PEG3-ASFs could basically achieve Na V Therefore, four Ab-PEG3-ASFs except 15F9-PEG3-ASF were selected to set up concentration gradient assay to inhibit Na V The dose-effect curve is 1.7.

[0680] 4 Ab-PEG3-ASF to Na V The inhibition curve of 1.7 is as follows Fig.26 As shown in B, the IC values ​​of each Ab-PEG3-ASF were obtained by fitting calculation. 50 The values ​​are shown in Table 9. The worst inhibitory activity is 15F7-PEG3-ASF, with an IC 50 The IC values ​​of the remaining three Ab-PEG3-ASF were 50 All of them were lower than 0.2 nM, which was significantly higher than the inhibitory activity of ASF-PEG3-DBCO, indicating that the optimized library screening could obtain Ab-ASF with stronger inhibitory activity than ASF small molecules.

[0681] Table 9: Na V 1.7 IC of Selective Ab-ASF 50 statistics

[0682] Ab-ASF <![CDATA[IC 50 ±SEM]]> 15F7-PEG3-ASF 6.484±1.199nM 15A10-PEG3-ASF 138.2±41.36pM 15C12-PEG3-ASF 39.45±9.684pM

[0683] 7. Specificity test of Ab-PEG3-ASF inhibitory activity

[0684] To verify the Na V 1.7 The screened Ab-PEG3-ASF has higher specificity than ASF molecules. The Ab-PEG3-ASF was tested for Na V 1.2 and Na V 1.6 inhibitory activity. HEK293-Na V 1.6 Cells and HEK293-Na V 1.2 The cells were incubated with the corresponding concentrations of Ab-PEG3-ASF at room temperature for 15 minutes and then the inhibitory activity was tested. The three concentrations were 10 nM, 100 nM and 500 nM. The data points are expressed as mean ± SD, n = 2-4. The inhibitory activity test results are shown in Fig. 27 As shown in the experiment, the Na V 1.2 and Na V The inhibitory activity of 1.6 was 0.04, and none of the four Ab-PEG3-DBCOs had Na V 1.2 Inhibitory activity and Na V 1.6 also showed no inhibitory activity, indicating that the Ab-ASF obtained by competitive library screening has specific Na V 1.7 Inhibitory activity.

[0685] In summary, targeting Na V The competitive library screening of 1.7 yielded five repeat sequences, including the non-competitive library screening for Na V 1.7 selectively inhibited H3 and A4 clones, as well as two newly discovered repeat sequences 15F7 and 15A10. In addition, the sequencing results also found a clone 15C12 with a similar sequence to H3. Activity test experiments confirmed that the Ab-PEG3-ASF prepared from these four new clones all had Na1.7 inhibitory activity, among which 15F7-PEG3-ASF had the worst inhibitory activity, with an IC 50 The inhibitory activity of the remaining three clones was stronger than that of the small molecule itself. Moreover, both the H3 and A4 clones and the newly discovered four clones had strong inhibitory activity against Na VThe inhibitory activity of 1.7 is very specific, even at a concentration of 500 nM, it is still effective against Na V 1.2 and Na V 1.6 had no inhibitory activity. At the same time, no Na V 1.2 The inhibitory activity of clone G3 was significantly higher than that of clone G3, indicating that competitive screening is helpful in obtaining highly selective clones. V Competitive library screening of 1.7 obtained Ab-PEG3-ASF with higher inhibitory activity and specificity than ASF small molecule, which once again proved that the smacAb screening strategy is suitable for targeting Na V 1.7 Screening of functional antibodies.

[0686] In addition, further attempts were made to use other coupling methods to couple small molecules with antibodies to prepare smacAb. In this example, Na V 1.7 H3-PEG3-ASF was used as a model smacAb to test whether active H3-PEG3-ASF could be prepared by Michael addition reaction Mal In the experiment, the H3 coupling site, i.e. the amino acid 91 of the light chain, was mutated to a thiol-containing Cystine, and expressed as a complete antibody H3-Cys through the HEK293F cell system. At the same time, the ASF molecule was modified with the reactive group PEG3-maleimide to prepare ASF-PEG3-Mal. H3-Cys and ASF-PEG3-Mal were reacted by Michael addition to prepare H3-PEG3-ASF. Mal The patch clamp technique was used to detect the effect of Na V The inhibitory activity of H3-PEG3-ASF was 1.7. Mal The activity test results are as follows Fig.37 The results showed that the coupling reaction of cysteine ​​and maleimide is also suitable for the construction of SmacAb.

[0687] In summary, the smacAb corresponding to H3 was successfully prepared through the Michael addition reaction, and the prepared smacAb still has a certain inhibitory activity. Therefore, it can be considered that the preparation process of smacAb is compatible with other common site-directed coupling technologies.

[0688] Example 6: Screening and identification of smacAbs with NaV1.2 and NaV1.6 as target proteins

[0689] 1. ASF inhibits Na1.2 and Na V 1.6 Activity Test

[0690] In the targeted Na V 1.2 and Na VBefore creating the Fab-ASF library of 1.6, it is necessary to confirm that the DBCO-modified ASF small molecule still has Na V 1.2 and Na V 1.6 Inhibitory activity. Specifically, HEK293 overexpressing cell lines were incubated with small molecules at various concentrations at room temperature for 15 min, and then the activity was tested. Data are shown as mean±SD, n=2-4, and graphpad fitting and plotting were performed. V 1.2 and HEK293-Na V 1.6 Testing the effect of ASF-DBCO on Na V 1.2 and Na V The inhibitory activity of 1.6. Fig.28 As shown, ASF-PEG3-DBCO and ASF-PEG8-DBCO molecules have a V 1.2 all had inhibitory activity, but the extension of the PEG chain had an effect on the Na V 1.2 The inhibitory activity of ASF-PEG3-DBCO was adversely affected. V 1.6 still has inhibitory activity, but IC 50 As high as 683.2 nM, while ASF-PEG8-DBCO had a V 1.6 had no inhibitory activity, indicating that the PEG chain reduced the activity of ASF-DBCO on Na V 1.6 inhibitory activity, with Na V 1.2 The phenomena of the inhibitory activity test experiment are consistent.

[0691] In summary, the ASF-PEG3-DBCO molecule still retains the Na V 1.2 and Na V The inhibitory activity of ASF-PEG8-DBCO was only 1.6. However, the long PEG chain led to a decrease in inhibitory activity. V 1.2 Has lower activity than ASF-PEG3-DBCO but without Na V The inhibitory activity was 1.6.

[0692] 2. Na V 1.2 Screening and identification of smacAb

[0693] Targeting Na V 1.2 Fab-ASF library screening: ASF-PEG3-DBCO and ASF-PEG8-DBCO were selected as small molecules with different PEG chain lengths and coupled with the newly prepared Fab-UAA phage to prepare Fab-PEG3-ASF library and Fab-PEG8-ASF library for screening Na V1.2. Library screening reference Na V 1.7 Screening Process ( Fig.18 ) was performed using HEK293-Na V 1.2 Overexpression cell lines were used as target cells, HEK293 cells were used as negative cells for non-competitive screening, and HEK293-Na V 1.6 Cells and HEK293-Na V 1.7 cells were used as negative cells and four rounds of screening were performed.

[0694] First, we targeted Na V 1.2 Non-competitive library screening. Targeting Na V The eluted phages from the fourth round of non-competitive library screening in 1.2 were used to infect TG1 bacterial solution and spread on plates to form monoclonal colonies. Single clones were randomly selected for Sanger sequencing, and the Fab variable region genes were translated into amino acid sequences for comparison, and the number of sequence repetitions was counted. The statistical results of clone enrichment of library screening products are shown in Figure 1. Fig.29 As shown, two repeating sequences, G3 and F3, were observed in the PEG3 library screening products, but these two repeating sequences were Na V 1.7 Selective antibody sequences, where F3-PEG3-ASF has no Na V 1.2 Inhibitory activity, G3-PEG3-ASF has a weaker inhibitory effect on Na V 1.2 activity. These results indicate that the screening of the Fab-PEG3-ASF library has not yet been able to obtain V 1.2 Selective inhibition of smacAb. Four repetitive sequences were detected in the PEG8 library screening products, three of which were F3, G3 and H3 sequences, and their corresponding Ab-PEG3-ASF was Na V 1.7 selective smacAb. At the same time, a new repeat sequence 5E4 (repeated 5 times) was also discovered. In summary, by targeting Na V 1.2 Non-competitive library screening, the repeated sequences obtained in the PEG3 group are all Na V 1.7 selective clones F3 and G3. Although the PEG8 group detected a new clone 5E4, the clone with the most repetitions in the group was H3, indicating that the screening results of the PEG8 group were not ideal. Therefore, it may be difficult to detect clones with Na V 1.2 Ab-ASF with specific inhibitory activity, considering enrichment of Na by competitive screening strategy V 1.2 Selectivity.

[0695] In order to obtain Na V1.2 Selective inhibition of the activity of Ab-ASF, targeting Na V Competitive library screening of 1.2. Targeting Na V The eluted phages from the fourth round of competitive library screening in 1.2 were used to infect TG1 bacterial solution and spread on plates to form monoclonal colonies. Single clones were randomly selected for Sanger sequencing, and the Fab variable region genes were translated into amino acid sequences for comparison, and the number of sequence repetitions was counted. The sequence enrichment statistics of the competitive library screening products are shown in Figure 1. Fig.30 As shown in Figure 2, the sequence enrichment of the Fab-PEG3-ASF library screening products was not high enough, and the only repeating sequence was Na V 1.7 specific F3 clone. The sequence enrichment of the clones of the Fab-PEG8-ASF library screening product was relatively high, and two repetitive sequences were detected, one of which was Na V 1.7 was the selectively cloned H3, and the other was the new clone 14B8. In addition, a clone 14C7 was found that was similar to the sequence of 14B8.

[0696] Analysis of Targeted Na V The results of clone enrichment of non-competitive screening and competitive screening in 1.2 were used to explore whether competitive screening is beneficial for enriching clones of target proteins. Through the screening of Fab-PEG3-ASF library, two Na V 1.7 Selective clones F3 and G3, only clone F3 appeared in the competitive screening products. Through the screening of Fab-PEG8-ASF library, three Na V 1.7 Selective clones, including F3, G3 and H3, only clone H3 appeared in the competitive screening products. These results show that in the screening process of PEG3 library and PEG8 library, the competitive library screening strategy eliminated some Na V 1.7 Selective cloning, thereby increasing Na V 1.2 Probability of selective clones being detected Therefore, clones were selected from the competitive screening products, expressed as Ab-ASF and tested for their inhibitory activity and selectivity.

[0697] Observe the sequence enrichment of the competitive library screening products and select clones for subsequent experiments. Most of the clone sequences of the PEG3 library screening products are not repetitive sequences, but there is a clustering phenomenon of CDRs. Therefore, one clone was selected from each of the several sequences with high CDRs enrichment for subsequent experiments, including 12B5, 12D4, 14H3, 14H4 and 14E6 (wherein the VL CDR3 sequence is shown in SEQ ID NO: 5-29, the VL CDR1-2 sequence is shown in Table 10, and the VH CDR1-3 sequence is shown in Table 11) for a total of 5 clones. For the screening products of the PEG8 library, since it is not certain whether H3-PEG8-ASF has Na V 1.2 Selective inhibition activity, so clones H3, 14B8 and 14C7 (wherein the VL CDR3 sequences are shown in SEQ ID NOs: 5-29, the VL CDR1-2 sequences are shown in Table 10, and the VH CDR1-3 sequences are shown in Table 11) were selected for subsequent experiments.

[0698] Table 10: VL CDR1-2

[0699]

[0700]

[0701] Table 11: VH CDR1-3

[0702]

[0703]

[0704] The amino acid sequences of the heavy chain variable region and light chain variable region of each antibody are as follows:

[0705]

[0706] >14H3.VL

[0707]

[0708] >14H4.VL

[0709]

[0710] >12D4.VL

[0711]

[0712] >14E6.VL

[0713]

[0714] >14C7.VL

[0715]

[0716] >14B8.VL

[0717]

[0718]

[0719] >12B5.VH

[0720]

[0721] >12D4.VH

[0722]

[0723] >14H3.VH

[0724]

[0725] >14H4.VH

[0726]

[0727] >14E6.VH

[0728]

[0729] >14C7.VH

[0730]

[0731]

[0732] >14B8.VH

[0733]

[0734] 3. Ab-ASF to Na V 1.2 Inhibitory activity test

[0735] Test the expression of Ab-ASF on Na V Since there were a large number of Ab-ASF, we first tested the inhibitory activity of 10nM and 100nM Ab-ASF on Na V 1.2 inhibitory activity. Specifically: HEK293-Na V 1.2 The cells were incubated with different concentrations of Ab-ASF at room temperature for 15 min before the inhibitory activity test was performed. The data points are represented as mean±SD, n=2-4, and Graphpad was used for fitting and plotting. The test results are shown in Fig.31 As shown in A, except for H3-PEG8-ASF, Ab-ASF has a certain Na V 1.2 Inhibitory activity, and Ab-PEG8-ASF all have weak inhibitory activity, which may be related to the weak activity of ASF-PEG8-DBCO. The Ab-ASFs with the best inhibitory activity are 14H4-PEG3-ASF and 14E6-PEG3-ASF, which can basically completely inhibit at 100nM. Therefore, 14H4-PEG3-ASF and 14E6-PEG3-ASF were selected to set up the inhibition experiment with concentration gradient, and the inhibition curves are shown in the figure. Fig.31 As shown in B, we obtained the IC values ​​of two Ab-PEG3-ASFs through fitting calculation. 50 IC of 14H4-PE3-ASF 50 The IC of 14E6-PEG3-ASF is 2.731 nM. 50 The ASF-PEG3-DBCO small molecule inhibited Na V 1.2 IC 50 was 7.384 nM, indicating that after targeting Na V Competitive library screening of 1.2 obtained Ab-PEG3-ASF, which has slightly stronger inhibitory activity than ASF small molecule.

[0736] 4. Specificity test of Ab-ASF inhibitory activity

[0737] In order to explore the V 1.2 Whether the inhibitory activity of the obtained Ab-ASF is selective was tested for Na V 1.2 The two Ab-ASFs with the strongest inhibitory activity against Na V 1.7 and Na V The inhibitory activity of Ab-ASF on Na V 1.6 and Na V 1.7 inhibitory activity of HEK293-Na V 1.6 Cells and HEK293-Na V 1.7 The cells were incubated with the corresponding concentrations of Ab-PEG3-ASF at room temperature for 15 min and then the inhibitory activity was tested. The three concentrations were 10 nM, 100 nM and 500 nM. The data points are expressed as mean ± SD, n = 2-4. The test results are shown in Fig.32 As shown, both Ab-ASFs showed no effect on Na V 1.6 inhibition. Na V The cell inhibition data of 1.7 showed that 14E6-PEG3-ASF did not have NaV 1.7 inhibitory activity, but 14H4-PEG3-ASF has a certain Na V 1.7 inhibitory activity, the inhibition rate was about 30% at a concentration of 100nM, and the inhibition rate was about 40% at a concentration of 500nM. Compared with the small molecule ASF-PEG3-DBCO, Ab-PEG3-ASF had a better inhibitory effect on Na V 1.7 decreased significantly, indicating that Ab-PEG3-ASF V The selectivity of 1.2 is significantly improved.

[0738] In summary, by targeting Na V The competitive library screening of 1.2 verified that the competitive library screening strategy can remove more clones that bind to non-target proteins during the screening process, thereby increasing the probability of detecting target protein clones. Through this screening strategy, two clones that bind to Na V 1.2 Ab-PEG3-ASF has stronger inhibitory activity than ASF-PEG3-DBCO small molecule. Both Ab-PEG3-ASFs showed no inhibitory activity against Na V 1.6 Inhibitory activity, 14E6-PEG3-ASF had an inhibitory effect on Na V 1.7 also had no inhibitory activity, and 14H4-PEG3-ASF had a weaker Na V 1.7 Inhibitory activity and IC calculation 50 These results show that the competitive library screening strategy was used to obtain a V 1.2 Selective inhibition of active smacAb, indicating that smacAb library screening can reverse the effects of ASF small molecules on Na V The results showed that the smacAb and smacAb library screening strategies are also applicable to targeting Na V The screening of inhibitory antibodies against 1.2 once again verified the applicability of this strategy for screening functional antibodies against complex membrane protein targets.

[0739] 5. Na V 1.6 Screening and identification of smacAb

[0740] Targeting Na V 1.6 Fab-ASF library screening: Na V The smacAb library screening experiment of 1.6 also used Fab-PEG3-ASF and Fab-PEG8-ASF libraries for screening. V1.6 Overexpression cell lines were used as target cells, HEK293 was used as negative cells for non-competitive screening, and HEK293-Na was used as negative cells for competitive library screening. V 1.2 Cells and HEK293-Na V 1.7 cells were used as negative cells and screened for 4 rounds. The products of the 4th round of screening were randomly selected to observe the clone enrichment, and then clones were selected to express and prepare smacAb and test its inhibitory activity and selectivity.

[0741] Targeting Na V The eluted phages from the fourth round of non-competitive library screening in 1.6 were used to infect TG1 bacterial solution and spread on plates to form monoclonal colonies. Single clones were randomly selected for Sanger sequencing, and the Fab variable region genes were translated into amino acid sequences for comparison, and the number of sequence repetitions was counted. The statistical results of clone enrichment in non-competitive library screening are shown in Figure 1. Fig.33 As shown, 3 of the 4 repeat sequences obtained by sequencing of the PEG3 screening group were Na V 1.7 selective clones, and only one unique clone 5A5 remained. Only two duplicate clones were detected in the PEG8 screening group, and both clones were Na V 1.7 Selective cloning. These results indicate that targeting Na V 1.6 Non-competitive library screening products enriched a large amount of Na V 1.7 Selective cloning, so try competitive library screening and see if you can reduce Na V 1.7 Emergence of selective clones.

[0742] In order to obtain Na V 1.6 Selective inhibitory activity of smacAb, targeting Na V 1.6 Competitive library screening. V The inhibitory activity of 1.6 was very weak and the new clones enriched by the non-competitive screening strategy appeared in the screening products of the PEG3 library, targeting Na V The competitive library screening experiment of 1.6 was performed using the Fab-PEG3-ASF library. Monoclonal colonies of competitive library screening products were randomly selected for Sanger sequencing to analyze the variable region sequence of Fab and count the sequence enrichment. The sequence enrichment statistics are shown in Fig.34 As shown, there are 5 repeat sequences in the library screening products, 3 of which are Na V 1.7 Selective F3, H3 and A4 clones were selected, and two new repetitive sequences 15A2 and 15C1 were discovered.

[0743] Analysis and comparison of targeted Na V1.6 The results of non-competitive screening and competitive screening of clone enrichment, to explore whether competitive screening is conducive to enrichment of Na V 1.6 Selective cloning. Through the screening of Fab-PEG3-ASF library, three Na V 1.7 Selective clones F3, G3 and A4, three Na V 1.7 Selective clones F3, H3 and A4. These results show that targeting Na V In the library screening process of 1.6, the competitive library screening strategy enriched Na V 1.7 Selective cloning has slightly changed, but has not significantly reduced the Na in the screening product V 1.7 Types of Selective Clones In general, the competitive screening strategy of the library was applied to target Na V When screening the library of 1.6, the observed results were inconsistent with expectations. It is believed that this may be because ASF binds to Na V The binding pocket of 1.6 and Na V 1.2 and Na V The binding pocket of 1.7 is quite different, resulting in Na V 1.2 and Na V 1.7 failed to play a competitive binding role in the screening process. V The weak affinity of 1.6 may have led to the presence of more non-target clones in the screening products, and it is necessary to increase the number of clones for activity testing to obtain smacAb with selective inhibitory activity.

[0744] Therefore, clones were selected from the screening products of the two screening strategies based on the principle of repetitive sequence priority for subsequent experiments. First, repetitive clones 5A5, 15C1 and 15A2 (wherein the VL CDR3 sequences are as shown in SEQ ID NO: 5-29, the VL CDR1-2 sequences are shown in Table 12, and the VH CDR1-3 sequences are shown in Table 13) were selected, and then, considering the sequence similarity of non-repetitive sequences, three non-repetitive sequence clones including 15F6, 15G4 and 15D3 (wherein the VL CDR3 sequences are as shown in SEQ ID NO: 5-29, the VLCDR1-2 sequences are shown in Table 12, and the VH CDR1-3 sequences are shown in Table 13) were selected for the next experiment.

[0745] Table 12: VL CDR1-2

[0746]

[0747] Table 13: VH CDR1-3

[0748]

[0749]

[0750] The amino acid sequences of the heavy chain variable region and light chain variable region of each antibody are as follows:

[0751]

[0752] >15D3.VL

[0753]

[0754] >15C1.VL

[0755]

[0756] >15G4.VL

[0757]

[0758] >15A2.VL

[0759]

[0760] >5A5.VL

[0761]

[0762] >15F6.VH

[0763]

[0764] >15D3.VH

[0765]

[0766] >15C1.VH

[0767]

[0768]

[0769] >15G4.VH

[0770]

[0771] >15A2.VH

[0772]

[0773] >5A5.VH

[0774]

[0775] 6. Ab-ASF to Na V 1.6 Inhibitory activity test

[0776] In order to test the screened Ab-ASF against Na V The inhibitory activity of each Ab-PEG3-ASF on Na V The inhibitory effect of 1.6. The test results are as follows Fig.35 As shown in A, each clone was sensitive to Na at 100 nM concentration. V 1.6 all showed a certain degree of inhibitory activity, among which 5A5-PEG3-ASF and 15G4-PEG3-ASF had stronger inhibitory activity, and their IC 50 The concentration of 5A5-PEG3-ASF with the best inhibitory activity was set to detect its effect on Na V The inhibition effect is 1.6, and the inhibition curve is as follows Fig.35 B shows the IC of 5A5-PEG3-ASF. 50 is 28.63nM. ASF-PEG3-DBCO small molecule inhibits Na V 1.6 IC 50 The inhibitory activity of 5A5-PEG3-ASF was 683.2 nM, which was more than 20 times higher than that of ASF-PEG3-DBCO.

[0777] 7. Specificity test of Ab-ASF inhibitory activity

[0778] To verify the specificity of the inhibitory activity of 5A5-PEG3-DBCO, its inhibitory activity against Na V 1.2 and Na V 1.7 inhibitory activity. Specifically: HEK293-Na V 1.2 Cells or HEK293-Na V 1.7 The cells were incubated with various concentrations of Ab-ASF at room temperature for 15 min before the inhibitory activity was detected. The data points are expressed as mean ± SD, n = 2-4. Fig.36 As shown, the highest concentration tested in the experiment was 500 nM, and 5A5-PEG3-ASF showed no effect on Na V 1.2 or Na V 1.7 inhibitory activity, indicating that 5A5-PEG3-ASF has Na V Selective inhibitory activity of 1.6.

[0779] In summary, the smacAb design and smacAb library screening strategy were used to obtain V1.6 Selective inhibition of active smacAb, again proving that smacAb library screening can reverse the effects of ASF small molecules on Na V The results showed that the smacAb and smacAb library screening strategies are also applicable to targeting Na V The screening of inhibitory antibodies with a concentration of 1.6 once again verified the applicability of this strategy for screening functional antibodies against complex membrane protein targets.

[0780] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0781] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An antibody conjugate, characterized in that: The antibody conjugate includes a covalent conjugate formed by site-specific conjugation between an antibody or an antigen-binding fragment thereof and a small molecule compound, wherein the small molecule compound and the antibody or an antigen-binding fragment thereof jointly participate in the binding with the target antigen.

2. A CD73 antibody or an antigen-binding fragment thereof, characterized in that: The CD73 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3; The VLCDR1 comprises any one of SEQ ID NOs: 30-39, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 30-39; The VLCDR2 comprises any one of SEQ ID NOs: 40-45, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 40-45; The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid; The VHCDR1 comprises any one of SEQ ID NOs: 46-55, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 46-55; The VHCDR2 comprises any one of SEQ ID NOs: 56-64, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 56-64; The VHCDR3 comprises any one of SEQ ID NOs: 65-72, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 65-72.

3. A Na v 1.2 An antibody or an antigen-binding fragment thereof, characterized in that: The Na v 1.2 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3; The VLCDR1 comprises any one of SEQ ID NOs: 94, 95, 151-156, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 94, 95, 151-156; The VLCDR2 comprises any one of SEQ ID NOs: 102, 103, 157-163, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 102, 103, 157-163; The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid; The VHCDR1 comprises any one of SEQ ID NOs: 110, 111, 164-170, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 110, 111, 164-170; The VHCDR2 comprises any one of SEQ ID NOs: 116, 117, 171-177, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 116, 117, 171-177; The VHCDR3 comprises any one of SEQ ID NOs: 125, 126, 178-183, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 125, 126, 178-183.

4. A Na v 1.6 An antibody or an antigen-binding fragment thereof, characterized in that: The Na v 1.6 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3; The VLCDR1 comprises any one of SEQ ID NOs: 93, 95, 96, 198-203, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 93, 95, 96, 198-203; The VLCDR2 comprises any one of SEQ ID NOs: 101, 103, 104, 204-209, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101, 103, 104, 204-209; The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid; The VHCDR1 comprises any one of SEQ ID NOs: 109, 111, 210-215, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109, 111, 210-215; The VHCDR2 comprises any one of SEQ ID NOs: 115, 117, 118, 216-221, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115, 117, 118, 216-221; The VHCDR3 comprises any one of SEQ ID NOs: 124, 126, 127, 222-227, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 124, 126, 127, 222-227.

5. A Na v 1.7 An antibody or an antigen-binding fragment thereof, characterized in that: The Na v 1.7 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3; The VLCDR1 comprises any one of SEQ ID NOs: 93-97 or 99-100, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 93-97 or 99-100; The VLCDR2 comprises any one of SEQ ID NOs: 101-105 or 107-108, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 101-105 or 107-108; The VLCDR3 comprises any one of SEQ ID NOs: 5-29, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 5-29, wherein * is a natural amino acid or a non-natural amino acid; The VHCDR1 comprises any one of SEQ ID NOs: 109-112 or 114, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 109-112 or 114; The VHCDR2 comprises any one of SEQ ID NOs: 115-120 or 122-123, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 115-120 or 122-123; The VHCDR3 comprises the sequence shown in any one of SEQ ID NO: 124-129 or 131-132, or has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the sequence shown in any one of SEQ ID NO: 124-129 or 131-132.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CD73 antibody or antigen-binding fragment thereof according to claim 2, the Na v 1.2 Antibodies or antigen-binding fragments thereof, Na according to claim 4 v 1.6 Antibody or antigen-binding fragment thereof or Na according to claim 5 v 1.7 Antibodies or antigen-binding fragments thereof.

7. Application of small molecule compounds, characterized in that: The applications described include: A) Construction of smacAb library; Preferably, the small molecule compound is site-specifically coupled to the antibody or antigen-binding fragment thereof in the antibody or antigen-binding fragment thereof library to form a covalent conjugate, namely, the smacAb library; B) smacAb screening; Preferably, the small molecule compound is site-specifically coupled to an antibody or an antigen-binding fragment thereof in a library of antibodies or antigen-binding fragments thereof to form a covalent conjugate, namely, a smacAb library, and smacAb is obtained by target antigen screening; C) Increase the binding strength or affinity between the antibody and the target antigen; Preferably, the small molecule compound and the antibody are site-specifically coupled to form a covalent conjugate, wherein the small molecule compound and the antibody jointly participate in the binding with the target antigen.

8. A smacAb library, characterized in that The smacAb library comprises forming a covalent conjugate by site-specifically coupling a small molecule compound with an antibody or an antigen-binding fragment thereof in the antibody or antigen-binding fragment library.

9. Use of the smacAb library according to claim 8 in antibody or smacAb screening.

10. Use of the antibody conjugate according to claim 1, the antibody or antigen-binding fragment thereof according to any one of claims 2 to 5, the nucleic acid molecule according to claim 6 or the smacAb library according to claim 8 in preparing a product for diagnosing and / or treating a disease.

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