Antibodies or their antigen-binding fragments, antibody-drug conjugates and their applications

Abstract

This invention relates to antibodies or their antigen-binding fragments, antibody-drug conjugates, and applications thereof. Specifically, the invention provides SEZ6 antibodies, their antigen-binding fragments, and SEZ6 antibody-drug conjugates, nucleic acid molecules encoding the antibody or its antigen-binding fragment, vectors and host cells containing the nucleic acid molecule, and methods for producing antibody-conjugate toxins. Further, the invention provides pharmaceutical compositions containing the antibody, its antigen-binding fragment, or antibody-conjugate toxin, and related pharmaceutical applications.

Description

[Technical Field]

[0001] The present invention belongs to the field of antibodies and specifically relates to antibodies that specifically bind to SEZ6 in mammals, particularly humans, as well as methods for preparing them, their conjugates, and applications. The drug is a linker-linked anti-SEZ6 antibody and the antitumor drug 9106. [Background technology]

[0002] Antibody-drug conjugates (ADCs) conjugate cytotoxins to antibodies that selectively bind to the surface of cancer cells. Through antigen-antibody mediated internalization, the cytotoxins are selectively delivered into tumor cells, accumulating to a certain level and thereby killing the tumor cells. An ADC consists of three parts: the antibody, the linker, and the toxin. Target selection of ADCs is crucial because they effectively improve the precise targeting of the toxin to the tumor and expand the therapeutic window. It is necessary that the ADC is specifically highly expressed in tumors and not expressed in normal tissues.

[0003] Small cell lung cancer (SLC) is a highly malignant tumor, with approximately 250,000 cases diagnosed worldwide annually, accounting for about 15% of all lung cancers. Of these, 70% of patients already have metastases at the time of diagnosis. Tumor development is closely linked to smoking, and it is associated with mutations in multiple tumor suppressor genes. The median survival time for diagnosed patients is only 8-12 months, with only a small number surviving for more than 5 years. The standard first-line treatment for SLC is combination therapy with platinum-based drugs and etoposide. While patients show a high response rate to first-line chemotherapy, there is a lack of effective new treatments for recurrence and resistance development. The response rate to second-line treatment with topotecan is low, and there has been no significant improvement in survival time. New breakthroughs and advances are urgently needed in the treatment of SLC. (Nature Reviews Clinical Oncology volume 14, pages 549-561 (2017)).

[0004] Human SEZ6 (seizure-related protein 6, hereafter referred to as hSEZ6) is a single-pass transmembrane protein that is mainly expressed in brain tissue and hardly expressed in peripheral tissues. In terms of neurological diseases, knockout of mouse SEZ6 promotes the formation of short and numerous synapses (Neuron 56, 621-639, November 21, 2007), and SEZ6 is a substrate of BACE1. Therefore, soluble SEZ6 in cerebrospinal fluid can be used as a label for Alzheimer's disease (Molecular Neurodegeneration 2016 11:67).

[0005] SEZ6 is specifically highly expressed in neuroendocrine tumor tissue as a tumor-specific marker. Immunohistochemical analysis of clinical specimens has shown that SEZ6 is highly expressed in small cell lung cancer, and that its expression levels are significantly higher than in normal tissue. In 174 tumor tissue sections from small cell lung cancer patients, approximately 70% of patients' tumors showed positive SEZ6 expression (Published Patent: US2016287720A1).

[0006] Based on the specific high expression of SEZ6 in tumors, AbbVie Pharmaceuticals has developed ABBV-011, an ADC against SEZ6, for the treatment of small cell lung cancer (published patent: WO2013126810A). It utilizes calicheamicin as the toxin, has a DAR value of 2, and employs an indegradable linker. This ADC entered Phase I clinical trials in 2019 (NCT03639194).

[0007] This patent describes how to obtain an innovative ADC drug by conjugating a SEZ6 antibody with high affinity and high endocytosis activity through mouse and camel immunization with a toxin compound 169106 (published patent: WO2020063676A). 169106 is a topoisomerase I inhibitor (also referred to herein as 9106), and previous studies have shown that this antibody-conjugated toxin possesses good drug-forming properties, in vivo antitumor activity, and safety. [Overview of the project]

[0008] This disclosure relates to anti-SEZ6 antibodies or antigen-binding fragments thereof, ADCs thereof, and their pharmaceutical uses. More specifically, it provides monoclonal antibodies or antigen-binding fragments that bind to the amino acid sequence or three-dimensional structure of the ECD region of SEZ6, and ADC drugs in which the same is conjugated with a cytotoxic substance exatecan analog.

[0009] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) or a heavy chain single domain (VHH), wherein the heavy chain variable region or heavy chain single domain comprises (1) heavy chains HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NO: 37, 38, and 39, or (2) heavy chains HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NO: 43, 44, and 45, or (3) heavy chains HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NO: 49, 50, and 51, or (4) heavy chains HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NO: 65, 66, and 67, or (5) heavy chains HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NO: 68, 69, and 70.

[0010] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are (1) heavy chains HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 37, 38, and 39, and light chains LCDR1, LCDR2, and LCDR3 shown in the amino acid sequences of SEQ ID NO: 40, 41, and 42, or (2) heavy chains HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 43, 44, and 45, and light chains LCDR1, LCDR2, and LCDR3 shown in the amino acid sequences of SEQ ID NO: 46, 47, and 48, or (3) heavy chains HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 49, 50, and 51, and SEQ ID NO:52, 53, and 54 contain the light chains LCDR1, LCDR2, and LCDR3 as shown in their amino acid sequences.

[0011] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises the amino acid sequence shown in SEQ ID NO:63, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or the amino acid sequence shown in SEQ ID NO:64, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0012] In some embodiments, the disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises the amino acid sequence shown in SEQ ID NO:63 or the amino acid sequence shown in SEQ ID NO:64.

[0013] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises the amino acid sequence shown in SEQ ID NO:63, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or the amino acid sequence shown in SEQ ID NO:64, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0014] In some embodiments, the disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises the amino acid sequence shown in SEQ ID NO:63 or SEQ ID NO:64.

[0015] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, or sequences having at least 85%, 90%, 95%, and 99% homology thereto, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or sequences having at least 85%, 90%, 95%, and 99% homology thereto.

[0016] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:35, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36.

[0017] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, or sequences having at least 85%, 90%, 95%, and 99% homology thereto, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or sequences having at least 85%, 90%, 95%, and 99% homology thereto.

[0018] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:35, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36.

[0019] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, or (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, or (3) the heavy chain variable region comprises SEQ ID (4) The light chain variable region includes the amino acid sequence shown in NO:31, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:34, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (4) the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:35, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:36, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0020] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30, or (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, or (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34, or (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36.

[0021] In some embodiments, the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are (1) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:29, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, the amino acid sequence of the light chain variable region shown in SEQ ID NO:30, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, or (2) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:31, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, the amino acid sequence of the light chain variable region shown in SEQ ID NO:32, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, or (3) SEQ ID (4) The amino acid sequence of the heavy chain variable region shown in NO:31, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, the amino acid sequence of the light chain variable region shown in SEQ ID NO:34, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (4) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:35, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, the amino acid sequence of the light chain variable region shown in SEQ ID NO:36, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0022] In some embodiments, the Disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region include (1) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:29 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:30, or (2) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:32, or (3) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:34, or (4) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:35 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:36.

[0023] In some embodiments, the aforementioned anti-SEZ6 antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), single-domain antibody (sdAb), dimerized V region (diabody), disulfide-bonded V region (dsFv), and antigen-binding fragment of a peptide containing a CDR.

[0024] In some embodiments, the anti-SEZ6 antibody or its antigen-binding fragment comprises an antibody constant region, where the heavy chain constant region is derived from human IgG1, IgG2, IgG3, or IgG4, or one having at least 95% sequence identity thereto, or a variant thereof, and / or the light chain constant region is derived from human antibody κ, λ chain, or one having at least 95% sequence identity thereto, or a variant thereof. Preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1, or one having at least 95% sequence identity thereto, or a variant thereof, and / or the light chain constant region is derived from human antibody κ chain, or one having at least 95% sequence identity thereto, or a variant thereof. More preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:33, and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:59.

[0025] In some embodiments, the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, where (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 55, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 56, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 58, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (3) the heavy chain comprises SEQ ID (4) The light chain contains the amino acid sequence shown in NO:57, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain contains the amino acid sequence shown in SEQ ID NO:60, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (4) the heavy chain contains the amino acid sequence shown in SEQ ID NO:61, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain contains the amino acid sequence shown in SEQ ID NO:62, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0026] In some embodiments, the anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 55 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 56, or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 58, or (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 60, or (4) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 61 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 62.

[0027] In some embodiments, the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, where the heavy chain and light chain are: (1) the heavy chain sequence shown in SEQ ID NO: 55 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain sequence shown in SEQ ID NO: 56 or a sequence having at least 85%, 90%, 95%, 99% homology thereto; (2) the heavy chain sequence shown in SEQ ID NO: 57 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain sequence shown in SEQ ID NO: 58 or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (3) the heavy chain sequence shown in SEQ ID NO: 57 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and SEQ ID (4) A sequence comprising the light chain sequence shown in NO:60 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or the heavy chain sequence shown in SEQ ID NO:61 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain sequence shown in SEQ ID NO:62 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0028] In some embodiments, the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain and light chain include (1) the heavy chain sequence shown in SEQ ID NO: 55 and the light chain sequence shown in SEQ ID NO: 56, or (2) the heavy chain sequence shown in SEQ ID NO: 57 and the light chain sequence shown in SEQ ID NO: 58, or (3) the heavy chain sequence shown in SEQ ID NO: 57 and the light chain sequence shown in SEQ ID NO: 60, or (4) the heavy chain sequence shown in SEQ ID NO: 61 and the light chain sequence shown in SEQ ID NO: 62.

[0029] In some embodiments, the aforementioned anti-SEZ6 antibody or antigen-binding fragment thereof comprises a heavy chain, wherein (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:71 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:72 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0030] In some embodiments, the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain, wherein (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:71, or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:72.

[0031] In some embodiments, the aforementioned anti-SEZ6 antibody or antigen-binding fragment thereof comprises a heavy chain and includes (1) the heavy chain sequence shown in SEQ ID NO:71 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (2) the heavy chain sequence shown in SEQ ID NO:72 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.

[0032] In some embodiments, the aforementioned anti-SEZ6 antibody or antigen-binding fragment thereof comprises a heavy chain and includes (1) the heavy chain sequence shown in SEQ ID NO:71, or (2) the heavy chain sequence shown in SEQ ID NO:72.

[0033] In another embodiment, the present disclosure provides an isolated anti-SEZ6 antibody or its antigen-binding fragment that competes for binding to SEZ6 with any of the antibodies or its antigen-binding fragments described in the preceding paragraph.

[0034] In another embodiment, the present disclosure provides an isolated nucleic acid which encodes an anti-SEZ6 antibody or an antigen-binding fragment thereof as described in any of the preceding items.

[0035] In another embodiment, the present disclosure provides a vector comprising the nucleic acids described above.

[0036] In another embodiment, the present disclosure provides a host cell which includes the vector described above.

[0037] In one embodiment, the present disclosure provides a method for preparing an anti-SEZ6 antibody or an antigen-binding fragment thereof, which comprises culturing the aforementioned host cells under conditions suitable for the expression of the anti-SEZ6 antibody or the antigen-binding fragment thereof.

[0038] In another embodiment, the Disclosure provides antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof, or tautomers, meso-forms, racemics, enantiomers, diastereomers, or mixtures thereof, comprising an anti-SEZ6 antibody or antigen-binding fragment thereof as described in any of the preceding paragraphs.

[0039] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the drug is selected from cytotoxic agents, radiolabels, fluorophores, chromophores, imaging agents, immunomodulators, angiogenesis inhibitors, cell proliferation inhibitors, apoptosis promoters, cytolytic enzymes, and any combination thereof, preferably, the drug is selected from DNA damaging agents, topoisomerase inhibitors, microtubule inhibitors, proteolytic agents, STING agonists, and any combination thereof.

[0040] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, having the structure shown in general formula (I), [ka] Here, Dr is selected from camptothecin, auristatin, maytansinoid, pyrrolobenzodiazepines (PBD), calicheamicin, duocarmycin, daunorubicin, doxorubicin, calicheamicin, anthramycin, neomycin, amanitin, hemiasterlin, eribulin, Tubulysin, and their analogs or derivatives. Preferably, Dr is selected from Exatecan, MMAE, MMAF, MMAD, SN-38, DM1, DM4, pyrrolobenzodiazepine (PBD) dimer, n is from 1 to 10, preferably, n is from 2 to 8, more preferably, n is from 3 to 5 or 4 to 8, n is a decimal or an integer, still more preferably, n is from 4 to 6, n is a decimal or an integer, still more preferably, n is from 5 to 6, n is a decimal or an integer, even more preferably, n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, L and Y are linker units, and Pc is the aforementioned anti-SEZ6 antibody or its antigen-binding fragment.

[0041] In some embodiments, it is the aforementioned antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, or its tautomer, meso form, racemate, enantiomer, diastereomer, or a mixture thereof, which has the structures shown in general formula (Pc-L-Y-Dr), general formula (II), general formula (III), and general formula (IV),

Chemical formula

[0042] In some embodiments, the antibody-drug conjugate described above or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, where n is 2 to 8, preferably 3 to 5 or 4 to 8, n is a decimal or integer, more preferably 4 to 6, n is a decimal or integer, even more preferably 5 to 6, n is a decimal or integer, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.

[0043] In some embodiments, the antibody-drug conjugate described in any of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)- and R a and R b R is the same or different, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group, 1 is a haloalkyl group or C 3-6 It is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group, or C 3-6 Selected from cycloalkyl groups, or R 1 and R 2 C 3-6 It forms a cycloalkyl group, and m is either 0 or 1.

[0044] In some embodiments, the antibody-drug conjugate described in any one of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein Y is [ka] Selected from.

[0045] In some embodiments, the antibody-drug conjugate described in any of the preceding paragraphs, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the O-terminus of Y is linked to a linker unit L.

[0046] In some embodiments, the antibody-drug conjugate described in any of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -and, L 1 teeth, [ka] and s 1 L is an integer between 2 and 8. 2 L is a chemical bond. 3 It is a tetrapeptide residue, L 4 -NR 5 (CR 6 R 7 )t- and R 5 , R 6 or R 7 t is either the same or different, and each is independently a hydrogen atom or an alkyl group, and t is either 1 or 2.

[0047] In some embodiments, the antibody-drug conjugate described in any of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -and, L1 teeth, [ka] and s 1 and s 2 These are independently selected from integers 0 to 8, preferably s 1 is 2, and s 2 is an integer between 2 and 8, more preferably s 1 is 2, and s 2 is 2, 3, 4, 5 or 6, L 2 L is a chemical bond. 3 It is a tetrapeptide residue, L 4 -NR 5 (CR 6 R 7 )t- and R 5 , R 6 or R 7 t is either the same or different, and each is independently a hydrogen atom or an alkyl group, and t is either 1 or 2.

[0048] In some embodiments, the antibody-drug conjugate described in any of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the linker unit -L- is the L 1 The end is connected to Pc, L 4 The end is connected to a Y.

[0049] In some embodiments, the antibody-drug conjugate described in any one of the preceding paragraphs or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein L 3 The tetrapeptide residue is an amino acid residue formed from two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and is preferably a tetrapeptide residue of GGFG.

[0050] In some embodiments, it is in the form of the antibody-drug conjugate according to any one of the foregoing items, or a pharmaceutically acceptable salt or solvate thereof, or a tautomerism, meso form, racemate, enantiomer, diastereoisomer, or a mixture thereof, wherein -L-Y- has the following structure:

Chemical formula

[0051] In some embodiments, it is in the form of the antibody-drug conjugate according to any one of the foregoing items, or a pharmaceutically acceptable salt or solvate thereof, or a tautomerism, meso form, racemate, enantiomer, diastereoisomer, or a mixture thereof, wherein -L-Y- is

Chemical formula

[0052] In some embodiments, it is in the form of the antibody-drug conjugate according to any one of the foregoing items, or a pharmaceutically acceptable salt or solvate thereof, or a tautomerism, meso form, racemate, enantiomer, diastereoisomer, or a mixture thereof, and it has the general formula (Pc-L a-Y-Dr) is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof. [ka] Here, W is C 1-8 Alkyl, C 1-8 Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups containing 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, where the C 1-8 Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups, L 2 -NR 4 (CH2CH2O) p1 CH2CH2C(O)-, -NR 4 (CH2CH2O) p1 CH2C(O)-, -S(CH2) p1 C(O)- or selected from chemical bonds, p 1 L is an integer between 1 and 20. 3 R is a peptide residue consisting of 2 to 7 amino acids, the amino acids may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking site, and the substituent is independently one or more groups selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group, 1 R is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. 2 is selected from a hydrogen atom, halogen, haloalkyl group, deuterated alkyl group, cycloalkyl group, heterocyclyl group, aryl group or heteroaryl group, or R 1 and R 2These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group, R 4 and R 5 R is the same or different and each is independently selected from a hydrogen atom, alkyl group, haloalkyl group, deuterated alkyl group and hydroxyalkyl group, 6 and R 7 Pc is the same or different and independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group and hydroxyalkyl group, m is an integer from 0 to 4, n is 1 to 10, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, n is a decimal or integer, even more preferably n is 4 to 6, n is a decimal or integer, even more preferably n is 5 to 6, n is a decimal or integer, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, and Pc is an anti-SEZ6 antibody or its antigen-binding fragment as described in any one of the preceding items.

[0053] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LY-Dr) described above, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, is in the form of the general formula (Pc-LY-Dr). b -Y-Dr) is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof. [ka] Here, s 1 is an integer between 2 and 8, and R 1 , R 2 , R 5 ~R 7 m and n are defined as in the general formula (Pc-La-Y-Dr).

[0054] In some embodiments, the antibody-drug conjugate described above or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the antibody-drug conjugate is [ka] [ka] [ka] [ka] [ka] [ka] [ka] Selected from the range, n is 1 to 10, preferably 2 to 8, more preferably 3 to 5 or 4 to 8, n is a decimal or integer, even more preferably 4 to 6, n is a decimal or integer, even more preferably 5 to 6, n is a decimal or integer, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. Pc is the aforementioned anti-SEZ6 antibody or its antigen-binding fragment.

[0055] In some embodiments, the antibody-drug conjugate described above or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the antibody-drug conjugate is [ka] [ka] [ka] Selected from, where n is 1 to 10, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, n is a decimal or an integer, even more preferably n is 4 to 6, n is a decimal or an integer, even more preferably n is 5 to 6, n is a decimal or an integer, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.

[0056] This disclosure is based on the general formula (Pc-L a The present invention further provides a method for producing an antibody-drug conjugate shown in -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, which, [ka] After reducing Pc, the general formula (L a -Y-Dr) is conjugated and the general formula (Pc-L a The process includes the step of obtaining the compound shown in -Y-Dr), where Pc is the anti-SEZ6 antibody or its antigen-binding fragment as described in any one of the preceding items. W, L 2 , L 3 , R 1 , R 2 , R 5 ~R 7 m and n are given by the general formula (Pc-L a As defined in -Y-Dr).

[0057] In another embodiment, another method is provided, where the general formula L a -Y-Dr is a general formula L b - Compounds shown in Y-Dr, [ka] or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, where R1 , R 2 , R 5 ~R 7 , s 1 And m is the general formula Pc-L b -As defined in Y-Dr.

[0058] In one preferred embodiment of the present disclosure, the general formula (Pc-L) described herein is used. a -Y-Dr) or general formula (Pc-L b According to the method for producing antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof shown in -Y-Dr), where the general formula (L a -Y-Dr) or compounds of the general formula (L b The compound shown in -Y-Dr) is [ka] Selected from.

[0059] In another embodiment, the Disclosure provides a kit comprising an anti-SEZ6 antibody or its antigen-binding fragment as described herein, or an antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described herein, or its tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

[0060] In another embodiment, the Disclosure provides a pharmaceutical composition comprising an anti-SEZ6 antibody or its antigen-binding fragment as described herein, or an antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described herein, or its tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, and one or more pharmaceutically acceptable carriers.

[0061] In another aspect, the Disclosure provides uses in the manufacture of drugs for treating SEZ6-mediated diseases or disorders, such as anti-SEZ6 antibodies or antigen-binding fragments thereof, antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof, or tautomers, meso-forms, racemics, enantiomers, diastereomers, or mixtures thereof, or pharmaceutical compositions comprising the same, wherein the SEZ6-mediated disease or disorder is SEZ6-high-expression cancer.

[0062] In another aspect, the Disclosure provides uses in the manufacture of drugs for treating or preventing tumors or cancers, of which cancers are preferably lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer.

[0063] The active compound can be prepared in a form suitable for administration by any appropriate route, preferably in the form of a unit dose or in a form that can be self-administered by a patient as a single dose. The unit dose of the compound or composition of this disclosure may be expressed as a tablet, capsule, cachet, bottled liquid, powder, granule, lozenge, suppository, rehydration powder or liquid formulation.

[0064] The dose of the compound or composition used in the therapeutic method disclosed herein generally depends on the severity of the disease, the patient's weight, and the relative potency of the compound. However, as general guidance, an appropriate unit dose may be between 0.1 and 1000 mg.

[0065] The pharmaceutical compositions of this disclosure may contain one or more excipients in addition to the active compound, the excipients being selected from components such as fillers (diluents), adhesives, wetting agents, disintegrants, or other excipients. Depending on the administration method, the composition may contain 0.1 to 99% by weight of the active compound.

[0066] The anti-SEZ6 antibody or its antigen-binding fragment exhibits no nonspecific binding, superior levels of binding to the antigen, and a unique binding epitope. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, or its tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, exhibits excellent in vitro cytotoxicity and in vivo tumor inhibitory effects, and has excellent in vivo pharmacokinetic parameters.

[0067] [Detailed description of the invention] 1. Terminology Unless otherwise limited, all technical and scientific terms used herein are consistent with those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but this specification describes preferred methods and materials. When seeking the description and protection of this disclosure, use the following terms as defined below.

[0068] When a trade name is used in this disclosure, the applicant intends to include formulations of the trade name product, non-patented drugs and active drug portions of the trade name product.

[0069] Unless otherwise stated, terms used in the specification and claims have the following meanings:

[0070] The term "drug" refers to cytotoxic drugs, which are represented by Dr and possess chemical molecules that potently inhibit the normal growth of tumor cells. While cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations, they lack specificity, causing serious side effects by simultaneously killing tumor cells and inducing apoptosis in normal cells. The term also includes toxins, such as low-molecular-weight toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioisotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 The material comprises radioactive isotopes of Lu, toxic drugs, chemotherapeutic agents, antibiotics, and lysosomes, preferably toxic drugs.

[0071] The term “linker unit (or linking fragment)” refers to a chemical structural fragment or linkage in which one end is linked to an antibody and the other end is linked to a drug, which may be linked to another linker before being linked to the drug. A preferred arrangement of the present disclosure is L and L 1 ~L 4 It is expressed as, where L 1 The end is linked to the antibody, L 4 The terminal end is linked to structural unit Y and then to the drug (Dr).

[0072] The linker comprises an elongator, a spacer, and an amino acid unit, and can be synthesized by methods known in the art, for example, as described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of the drug in the cell. For example, an acid-stable linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020).

[0073] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linking unit. In this disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), in which a clonal antibody or antibody fragment is linked to a biologically active toxic drug via a stable linking unit.

[0074] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0075] The term "antibody" refers to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in amino acid composition and sequence, resulting in different antigenicity. This allows for the classification of immunoglobulins into five classes, or sometimes referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig of the same class may be further divided into various subclasses based on differences in the amino acid composition of their hinge region and the number and position of heavy chain disulfide bonds; for example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are divided into κ or λ chains based on their constant region. Each of the five classes of Ig may possess either a κ or λ chain.

[0076] The approximately 110 amino acids near the N-terminus of the antibody heavy and light chains exhibit significant sequence changes and are called variable regions (Fv regions), while the other amino acids near the C-terminus have relatively stable sequences and are called constant regions. The variable regions include three hypervariable regions (HVRs) and four relatively conserved skeletal regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino group terminus to the carboxyl group terminus. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragments described herein conform to the Kabat numbering rules (LCDR1-3, HCDR2-3) whose number and position are known, or conform to the Kabat and Chothia numbering rules (HCDR1).

[0077] "Fully humanized antibodies," also known as "fully humanized monoclonal antibodies," are antibodies in which both the variable and constant regions are derived from humans, and immunogenicity and toxic side effects have been eliminated. The development of monoclonal antibodies has progressed through four stages: mouse-derived monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully humanized monoclonal antibodies. Related technologies for the preparation of fully humanized antibodies mainly include human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology. The "fully human antibodies" in this disclosure were obtained using phage display technology. Phage display technology involves isolating B cells from human PBMCs, spleen, and lymph node tissue, constructing a natural single-stranded phage human antibody library, or screening antibodies obtained by immunizing transgenic mice capable of expressing human antibody light and heavy chains.

[0078] The term "antigen-binding fragment" refers to one or more fragments in an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks on a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single domains or dAb fragments consisting of a VHH domain (Ward et al., (1989) Nature 341:544-546); and (vi) combinations of two or more isolated CDRs that can be linked by isolated complementarity-determining regions (CDRs) or (vii) optionally synthesized linkers. Although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via a synthesized linker using a recombination method, thereby allowing the VL and VH regions to pair up and form a single monovalent protein chain (called single-stranded Fv (scFv), see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-stranded antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be screened for functionality in the same manner as for complete antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of complete immunoglobulins. The antibody may be an antibody of a different isotype, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0079] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by treating an IgG antibody molecule with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). In this fragment, approximately half of the N-terminal side of the H chain and the entire L chain are linked via disulfide bonds.

[0080] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity and containing two Fab regions linked at the hinge position. It is obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

[0081] Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of F(ab')2 described above.

[0082] Furthermore, Fab' can be produced by inserting the DNA encoding the antibody's Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the vector into a prokaryote or eukaryote to express Fab'.

[0083] "Single-chain antibody," "single-chain Fv," or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) linked via a linker. Such scFv molecules may have the common structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeated GGGGS amino acid sequence or a variant thereof, for example, using 1 to 4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers available in this disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immuno l. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0084] The terms "single-domain antibody," "single-domain antibody," or "sdAb" refer to a novel type of antibody that lacks the CH1 region of the heavy chain and the light chain, containing only a single heavy chain variable region while retaining complete antigen-binding activity. It is also called a nanobody. Like complete antibodies, it can selectively bind to specific antigens. Compared to the 150-160 kDa mass of complete antibodies, single-domain antibodies are significantly smaller, at approximately 12-15 kDa. The first single-domain antibody was artificially created from camel heavy chain antibodies and is called the "VHH segment."

[0085] The terms "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH segment," and "nano antibody" are interchangeable, and a single-domain antibody is the variable region of a heavy chain antibody. Generally, a single-domain antibody contains three CDRs and four FRs.

[0086] Single-domain antibodies may be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelid species (e.g., camels, dromedaries, llamas, and guanacos). Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. A single-domain antibody may contain only the variable domain of an immunoglobulin chain, which has CDR1, CDR2, and CDR3 as well as a framework region.

[0087] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR is used only for the light chain variable domains CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) and the heavy chain variable domains CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).

[0088] The term "antibody framework" refers to a portion of the variable domain (VL) or VH that functions as a scaffold for the antigen-binding loop (CDR) of the variable domain. Essentially, it is a variable domain without a CDR.

[0089] The term "epitope" or "antigen-determining cluster" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0090] The terms "specific binding," "selective binding," and "specifically binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Generally, antibodies bind with affinity (KD) less than approximately 10⁻⁷M, for example, approximately 10⁻⁸M, 10⁻⁹M, or less than 10⁻¹⁰M.

[0091] The term "nucleic acid" refers to DNA and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. When nucleic acids are functionally related to another nucleic acid sequence, they are "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operablely linked to the coding sequence.

[0092] The term “expression vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it can be ligated. In one embodiment, the vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which another DNA segment can be ligated. In another embodiment, the vector is a viral vector, in which another DNA segment can be ligated to a viral genome. The “vectors” disclosed herein can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors) or replicate together with the host genome by being incorporated into the host cell's genome after introduction into the host cell (e.g., non-episomal mammalian vectors).

[0093] Methods for producing and purifying antibodies and antigen-binding fragments that are well known in the prior art are as described in Chapters 5-8 and 15 of the Cold Spring Guidelines for Antibody Experimental Techniques. Antigen-binding fragments can similarly be produced by conventional methods. The antibodies or antigen-binding fragments described in the invention are obtained by adding one or more human FR regions to a non-human CDR region using genetic engineering methods. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr, or from the Journal of Immunoglobulins, 2001, ISBN 012441351, through alignment with the IMGT Human Antibody Variable Region Germline Gene Databank and MOE software.

[0094] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include budding yeasts (Saccharomyces cerevisiae) and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0095] The manipulated antibodies or antigen-binding fragments of this disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombinant into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As one more preferred conventional technique, mammalian expression systems induce glycosylation of antibodies, particularly at the highly conserved N-terminal region of the Fc area. Positive clones are expanded and cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium secreted by the antibodies can be purified by conventional techniques. For example, purification by an A or G Sepharose FF column containing a prepared buffer. Nonspecifically bound components are washed away. Further elution of bound antibodies by pH gradient elution, and antibody fragments are detected and collected by SDS-PAGE. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, e.g., molecular sieves, ion exchange. The resulting product must be immediately frozen, for example, at -70°C, or freeze-dried.

[0096] The term "peptide" refers to a compound fragment that interposes between amino acids and proteins. It consists of two or more amino acid molecules interconnected via peptides, forming structural and functional fragments of proteins. For example, hormones and enzymes are essentially peptides.

[0097] The term "sugar" refers to biomacromolecules composed of three elements: C, H, and O, and may be further classified into monosaccharides, disaccharides, polysaccharides, etc.

[0098] The term "fluorescent probe" refers to a fluorescent molecule that exhibits characteristic fluorescence in the ultraviolet-visible-near-infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, and polarization, etc.) change sensitively in response to changes in the properties of its environment, such as polarity, refractive index, and viscosity. It can change one or more fluorescence properties by non-covalently interacting with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, and is useful for studying the properties and behavior of macromolecular materials.

[0099] The term "toxic drug" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. This includes toxins and other compounds available for use in tumor treatment.

[0100] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, and 5-methylhexyl group. Examples include 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof.More preferably, the lower alkyl group contains 1 to 6 carbon atoms, and non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking site, and the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0101] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, where the alkyl group is as defined above.

[0102] The term "alkylene group" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and is a linear or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms (1, 2, 3, 4, 5, or 6 carbon atoms). Non-restrictive examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking site, and the substituent is preferably independently and optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0103] The term "alkoxy group" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where the definitions of alkyl and cycloalkyl groups are as described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. Alkoxy groups may be optionally substituted or unsubstituted, and if substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0104] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where the ring of a cycloalkyl group contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (3, 4, 5, 6, 7, or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, while polycyclic cycloalkyl groups include spirocyclic, fused, and crosslinked cyclic cycloalkyl groups.

[0105] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which contains 3 to 20 ring atoms, where one or more ring atoms are nitrogen, oxygen, or S(O). mThe heteroatom is selected from (where m is an integer 0, 1, or 2) but does not contain the -OO-, -OS-, or -SS- ring portion, and the other ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, where 1 to 4 are heteroatoms (1, 2, 3, or 4 heteroatoms), and more preferably the cycloalkyl ring contains 3 to 10 ring atoms (3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of monocyclic heterocyclil groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholino, thiomorpholino, homopiperazinyl, etc. Polycyclic heterocyclil groups include spiro-rings, fused rings, and bridging ring heterocyclil groups.

[0106] The term "spiroheterocyclyl group" refers to a 5-20 membered polycyclic heterocyclyl group in which monocyclic rings share one atom (called a spiro atom), where one or more ring atoms are nitrogen, oxygen, or S(O). m A heteroatom selected from (where m is an integer from 0 to 2), and the other ring atoms are carbon. It may contain one or more double bonds, but there are no rings having a fully conjugated π-electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of covalent spiroatoms between the rings, the spiroheterocyclyl group is divided into a monospiroheterocyclyl group, a bisspiroheterocyclyl group, or a polyspiroheterocyclyl group, preferably a monospiroheterocyclyl group and a bisspiroheterocyclyl group. More preferably, a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl group. Non-limiting examples of spiroheterocyclyl groups are: [ka] Includes.

[0107] The term "condensed heterocyclyl group" refers to a 5-20 membered polycyclic heterocyclyl group in which each ring in the system shares one adjacent pair of carbon atoms with the other rings in the system, and one or more rings may contain one or more double bonds, but none of the rings have a fully conjugated π-electron system, where one or more ring atoms are nitrogen, oxygen, or S(O) m (where m is an integer 0, 1, or 2) the heteroatom is selected from the above, and the other ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered rings). Depending on the number of rings that make up the group, it may be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclyl group, preferably bicyclic or tricyclic, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups are: [ka] Includes.

[0108] The term "bridged heterocyclyl group" refers to a 5-14 member polycyclic heterocyclyl group in which any two rings share two atoms that are not directly linked, and which may contain one or more double bonds, but which have no rings having a fully conjugated π-electron system, where one or more ring atoms are nitrogen, oxygen, or S(O). m (where m is an integer 0, 1, or 2) the heteroatom is selected from the above, and the other ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered rings). Depending on the number of rings that make up the group, it may be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridging heterocyclyl group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridging heterocyclyl groups are: [ka] Includes.

[0109] The heterocyclyl ring may be condensed onto an aryl group, a heteroaryl group, or a cycloalkyl group, where the ring linked to the basic skeleton is a heterocyclyl group, and non-limiting examples include: [ka] This includes, among others.

[0110] The heterocyclyl group may be optionally substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0111] The term "aryl group" refers to a 6-14 membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6-10 membered (6, 7, 8, 9, or 10 membered), such as a phenyl group and a naphthyl group, preferably a phenyl group. The aryl group ring can be fused onto a heteroaryl group, heterocyclyl group, or cycloalkyl group ring, where the ring linked to the basic skeleton is an aryl group ring, and non-limiting examples include: [ka] Includes.

[0112] The aryl group may be substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0113] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3, or 4 heteroatoms) and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered (5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered heteroaryl group), more preferably 5-membered or 6-membered, and includes, for example, a furyl group, thienyl group, pyridyl group, pyrrolyl group, N-alkylpyrrolyl group, pyrimidinyl group, pyrazinyl group, imidazolyl group, tetrazolyl group, etc. The ring of the heteroaryl group may be condensed on an aryl group, heterocyclyl group, or cycloalkyl group, where the ring linked to the basic skeleton is the ring of the heteroaryl group, and non-limiting examples are: [ka] Includes.

[0114] The heteroaryl group may be optionally substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0115] The term "amino protecting group" refers to protecting an amino group with an easily detachable group in order to retain the amino group without alteration when reactions occur at other parts of the molecule. Non-limiting examples include the 9-fluorenyl methoxycarbonyl group, the tert-butoxycarbonyl group, the acetyl group, the benzyl group, the allyl group, and the p-methoxybenzyl group. These groups may be optionally substituted with 1 to 3 substituents (1, 2, or 3 substituents) selected from halogens, alkoxy groups, or nitro groups. The amino protecting group is preferably the 9-fluorenyl methoxycarbonyl group.

[0116] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.

[0117] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.

[0118] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxyl groups, where the alkyl group is as defined above.

[0119] The term "hydroxyl group" refers to the -OH group.

[0120] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0121] The term "amino group" refers to -NH2.

[0122] The term "nitro group" refers to -NO2.

[0123] The term "cyano group" refers to -CN.

[0124] The term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are defined above.

[0125] The term "carboxylate group" refers to -C(O)O(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are defined above.

[0126] This disclosure further includes compounds of formula (I) in various deuterated forms. Each available hydrogen atom bonded to a carbon atom can be independently substituted with a deuterium atom. Those skilled in the art can synthesize compounds of formula (I) in deuterated forms by referring to relevant literature. When preparing compounds of formula (I) in deuterated forms, commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques with deuterating reagents, which include, but are not limited to, borane deuterated, borane-tetrahydrofuran trihydrogenated solution, lithium aluminum deuterated, iodoethane deuterated, and iodomethane deuterated.

[0127] "Optional" or "optionally" means that the event or environment described later may occur but is not required, and the description includes cases where the event or environment occurs or does not occur. For example, "optionally alkyl-substituted heterocyclyl group" means that the alkyl group may be present but is not required, and the description includes cases where the heterocyclyl group is substituted with an alkyl group and cases where the heterocyclyl group is not substituted with an alkyl group.

[0128] "Substituting" means that one or more hydrogen atoms in a group, preferably up to five, more preferably one, two, or three, are substituted independently of each other by a corresponding number of substituents. Needless to say, substituents exist only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions (experimentally or theoretically) with little effort. For example, an amino or hydroxyl group with free hydrogen can become unstable if bonded to a carbon atom with an unsaturated (e.g., olefin) bond.

[0129] The term "pharmaceutical composition" means a mixture of one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to a living organism, to facilitate the absorption of the active ingredient, and thereby to exert biological activity.

[0130] The term "pharmaceutically acceptable salt" means a salt of the antibody-drug conjugate of the present disclosure, or a salt of a compound described herein, which is safe and effective when used in a mammal and has the desired biological activity, and the antibody-drug conjugate of the present disclosure contains at least one amino group and can therefore form salts with acids, and non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbic acid, oxalate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0131] The term "solvate" refers to the antibody-drug conjugate of this disclosure forming a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0132] The term "drug load" refers to the average number of cytotoxic drugs loaded onto each antibody in the molecule of formula (I), and may be expressed as a ratio of drug amount to antibody amount. The drug load may range from 0 to 12, preferably 1 to 10, cytotoxic drugs (D) to each antibody (Pc). In embodiments of this disclosure, the drug load is represented as n and may also be called the DAR value, and is exemplary the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Conventional methods, such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC analysis, can identify the average number of drugs on each ADC molecule after the conjugation reaction.

[0133] In one embodiment of this disclosure, a cytotoxic drug is conjugated to the N-terminal amino group and / or the ε-amino group of a lysine residue of an antibody via a linking unit, and generally, the number of drug molecules that can be conjugated with the antibody in the conjugation reaction is smaller than the theoretical maximum.

[0134] The loading of antibody-cytotoxic drug conjugates can be controlled using the following non-limiting methods: (1) Controlling the molar ratio of the linking reagent to the monoclonal antibody, (2) Controlling the reaction time and temperature, (3) Includes selecting different reaction reagents.

[0135] The manufacture of ordinary pharmaceutical compositions is based on the Chinese Pharmacopoeia.

[0136] The term "carrier" as used in this disclosure refers to a system that alters the method of drug administration to the human body and its distribution within the body, controls the rate of drug release, and delivers the drug to a target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, mitigate side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structure and form various forms of aggregates, preferred examples of which include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and also possess excellent permeability to membranes, making them excellent drug carriers.

[0137] The term "excipient" refers to any substance added to a pharmaceutical preparation other than the active ingredient, and may also be called an excipient. For example, adhesives, fillers, disintegrants, and lubricants in tablets; matrix components in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solvent co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations may all be called excipients.

[0138] The term "diluent," also known as a filler, primarily serves to increase the weight and volume of tablets. Adding a diluent not only ensures a consistent volume but also reduces variations in the dosage of the main ingredient and improves the compressibility of the drug. If the drug in the tablet contains oily components, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating the manufacturing process. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0139] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. The acceptable vehicles and solvents used may include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and treated to form a microemulsion. The injection solution or microemulsion can be injected into the patient's bloodstream by a large local injection. Alternatively, it is preferable to administer the solution or microemulsion in a manner that can maintain a steady circulating concentration of the compound of this disclosure. To maintain such a steady concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

[0140] The pharmaceutical composition may be in the form of sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. The sterile injection formulation may be a sterile injection solution or suspension prepared in a parenterally acceptable, non-toxic diluent or solvent, such as a solution prepared in 1,3-butylene glycol. Sterile fixative oils are readily available as solvents or suspension media. For this purpose, any formulated fixative oil containing synthetic monoglycerides or diglycerides may be used. Fatty acids, such as oleic acid, can also be used in the preparation of injections.

[0141] This disclosure relates to a cleavable linking arm of a specific structure and an active substance of a specific structure, as well as an antibody-drug conjugate (ADC) comprising the linking arm, the active substance and the antibody. Such an ADC is a complex formed by linking a single toxic substance to an antibody via a spacer. The antibody-drug conjugate (ADC) exerts an antitumor effect by releasing an active molecule by degradation in vivo. [Brief explanation of the drawing]

[0142] [Figure 1]This is the result of detecting the activity of recombinant human SEZ6 protein. [Figure 2A] This is the result of detecting human SEZ6 expression in stable transfection cell lines. [Figure 2B] This is the result of detecting monkey SEZ6 expression in stable transfection cell lines. [Figure 3A] This is the result of detecting the binding activity of mouse-derived chimeric antibodies to human SEZ6 on the cell membrane. [Figure 3B] This is the result of detecting the binding activity of mouse-derived chimeric antibodies to monkey SEZ6 on the cell membrane. [Figure 4] This report presents the results of detecting the internalization of mouse-derived chimeric antibodies and their immunotoxic activity. [Figure 5] This is the result of detecting the binding activity of humanized antibodies against human cell membrane proteins. [Figure 6A] This shows the results of detecting the internalization of humanized antibodies and their immunotoxic activity on H69 cells. [Figure 6B] This shows the results of detecting the internalization of humanized antibodies and their immunotoxic activity on H82 cells. [Figure 7A] This is the result of detecting the binding activity of chimeric nanoantibodies to human SEZ6 on the cell membrane. [Figure 7B] This is the result of detecting the binding activity of chimeric nanoantibodies to monkey SEZ6 on the cell membrane. [Figure 8] This shows the results of internalization of chimeric nanoantibodies on H69 cells and detection of immunotoxicity. [Figure 9] This is the result of detecting the binding activity of humanized nanoantibodies to the human SEZ6 protein on the cell membrane. [Figure 10] This shows the results of detecting the internalization of humanized nanoantibodies and their immunotoxic activity on H69 cells. [Figure 11] This shows the results of detecting the binding activity of PR3178 and PR0071 to SEZ6 homologous family proteins. [Figure 12] These are the results of identifying the affinity epitopes PR3178 and PR0071 for SEZ6. [Figure 13A] This describes the in vitro inhibitory activity of PR3178-9106 and PR0071-9106 in H69 cells. [Figure 13B] This describes the in vitro inhibitory activity of PR3178-9106 and PR0071-9106 in H82 cells. [Figure 14] This describes the antitumor activity of PR3178-9106 and PR0071-9106 in an H69 xenograft model. [Figure 15] This study evaluates the single-dose pharmacokinetics of PR3178-9106 and PR0071-9106 in SCID mice. [Modes for carrying out the invention]

[0143] Further details will be provided below, along with examples, but these examples are not intended to limit the scope.

[0144] For experimental methods where specific conditions are not specified in the examples or test cases, the methods are generally carried out according to conventional conditions or conditions suggested by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Laboratory, Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents whose specific source is not specified are common reagents purchased from the market.

[0145] Example 1. Detection of recombinant SEZ6 protein activity Recombinant human SEZ6-Fc fusion protein is formed by fusing a human IgG1 Fc-tagged protein to the C-terminus of the human SEZ6 protein ECD region (see GenBank databank, accession number: NP_849191, SEQ ID NO: 1). Recombinant human SEZ6-His6 protein is formed by fusing polyhistidine-tagged His6 to the C-terminus of the human SEZ6 extracellular domain (ECD) (see GenBank databank, accession number: NP_849191). Recombinant monkey SEZ6-His6 protein is formed by fusing a His tag to the C-terminus of the monkey SEZ6 ECD region (see GenBank databank, accession number: XP_005583333.2, SEQ ID NO: 2). The recombinant mouse SEZ6-His6 protein was created by fusing the His tag to the C-terminus of the mouse SEZ6 ECD region (positions 1-922) (refer to the GenBank databank, accession number: NP_067261, SEQ ID NO: 3). The extracellular domain sequence is marked with an underline.

[0146] [Table 1-1] [Table 1-2] [Table 1-3]

[0147] To detect the good immunohistochemical activity of recombinant proteins, validation was performed using the ELISA (enzyme-linked immunosorbent assay) method. Specifically, the procedure was as follows: Recombinant human SEZ6-His6 protein was diluted to 1 μg / mL in PBS and added to an ELISA microwell plate at a rate of 100 μL per well. The plate was incubated overnight at 4°C, and an ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w / v) and pH 7.4) was added. The plate was blocked at 37°C for 2 hours. Then, gradient-diluted anti-SEZ6 detection antibody hSC200 (hSC17,200, heavy chain SEQ ID NO: 6, light chain SEQ ID NO: 7) was added in sequence. The plate was incubated at 37°C for 1 hour, washed 2-3 times with plate washing solution, and a horseradish peroxidase-labeled (HRP) secondary antibody was added. The plate was incubated at 37°C for 1 hour and washed 2-3 times with plate washing solution. TMB substrate was added at a rate of 100 μL per well and incubated at room temperature for 15 minutes, followed by the addition of 50 μL of stop solution (2M HCl) per well. The OD450 nm value was read using an ELISA plate reader (SpectraMax M5e), and the results are shown in Figure 1. Recombinant human SEZ6-His6 protein can bind to the SEZ6 antibody hSC200 with high affinity and is usable for subsequent in vivo protein immunization.

[0148] The hSC200 sequence disclosed in WO2019232241 (AbbVie Stemcentrx LLC) is as follows: [ka]

[0149] Example 2. Establishment of a stable cell line expressing recombinant human SEZ6. A constructed overexpression cell line was used in the screening process to enable screening of candidate antibody molecules with affinity for membrane proteins. Specifically, CHO-K1 cells were transfected with a plasmid encoding recombinant human SEZ6 protein or a plasmid encoding cynomolgus monkey SEZ6 protein, and then cell lines stably expressing recombinant human or cynomolgus monkey SEZ6 protein were obtained by screening. The nucleotide sequences encoding human or cynomolgus monkey SEZ6 protein were cloned into a lentiviral expression vector and ligated to prepare a plasmid. CHO-K1 cell lines were transfected using a well-known lentiviral transfect method, selectively cultured for two weeks in F-12 medium containing puromycin (purchased from Gibco), subcloning was performed in 96-well culture plates using the finite dilution method, and the cells were cultured in a cell incubator. After two weeks, the selected monoclonals were detected and screened by flow cytometry. The results in Figure 2 demonstrate that the cell line can stably express human SEZ6 or monkey SEZ6 at high levels, making it usable in subsequent antibody screening processes.

[0150] Example 3. Obtaining mouse-derived anti-SEZ6 monoclonal antibodies from recombinant SEZ6 protein-immunized mice. To screen for antibodies that can specifically bind to human and monkey SEZ6 membrane proteins, this experiment immunized 6-8 week old Balb / c and SJL / J mice using full-length human SEZ6 expression plasmid, recombinant human SEZ6-Fc, and human SEZ6-His6 proteins as immunogens, and also immunized Balb / c and SJL / J mice using recombinant human SEZ6-His6 as an immunogen. The protein dose for the initial immunization was 50 μg per mouse. Two weeks after the initial immunization, a booster immunization was performed with an immunosuppressant dose of 25 μg per mouse, with each booster immunization performed at two-week intervals.

[0151] Serum samples were collected one week after each booster immunization, and antibody titers in mouse serum were detected by ELISA. Plates were coated with 1 μg / mL recombinant human SEZ6-His6, left overnight at 4°C, blocked for 1 hour with 1% BSA-containing PBST buffer, and washed three times. In the blocking buffer, a 10-fold gradient dilution was started at 1:100 against mouse serum, incubated at 37°C for 1 hour, washed three times, and incubated with anti-mouse IgG-Fc secondary antibody (HRP-labeled) for 1 hour. Washed three times with PBST, 100 μL of TMB substrate was added per well, the reaction was terminated with 2 M HCl after 15 minutes, and the absorbance at 450 nm was measured. Immune mouse serum showed varying degrees of binding to the immunogen, with the maximum dilution factor of serum being 1:10. 5 In that case, an antigen-antibody reaction could still be observed.

[0152] In the final immunization, 25 μg of recombinant human SEZ6-His6 protein was intraperitoneally injected. After 5 days, the mice were euthanized, the spleens were removed, and splenocytes were recovered by grinding. A final concentration of 1% (w / w) NH4OH was added to cleave the erythrocytes mixed with the splenocytes, obtaining a splenocyte suspension. The cells were centrifuged three times at 1000 rpm. Mouse splenocytes and mouse myeloma cells SP2 / 0 were mixed in a viable cell ratio of 5:1, and cell fusion was performed using a high-efficiency electrofusion method. The fused cells were diluted in 20% fetal bovine serum and 1×HAT (w / w)-containing DMEM medium in a 96-well cell culture plate, with 200 μL per well, for a total of 1×10⁶ cells. 5The cells were placed in a 37°C incubator with 5% (v / v) CO2. After 14 days, the fused cells were screened using ELISA and FACS. Positive clones with an OD450nm > 1.0 and a proportion of FACS-positive cells greater than 20% were amplified into 24-well cell plates and continuously cultured at 37°C under 5% (v / v) CO2 conditions in DMEM medium containing 10% (w / w) HT fetal bovine serum. After 3 days, the culture medium from the 24-well cell plates was taken, the supernatant was collected by centrifugation, and the binding activity to the SEZ6 protein and SEZ6-positive cells was determined by ELISA and FACS. Positive clones were obtained through two monoclonalizations and sequenced. The variable region sequences of the mouse-derived antibodies are shown in Table 2.

[0153] [Table 2-1] [Table 2-2]

[0154] Recombinant chimeric antibodies were obtained by replacing the constant region of mouse-derived monoclonal antibodies. Subsequently, the nucleotide sequence encoding the variable region of the mouse-derived monoclonal antibody was cloned into a pTT5 vector containing the protein sequence encoding the human heavy and light chain constant region (Human IgG1, kappa). After synthesizing the gene sequence, HEK293 cells were transfected, and after 5 days, the cells were removed by centrifugation, and the cell culture medium was collected and filtered. The supernatant of the harvested cell culture medium was placed on a Protein A column (MabSelect SuRe, GE), and the glycine-bound antibody was eluted. The eluate was neutralized with 1M Tris, and the buffer was replaced with PBS to obtain chimeric antibodies.

[0155] Example 4. Obtaining anti-SEZ6 nanoantibodies from recombinant SEZ6 protein-immunized camels To screen for more antibodies that specifically bind to human and monkey SEZ6 membrane proteins, this experiment added an immunized camel species. The specific method is as follows: Two healthy camels were immunized with recombinant human SEZ6-Fc, and after 4-5 immunizations, 200 ml of peripheral blood was collected from each camel, and PBMCs were isolated. RNA was extracted with TriZol, reverse transcription was performed, cDNA was prepared, and the antibody gene was amplified by PCR. Enzymatic cleavage, ligation, and transformation were performed to create a VHH phage display library (library volume 1.2 × 10⁶). 9 We constructed a gene (which is...). Random sequencing results showed that the positive rate for antibody gene insertion reached 96%.

[0156] The previously constructed immunoantibody library was screened with biotinylated recombinant human SEZ6-His6 protein, affinity panning was performed 3-5 times, monoclonals were randomly selected to prepare phages, and phage detection was performed using ELISA. Positive clones were selected and sequenced for analysis. VHH-hFc expression vectors were constructed from clones with unique sequences, transiently expressed in 293 cells, and then detected by ELISA and FACS. The amino acid sequences of some VHH antibodies are shown in Table 3.

[0157] [Table 3]

[0158] Example 5. Humanization of antibodies To reduce the immune response in humans caused by non-human species, it is necessary to humanize mouse and camel-derived antibodies, and the specific method is as follows: For the humanized antibody, the structure of the mouse-derived monoclonal antibody was predicted by homology modeling, then the mouse anti-CDR was chimeric into an appropriate human GermLine framework (Bioinformation.2014, 10(4):180-186, Methods Mol Biol.2019, 1904:213-230), reverse mutations were introduced in sites that may affect antibody-antigen binding, and finally, the nucleotide sequence encoding the variable region of the humanized monoclonal antibody was cloned into a pTT5 vector containing protein sequences encoding the human heavy and light chain constant regions (Human IgG1, kappa). HEK293 cells were then transfected in a manner similar to that described above to produce humanized antibodies.

[0159] The sequence of the constant region of human IgG1 is as follows: (SEQ ID NO:33) [ka]

[0160] The sequence of the Kappa constant region is as follows: (SEQ ID NO: 59) [ka]

[0161] Refer to Table 4 for the selection of the variable region framework used for humanization of mouse-derived antibodies, Table 5 for the humanized antibody variable region sequences, Table 6 for the heavy chain CDRs (HCDR1 / HCDR2 / HCDR3) and light chain CDRs (LCDR1 / LCDR2 / LCDR3), and Table 7 for the full-length amino acid sequences of the humanized antibodies. Here, PR3178 is the humanized antibody of cAb3009, PR3121 and PR3123 are the humanized antibodies of cAb3014, and PR3132 is the humanized antibody of cAb3056.

[0162]

Table 4

[0163]

Table 5

[0164]

Table 6

[0165]

Table 7-1

Table 7-2

[0166] The variable region framework used for antibody humanization of nanobodies refers to Table 8, the humanized antibody variable region sequence refers to Table 9, the heavy chain CDR (HCDR1 / HCDR2 / HCDR3) refers to Table 10, and the human IgG1-Fc fragment fused with the humanized antibody amino acid sequence refers to Table 11. Here, PR0069 is the humanized antibody of cAb025, and PR0071 is the humanized antibody of cAb314.

[0167]

Table 8

[0168]

Table 9

[0169]

Table 10

[0170]

Table 11

[0171] Example 6 Cellular SEZ6 Binding Activity of Anti-SEZ6 Antibodies To screen chimeric or humanized antibodies that can bind to human and monkey SEZ6 membrane proteins with high affinity, the fluorescence signal of cell surface-binding antibodies was detected to evaluate the binding strength of the antibodies based on the intensity of the fluorescence signal. Specifically, it was as follows. Serial-diluted antibody molecules and control molecules were incubated with 1x10 5 cells at 4°C for 1 hour, the excess antibodies were washed away, mouse-derived Alexa Flour 647-labeled anti-human Fc antibody was added, incubated at 4°C for 30 minutes, and after washing away the excess antibodies, the cells were resuspended in 200 μL of 1% BSA / PBS buffer, and the fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. Table 12 shows the binding characteristics of mouse-derived chimeric antibodies (Figure 3), Table 13 shows the binding characteristics of mouse-derived humanized antibodies (Figure 5), Table 14 shows the binding characteristics of nanomeric antibodies (Figure 7), and Table 15 shows the affinity characteristics of nanohumanized antibodies (Figure 9). According to the results, it was shown that the candidate antibodies had SEZ6 membrane protein binding activity similar to hSC200.

[0172]

Table 12

[0173]

Table 13

[0174]

Table 14

[0175]

Table 15

[0176] Example 7: Measurement of in vitro intracellular relocation activity of anti-SEZ6 antibody To evaluate the endocytotic activity of the antibody, a DT3C antibody internalization activity evaluation system was used. DT3C is a recombinant fusion protein with a molecular weight of 70 kD, formed by fusing Fragment A (toxin portion only) of diphtheria toxin with the 3C fragment (IgG binding portion) of Group G Streptococcus. This protein can bind to the IgG portion of the antibody with high affinity, enters the cell when endocytosis occurs in the antibody, and releases toxic DT under the action of intracellular furin. DT inhibits EF2-ADP ribosylation activity, blocks the protein translation process, and ultimately induces cell death. By using this system, antibody internalization and the cell-killing effect of immunotoxins can be observed simultaneously (Yamaguchi, M., Hama, H., et al., Biochemical and Biophysical Research Communications 454(2014)600-603).

[0177] The evaluation of DT3C internalization and immunotoxin activity depended on the following: Sterile filtered DT3C and the test chimeric antibody (DT3C molar concentration was twice that of the antibody molar concentration) were homogeneously mixed in a 1:1 volume, incubated at 37°C for 30 minutes, then gradient diluted with complete medium and added to cells (H69 or H82) (3000 cells / well), and incubated in a 5% carbon dioxide incubator at 37°C for 6 days. CellTiter-Glo was added, incubated at room temperature for 10 minutes in the dark, and chemiluminescence was read using PerkinElmer. As shown in Figure 4, mouse-derived chimeric antibodies cAb3009, cAb3014, and cAb3056 exhibited stronger internalization and immunotoxin-killing activity than the control molecule hSC200. Furthermore, the endocytosis activity of chimeric antibodies similar to the sequence cAb3003, cAb3005, cAb3006, and cAb3009 was significantly superior to that of the control molecule hSC200. The general formulas for CDRH1, CHDRH2, and CDRH3 in this sequence are XYEMH, GIDPEXXNTVYNQKFKX, and GDWYFDX, respectively, while the general formulas for CDRL1, CDRL2, and CDRL3 are KSSQSLLNSRTRENYLA, WASTRXX, and XQSYNLFT.

[0178] [Table 16]

[0179] [Table 17]

[0180] [Table 18]

[0181] As shown in Table 16 and Figure 6, the humanized anti-SEZ6 mouse-derived antibodies PR3178, PR3121, PR3123, and PR3132 exhibited internalization and immunotoxicity-mediated killing activity equivalent to or slightly stronger than the control molecule hSC200. As shown in Table 17 and Figure 8, the camel-derived chimeric antibodies cAb025 and cAb314 exhibited stronger internalization and immunotoxicity-mediated killing activity than the control molecule hSC200. As shown in Table 18 and Figure 10, the humanized anti-SEZ6 camel-derived antibodies PR0069 and PR0071 exhibited stronger internalization and immunotoxicity-mediated killing activity than the control molecule hSC200.

[0182] Example 8: Affinity detection of anti-SEZ6 antibodies against SEZ6L and SEZ6L2, which are homologous families of SEZ6. To screen for antibodies specifically compatible with SEZ6, the binding of antibodies to SEZ6 homologous family proteins was detected using the ELISA method. Specifically, the following procedure was performed: Recombinant human SEZ6-Fc fusion protein, SEZ6L-Fc protein, and SEZ6L2-Fc were each diluted to 1 μg / mL in PBS and added to an ELISA microwell plate at a concentration of 100 μL per well. The plate was incubated overnight at 4°C, an ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w / v) and pH 7.4) was added, and the plate was blocked at 37°C for 2 hours. Then, 10 nM detection antibodies were added to each, and the plate was incubated at 37°C for 1 hour. The plate was washed 2-3 times with plate washing solution, a secondary antibody labeled with horseradish peroxidase (HRP) was added, and the plate was incubated at 37°C for 1 hour. The plate was then washed 2-3 times with plate washing solution. TMB substrate was added at a rate of 100 μL per well, incubated at room temperature for 15 minutes, and then 50 μL of stop solution (2M HCl) was added per well. The OD450nm value was read using an ELISA plate reader (SpectraMax M5e).

[0183] The results are shown in Figure 11. Both PR3178 and PR0071 bound to the SEZ6 protein but not to the SEZ6L protein or SEZ6L2 protein, demonstrating that PR3178 and PR0071 possess good affinity specificity.

[0184] Example 9: Identification of affinity epitopes of anti-SEZ6 antibodies against SEZ6 The endocytosis activity of antibodies correlates with their antigen-affinity epitopes, and this experiment identified the antigen-affinity epitopes of antibodies using the ELISA method. Specifically, the following procedure was performed: Recombinant human SEZ6-SUSHI1 protein, SEZ6L-SUSHI2 protein, and SEZ6-N1 protein were each diluted to 1 μg / mL in PBS, added to an ELISA microwell plate at 100 μL per well, incubated overnight at 4°C, and then ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w / v) and pH 7.4) was added. After blocking at 37°C for 2 hours, 10 nM detection antibodies were added to each, incubated at 37°C for 1 hour, washed the plate 2-3 times with plate washing solution, added a secondary antibody labeled with horseradish peroxidase (HRP), incubated at 37°C for 1 hour, and washed the plate 2-3 times with plate washing solution. TMB substrate was added at a rate of 100 μL per well, incubated at room temperature for 15 minutes, and then 50 μL of stop solution (2M HCl) was added per well. The OD450nm value was read using an ELISA plate reader (SpectraMax M5e).

[0185] The results are shown in Figure 12. PR3178 bound to the SUSHI4 domain of the SEZ6 protein, while PR0071 bound to the non-SUSHI1, SUSHI4, and N1 single domains of SEZ6. All of the screened antibody molecules possessed unique affinity epitopes and exhibited superior endocytosis activity.

[0186] Example 10 Evaluation of the dynamics of antibody-SEZ6 interaction using surface plasmon resonance (SPR) technology SPR detection of affinity activity for PR3178 and PR0071 antibodies was performed using the BIAcore 8K (Cityva) system. The detection sensor chip protein A and related reagents were purchased from Cytiva. Antibodies PR3178, PR0071, and a control antibody were each diluted to 1 μg / ml in HBS-EP+ buffer, and the antibody was captured to a level of 200 RU at a flow rate of 10 μl / min. The his-tagged SEZ6 antigen was diluted in HBS-EP+ buffer at a constant ratio to concentration gradients of 0 nM, 3.125 nM, 6.25 nM, 12.5 nM (two repeats), 25 nM, 50 nM, 100 nM, and 200 nM. The flow rate was set to 30 μl / min during sample analysis. The binding time was 120 s and the dissociation time was 900 s. Regeneration was then performed using Gly-HCl buffer at pH 1.5 as the regeneration buffer, with a regeneration flow rate set to 30 μl / min, and regeneration was performed for 30 seconds. The response signal was plotted with analysis time as the x-coordinate and response value as the y-coordinate. The obtained data was fitted using BIAcore 8K analysis software, and dynamic constants such as the binding rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD) were determined using a 1:1 Langmuir binding model. The results showed that the PR3178 and PR0071 molecules bound to human and cynomolgus monkey antigens, and that their affinity was stronger than that of hSC200.

[0187] [Table 19]

[0188] Example 11: Conjugation of PR3178 and PR0071 with low molecular weight toxins In order to target the low molecular weight toxin 9106 to the tumor site, this experiment requires the conjugation of antibodies PR3178 and PR0071 with the low molecular weight toxin 9106. The specific experiment is as follows: Under conditions of 37°C, an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 75.4 μL, 753.5 nmol) was added to an aqueous solution of the antibody buffered in PBS (0.05 M PBS buffered aqueous solution with pH=6.5, 10.0 mg / mL, 5.0 mL, 342.5 nmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was then cooled to 25°C in a water bath.

[0189] Compound 9106 (3.7 mg, 3424.6 nmol), prepared according to WO2020063676A1, was dissolved in 250 μL of dimethyl sulfoxide and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted with HiPrep 26 / 10 (buffer solution: PBS pH 7.2~7.4, flow rate 10 mL / min), concentrated in an ultrafiltration tube to obtain PBS buffer solutions of PR3178-9106 and PR0071-9106 (PR3178-9106 concentration 1.0 mg / mL, 26 mg; PR0071-9106 concentration 2.0 mg / mL, 20 mg), and stored refrigerated at 4°C. The conjugation method between the anti-SEZ6 detection antibody hSC200 (hSC17.200, heavy chain SEQ ID NO: 6, light chain SEQ ID NO: 7) and 9106 was the same as described above.

[0190] The structures of the obtained antibody-drug conjugates PR3178-9106 and PR0071-9106 are shown below. When the drug load was calculated by RP-HPLC, the DAR values ​​for PR3178-9106 and PR0071-9106 were n=4.1 and n=3.6, respectively. [ka]

[0191] Example 12: Measurement of in vitro inhibitory activity of ADC against tumor cell line proliferation To detect the in vitro antitumor activity of the ADC molecule after conjugation, the in vitro activity of the ADC was reflected by inhibiting the proliferation of tumor cell lines. Specifically, the detection cells H69 and H82 were grown overnight in 96-well culture plates, with a density of 3000 cells per well. The following day, an isovolume gradient-diluted toxin drug conjugate was added. After 5 days, cell viability was determined using the CellTiter-Glo luminescence cell viability assay kit (Promega) as described in the manufacturer's instructions. Cell viability was evaluated as a percentage of untreated control cells. As shown in Figure 13, the ADC drug exhibited good killing activity in the H69 and H82 tumor cell lines.

[0192] [Table 20]

[0193] Example 13: Evaluation of in vivo antitumor activity of ADC To detect the in vivo antitumor activity of the ADC molecule, we used an H69 xenograft tumor model and reflected the antitumor activity of ADC by detecting changes in tumor growth size. Specifically, to produce xenografts, 5 x 10^6 H69 cells / 0.2 ml (PBS + gel) were subcutaneously inoculated into the right dorsal region of SCID mice, and the average tumor volume was 128 mm². 3 If the target was reached, the animals were divided into five groups of six according to tumor volume and body weight. Administration was started on the day of group division, with two intraperitoneal injections administered on Day 0 and Day 7. Tumor volume and body weight were detected twice a week, and the data were recorded.

[0194] Tumor volume V = 1 / 2 × a × b 2Here, a and b represent length and width, respectively. The relative tumor growth rate T / C (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes of the treatment group and control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Tumor inhibition rate TGI (%) = 1 - T / C (%). As described above, human IgG control antibody was used as a negative control. TGI represents the highest tumor growth inhibition rate during the experiment.

[0195] The results of the study are shown in Figure 14 and Table 21, and ADC showed significant antitumor activity against the proliferation of H69 xenograft tumors at equimodel dose levels.

[0196] [Table 21]

[0197] Example 14: Measurement of blood concentration of toxic drug conjugates To detect the pharmacokinetic characteristics of ADC in vivo in mice, the concentration of ADC in mouse serum after a single dose was calculated using ELISA. Specifically, SCID mice were used, and an equimolar dose of ADC was administered via tail vein injection as a single dose. Blood samples were collected at 15 min, 6 h, 24 h, 72 h, 144 h, 240 h, and 336 h after the initial dose. Blood concentrations were detected using ELISA. Plates were coated with anti-hFc and incubated overnight at 4°C. After washing once with 300 μL of PBST buffer, diluted test serum was added and incubated at room temperature for 1 hour. Serum from each tube was detected on two plates. One serum plate was washed three times with 300 μL of PBST buffer, then 100 μL of HRP-labeled sheep anti-human IgG Fc solution was added per well and incubated at room temperature for 1 hour. After washing five times with PBST buffer, a chromogenic solution was added, and after color development was complete, a stop solution was added, and the OD450 was read using a microplate reader. PK parameters were calculated using a non-compartment model in Phoenix software. Refer to Figure 15 and Table 22 for the results, which showed that PR3178-9106 had superior pharmacokinetic characteristics compared to hSC200-9106.

[0198] [Table 22]

[0199] The use and welfare of laboratory animals in this disclosure were carried out in accordance with the regulations of the International Association for the Evaluation and Approval of Laboratory Animals (AAALAC). The health status and mortality of the animals were monitored daily, and routine examinations included observing the effects of the test substance and drug on the animals' daily behavioral expressions, such as behavioral activity, weight changes, and external vital signs.

[0200] While specific embodiments of this disclosure have been described above, those skilled in the art will understand that these are merely illustrative examples and that many changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is limited by the attached claims.

Claims

1. An anti-SEZ6 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) or a heavy chain single domain (VHH), wherein the heavy chain variable region or the heavy chain single domain is (1) Heavy chain HCDR1, HCDR2, HCDR3, as shown in the amino acid sequences of SEQ ID NO: 37, 38 and 39, or (2) Heavy chain HCDR1, HCDR2, HCDR3, as shown in the amino acid sequences of SEQ ID NO: 43, 44 and 45, (3) Heavy chain HCDR1, HCDR2, HCDR3, or as shown in the amino acid sequences of SEQ ID NO: 49, 50 and 51 (4) Heavy chain HCDR1, HCDR2, HCDR3, or as shown in the amino acid sequences of SEQ ID NO: 65, 66 and 67 (5) An anti-SEZ6 antibody or its antigen-binding fragment, comprising the heavy chains HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 68, 69, and 70.

2. It includes a heavy chain variable region (VH) and a light chain variable region (VL), where the heavy chain variable region and the light chain variable region are (1) Heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 37, 38, and 39, and light chain LCDR1, LCDR2, and LCDR3 shown in the amino acid sequences of SEQ ID NO: 40, 41, and 42, or (2) Heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 43, 44, and 45, and light chain LCDR1, LCDR2, and LCDR3 shown in the amino acid sequences of SEQ ID NO: 46, 47, and 48, or (3) The anti-SEZ6 antibody or antigen-binding fragment thereof according to claim 1, comprising heavy chains HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 49, 50, and 51, and light chains LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 52, 53, and 54.

3. The heavy chain single domain includes the amino acid sequence shown in SEQ ID NO: 63, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or the amino acid sequence shown in SEQ ID NO: 64, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto. Preferably, the heavy chain single domain comprises the amino acid sequence shown in SEQ ID NO: 63 or the amino acid sequence shown in SEQ ID NO: 64, according to claim 1, an anti-SEZ6 antibody or antigen-binding fragment thereof.

4. It includes a heavy chain variable region and a light chain variable region, where, The heavy chain variable region includes the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 35, or sequences having at least 85%, 90%, 95%, and 99% homology thereto. The anti-SEZ6 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or a sequence having at least 85%, 90%, 95%, and 99% homology thereto.

5. It includes a heavy chain variable region and a light chain variable region, where, (1) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 29, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 32, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (3) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 34, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (4) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 35, or a sequence having at least 85%, 90%, 95%, and 99% homology thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 36, or a sequence having at least 85%, 90%, 95%, and 99% homology thereto. Preferably, (1) the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, or (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 32, or (3) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 34, or (4) The anti-SEZ6 antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

36.

6. The antigen-binding fragment is selected from antigen-binding fragments of peptides comprising Fab, Fab', F(ab')2, a single-chain antibody (scFv), a single-domain antibody (sdAb), a dimerized V region (diabody), a disulfide bond-stabilized V region (dsFv), and a CDR, according to any one of claims 1 to 5, for an anti-SEZ6 antibody or its antigen-binding fragment.

7. Includes the antibody constant region, Here, the heavy chain constant region is derived from human IgG1, IgG2, IgG3, IgG4 or their variants, and / or the light chain constant region is derived from human antibody κ, λ chain or their variants. Preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1 or a variant thereof, and / or the light chain constant region is derived from a human antibody κ chain or a variant thereof. More preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 33, and / or the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 59, the anti-SEZ6 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. It includes heavy chains and light chains, and here, (1) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 55, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain contains the amino acid sequence shown in SEQ ID NO: 56, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (2) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 57, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain contains the amino acid sequence shown in SEQ ID NO: 58, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (3) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 57, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, and the light chain contains the amino acid sequence shown in SEQ ID NO: 60, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, or (4) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 61, or a sequence having at least 85%, 90%, 95%, and 99% homology thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 62, or a sequence having at least 85%, 90%, 95%, and 99% homology thereto. Preferably, (1) the heavy chain contains the amino acid sequence shown in SEQ ID NO: 55, and the light chain contains the amino acid sequence shown in SEQ ID NO: 56, or (2) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 57, and the light chain contains the amino acid sequence shown in SEQ ID NO: 58, or (3) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 57, and the light chain contains the amino acid sequence shown in SEQ ID NO: 60, or (4) The anti-SEZ6 antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 61 and the light chain comprises the amino acid sequence shown in SEQ ID NO:

62.

9. It consists of heavy chains, and here, (1) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 71, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto, (2) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto. Preferably, (1) the heavy chain contains the amino acid sequence shown in SEQ ID NO: 71, or (2) The anti-SEZ6 antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

72.

10. An isolated nucleic acid encoding an anti-SEZ6 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A vector comprising the isolated nucleic acid described in claim 10.

12. A host cell comprising the vector according to claim 11.

13. A method for preparing an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in claim 12 under conditions suitable for the expression of the anti-SEZ6 antibody or the antigen-binding fragment thereof.

14. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, comprising an anti-SEZ6 antibody or its antigen-binding fragment and a drug according to any one of claims 1 to 9, wherein the drug is selected from cytotoxic agents, radiolabels, fluorophores, chromophores, imaging agents, immunomodulators, angiogenesis inhibitors, cell proliferation inhibitors, apoptosis promoters, cytolytic enzymes, and any combination thereof. Preferably, the drug is selected from DNA damaging agents, topoisomerase inhibitors, microtubule inhibitors, proteolytic agents, STING agonists, and any combination thereof, in the form of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

15. Having the structure shown in general formula (I), 【Chemistry 1】 Here, Dr is the drug described in claim 14, Preferably, Dr is selected from camptothecin, auristatin, meitansinoids, vinca alkaloids, pyrrolobenzodiazepines (PBDs), calicheamicin, duocalmycin, daunorubicin, doxorubicin, calicheamicin, anthramycin, neomycin, amanitin, hemiasterin, eribulin, tubullysin, and their analogs or derivatives. More preferably, Dr is selected from Exatecan, MMAE, MMAF, MMAD, SN-38, DM1, DM4, pyrrolobenzodiazepine (PBD) dimers and their analogs or derivatives. n is 1 to 10, n is a decimal or an integer, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, even more preferably n is 4 to 6, even more preferably n is 5 to 6, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. L and Y are linker units. Pc is an anti-SEZ6 antibody according to any one of claims 1 to 9 or an antigen-binding fragment thereof, in the form of an antibody-drug conjugate according to claim 14, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

16. It has the structure shown in general formula (Pc-L-Y-Dr), general formula (II), general formula (III), and general formula (IV), 【Chemistry 2】 Here, Y is selected from -O-(CR １ , ２ R ｂ ), ｍ -CR １ R ２ -C(O)-, -O-CR １ R ２ -(CR ａ R ｂ ), ｍ -O-CR １ R ２ -, -NH-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)- or -S-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)-; R ａ and R ｂ These are the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, hydroxyl groups, amino groups, cyano groups, nitro groups, hydroxyalkyl groups, cycloalkyl groups, or heterocyclyl groups. Or, R ａ and R ｂ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. R １ This is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. R ２ This is selected from a hydrogen atom, halogen, haloalkyl group, deuterated alkyl group, cycloalkyl group, cycloalkylalkyl group, alkoxyalkyl group, heterocyclyl group, aryl group, or heteroaryl group. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. Or, R ａ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. m is an integer between 0 and 4. n is 1 to 10, n is a decimal or an integer, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, even more preferably n is 4 to 6, even more preferably n is 5 to 6, and even more preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. L is a linker unit, Pc is an anti-SEZ6 antibody according to any one of claims 1 to 9 or an antigen-binding fragment thereof, in the form of an antibody-drug conjugate according to claim 14 or 15, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

17. Y is -O-(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)-, R ａ and R ｂ These are the same or different, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group. R １ is a haloalkyl group or C ３－６ It is a cycloalkyl group, R ２ is a hydrogen atom, a haloalkyl group, or C ３－６ Selected from cycloalkyl groups, Or, R １ and R ２ C ３－６ Forms a cycloalkyl group, The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 16, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein m is 0 or 1.

18. Y is, 【Transformation 3】 An antibody-drug conjugate according to claim 16 or 17, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, selected from among.

19. The O-terminus of Y is linked to a linker unit L, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 18, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

20. Linker unit -L- is -L １ -L ２ -L ３ -L ４ - and L １ teeth, 【Chemistry 4】 and s １ These are integers from 2 to 8, L ２ It is a chemical bond, L ３ It is a tetrapeptide residue, L ４ -NR ５ (CR ６ R ７ )t-, R ５ , R ６ or R ７ an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 19, wherein is the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

21. Linker unit -L- is -L １ -L ２ -L ３ -L ４ - and L １ teeth, 【Transformation 5】 and s １ and s ２ These are independently selected from integers 0 to 8, preferably s １ is 2, and s ２ is an integer from 2 to 8, more preferably s １ is 2, and s ２ is 2, 3, 4, 5 or 6, L ２ It is a chemical bond, L ３ It is a tetrapeptide residue, L ４ -NR ５ (CR ６ R ７ )t-, R ５ , R ６ or R ７ an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 19, wherein is the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

22. The aforementioned linker unit -L- is L １ The end is connected to Pc, L ４ An antibody-drug conjugate according to any one of claims 16 to 21, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, wherein the terminal is linked to Y.

23. Said L ３ The tetrapeptide residue is an amino acid residue formed from two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, preferably the tetrapeptide residue of GGFG, the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

24. The antibody-drug conjugate is 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 23, selected from, where Pc and n are as defined in claim 16, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

25. The antibody-drug conjugate is 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 24, selected from, where n is as defined in claim 16, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

26. A kit comprising an anti-SEZ6 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 14 to 25, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof.

27. A pharmaceutical composition comprising an anti-SEZ6 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9, or an antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described in any one of claims 14 to 25, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, and one or more pharmaceutically acceptable carriers.

28. Uses of an anti-SEZ6 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 14 to 25, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutical composition according to claim 27, in the manufacture of a drug for treating SEZ6-mediated diseases or disorders, Preferably, the SEZ6-mediated disease or disorder is a cancer with high SEZ6 expression.

29. An anti-SEZ6 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 14 to 25, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutical composition according to claim 27, for use in the manufacture of a drug for treating or preventing tumors or cancer, Preferably, the tumor or cancer here is selected from lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, and bone cancer.

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