Anti-b7h3 and DLL3 antibody, antibody-drug conjugate thereof and use thereof

Abstract

Provided is a bispecific antibody capable of specifically binding to B7H3 and DLL3. The bispecific antibody is capable of simultaneously targeting B7H3 and DLL3, and may represent a new strategy for tumor treatment. In addition, further provided are an antibody-drug conjugate (ADC) of the bispecific antibody, a polynucleotide encoding the bispecific antibody, a vector, a host cell, and a pharmaceutical composition containing the bispecific antibody or ADC thereof.

Description

Anti-B7H3 and DLL3 antibodies, their antibody-drug conjugates, and their applications

[0001] This disclosure claims priority to Chinese Patent Application No. 202510044326.2, filed on January 11, 2025, entitled "Anti-B7H3 and DLL3 Antibodies, Anti-Drug Conjugates Thereof and Their Applications", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibodies, and more specifically, to anti-B7H3 / DLL3 antibodies, antibody-drug conjugates thereof, and their applications. Background Technology

[0003] B7H3, also known as CD276, is a type I transmembrane protein and belongs to the B7 ligand family. B7H3 is widely expressed in normal tissues, but at limited levels, remaining at relatively low levels, such as in the liver, colon, and prostate. However, it is expressed at higher levels in tumor tissues and is closely related to cancer patient progression, survival, and prognosis. Studies have found high expression of B7H3 in various tumor tissues, including small cell lung cancer, prostate cancer, renal cell carcinoma, colorectal cancer, and squamous cell lung cancer. In tumor tissues, B7H3 is expressed in both tumor cells and stromal cells, such as tumor vascular endothelial cells, pericytes, and fibroblasts.

[0004] Delta-like ligand 3 (DLL3) is a member of the Notch ligand family. DLL3 is largely unexpressed in normal tissues, but its mRNA transcription level is high in tissues such as the nervous system, pancreas, and testes, while its protein expression level is low. DLL3 is highly expressed in small cell lung cancer samples, distributed in the cell membrane and cytosol, and shows a trend of correlation with disease grade, but this correlation is not significant. AbbVie reported that 83% of small cell lung cancer patient samples were DLL3 positive, with 32% of these samples showing strong DLL3 positivity (50% cellular positivity). CD3-DLL3 bispecific antibody products, such as AMG757, have provided strong proof-of-concept for the clinical efficacy of DLL3. In summary, both B7H3 and DLL3 are widely expressed in small cell lung cancer, and related drugs are in clinical research stages. Summary of the Invention

[0005] Given that both B7H3 and DLL3 have relatively high positivity rates in SCLC (>80%), but their expression levels are not high (over 70% of patients show IHC staining as 1+), and that B7H3 has a relatively slow endocytosis rate while DLL3 has a relatively fast endocytosis rate, this invention combines B7H3 and DLL3 to develop bispecific antibodies and their ADC drugs. It provides anti-B7H3 and DLL3 bispecific antibodies, their antibody-drug conjugates (ADCs), the nucleic acids encoding them, antibody preparation methods, pharmaceutical compositions containing said antibodies or ADCs, and related uses of the pharmaceutical compositions for the treatment of tumors.

[0006] In a first aspect, this disclosure provides a multispecific antibody or an antigen-binding fragment thereof, which includes a first antigen-binding domain that specifically binds to B7H3 and a second antigen-binding domain that specifically binds to DLL3.

[0007] In some specific embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from antibodies, antibody fragments, F(ab')2, Fab', Fab, Fv, scFv, nanobodies, or VHH.

[0008] In some specific embodiments, the multispecific antibody or its antigen-binding fragment comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein,

[0009] (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1;

[0010] (2) The first light chain includes the following structure: VL B7H3 -CL;

[0011] (3) The second heavy chain includes the following structure: VL DLL3 -CH1-Fc2;

[0012] (4) The second light chain includes the following structure: VH DLL3 -CL;

[0013] Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 These are the heavy chain variable region and light chain variable region that specifically bind to DLL3, respectively; Fc1 and Fc2 are each the Fc region of any antibody.

[0014] In some specific embodiments, the multispecific antibody or its antigen-binding fragment comprises three polypeptide chains: a first heavy chain, a first light chain, and a second heavy chain; wherein,

[0015] (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1;

[0016] (2) The first light chain includes the following structure: VL B7H3 -CL;

[0017] (3) The second heavy chain includes the following structure: VL DLL3 -(G4S)3-VH DLL3 -GGG-Fc2; or VHH DLL3 -GGG-Fc2;

[0018] Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 They specifically bind to the heavy chain variable region and light chain variable region of DLL3, respectively, and form scFv; VHH DLL3 These are nanobodies that specifically bind to DLL3; Fc1 and Fc2 are the Fc regions of any antibody.

[0019] In some specific embodiments, the scFv that specifically binds to DLL3 introduces a CC mutation; Fc1 and Fc2 are different, with Fc1 being a knock-Fc and Fc2 being a hole-Fc; preferably, the knock-Fc contains a T366W mutation, and / or the hole-Fc contains a T366S, L368A, and / or Y407V mutation; preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4.

[0020] In some specific embodiments, the first antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 20; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 21.

[0021] In some specific embodiments, the first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions:

[0022] Heavy chain CDR1, such as SEQ ID NO.27, 48, 69;

[0023] Heavy chain CDR2 such as SEQ ID NO.28, 49, 70;

[0024] Heavy chain CDR3 such as SEQ ID NO.29, 50, 71;

[0025] Light chain CDR1, such as SEQ ID NO.39, 60, 81;

[0026] Light chain CDR2 such as SEQ ID NO.40, 61, 82;

[0027] Light chain CDR3, such as SEQ ID NO.41, 62, 83.

[0028] In some specific embodiments, the VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 21.

[0029] In some specific embodiments, the second antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 22, 24 or 26; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 23 or 25;

[0030] Preferably, the second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably, it has conserved amino acid substitutions:

[0031] Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78;

[0032] Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79;

[0033] Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80;

[0034] Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87;

[0035] Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88;

[0036] Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89.

[0037] In some specific embodiments, the VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 23 or 25.

[0038] In some specific embodiments, the first heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 12; the first light chain comprises an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 13;

[0039] The second heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 14, 16, 17 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 14, 16, 17; the second light chain comprises an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 15.

[0040] In some specific embodiments, the multispecific antibody or its antigen-binding fragment is:

[0041] (1) Chimeric antibodies or fragments thereof;

[0042] (2) Humanized antibodies or fragments thereof; or,

[0043] (3) Fully human antibodies or fragments thereof.

[0044] In some specific embodiments, the multispecific antibody or its antigen-binding fragment is trivalent, tetravalent, pentavalent, or hexavalent; preferably, the multispecific antibody or its antigen-binding fragment is bivalent.

[0045] In some specific embodiments, the multispecific antibody or its antigen-binding fragment is further conjugated with a cytotoxic drug, which is selected from chemotherapeutic drugs or antibiotics; optionally, the cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents, or DNA topoisomerase inhibitors, wherein the tubulin inhibitors include dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins, the DNA damaging agents include PBD drugs, and the DNA topoisomerase inhibitors include camptothecin drugs.

[0046] In some specific embodiments, the cytotoxic drug is linked to the multispecific antibody or its antigen-binding fragment via a linker.

[0047] In a second aspect, this disclosure provides an isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the multispecific antibody or antigen-binding fragment thereof described in the first aspect of this disclosure.

[0048] In a third aspect, this disclosure provides a vector comprising the nucleic acid fragment described in the second aspect of this disclosure.

[0049] In a fourth aspect, this disclosure provides a host cell, characterized in that the host cell comprises the vector described in the third aspect of this disclosure; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0050] In a fifth aspect, this disclosure provides a method for preparing the multispecific antibody or antigen-binding fragment thereof described in the first aspect of this disclosure, the method comprising culturing the cells described in the fourth aspect of this disclosure, and isolating the multispecific antibody or antigen-binding fragment thereof expressed by the cells.

[0051] In a sixth aspect, this disclosure provides an antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt thereof, Pc-(LD). n (A)

[0052] in,

[0053] D is a cytotoxic drug;

[0054] L is the connecting subunit;

[0055] Pc is a multispecific antibody or its antigen-binding fragment, which contains a first antigen-binding domain that specifically binds to B7H3 and a second antigen-binding domain that specifically binds to DLL3.

[0056] Furthermore, n is a real number from 1 to 16.

[0057] In some specific embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or its pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment Pc is the antibody or antigen-binding fragment described in the first aspect of this disclosure.

[0058] In some specific embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from antibodies, antibody fragments, F(ab')2, Fab', Fab, Fv, scFv, nanobodies, or VHH.

[0059] In some specific embodiments, the multispecific antibody or its antigen-binding fragment comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein,

[0060] (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1;

[0061] (2) The first light chain includes the following structure: VL B7H3 -CL;

[0062] (3) The second heavy chain includes the following structure: VL DLL3 -CH1-Fc2;

[0063] (4) The second light chain includes the following structure: VH DLL3 -CL;

[0064] Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 These are the heavy chain variable region and light chain variable region that specifically bind to DLL3, respectively; Fc1 and Fc2 are each the Fc region of any antibody.

[0065] In some specific embodiments, the multispecific antibody or its antigen-binding fragment comprises three polypeptide chains: a first heavy chain, a first light chain, and a second heavy chain; wherein,

[0066] (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1;

[0067] (2) The first light chain includes the following structure: VL B7H3 -CL;

[0068] (3) The second heavy chain includes the following structure: VL DLL3 -(G4S)3-VH DLL3 -GGG-Fc2; or VHH DLL3 -GGG-Fc2;

[0069] Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 They specifically bind to the heavy chain variable region and light chain variable region of DLL3, respectively, and form scFv; VHH DLL3 These are nanobodies that specifically bind to DLL3; Fc1 and Fc2 are the Fc regions of any antibody.

[0070] In some specific embodiments, the scFv that specifically binds to DLL3 introduces a CC mutation; Fc1 and Fc2 are different, with Fc1 being a knock-Fc and Fc2 being a hole-Fc; preferably, the knock-Fc contains a T366W mutation, and / or the hole-Fc contains a T366S, L368A, and / or Y407V mutation; preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4.

[0071] In some specific embodiments, the first antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 20; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 21.

[0072] In some specific embodiments, the first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions:

[0073] Heavy chain CDR1, such as SEQ ID NO.27, 48, 69;

[0074] Heavy chain CDR2 such as SEQ ID NO.28, 49, 70;

[0075] Heavy chain CDR3 such as SEQ ID NO.29, 50, 71;

[0076] Light chain CDR1, such as SEQ ID NO.39, 60, 81;

[0077] Light chain CDR2 such as SEQ ID NO.40, 61, 82;

[0078] Light chain CDR3, such as SEQ ID NO.41, 62, 83.

[0079] In some specific embodiments, the VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 21.

[0080] In some specific embodiments, the second antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 22, 24 or 26; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 23 or 25;

[0081] Preferably, the second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably, it has conserved amino acid substitutions:

[0082] Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78;

[0083] Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79;

[0084] Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80;

[0085] Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87;

[0086] Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88;

[0087] Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89.

[0088] In some specific embodiments, the VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 23 or 25.

[0089] In some specific embodiments, the first heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 12; the first light chain comprises an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 13;

[0090] The second heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 14, 16, 17 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 14, 16, 17; the second light chain comprises an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 15.

[0091] In some specific implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt thereof, wherein the cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics.

[0092] In some specific implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt thereof, wherein the cytotoxic drug D is selected from tubulin inhibitors, DNA damaging agents, or DNA topoisomerase inhibitors, wherein the tubulin inhibitors include, but are not limited to, dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins, wherein the DNA damaging agents include, but are not limited to, PBD drugs, and wherein the DNA topoisomerase inhibitors include, but are not limited to, camptothecin drugs.

[0093] In some specific embodiments, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

[0094] In some specific embodiments, the cytotoxic drug D is selected from compounds of formula (DI).

[0095] in,

[0096] R 1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more D atoms;

[0097] R 4 Selected from H or C1-C3 alkyl groups;

[0098] R 5 Selected from H, halogens, CN, =O, OH, NH2, or C1-C3 alkyl groups;

[0099] R 6 Selected from H or C1-C3 alkyl groups;

[0100] R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

[0101] In some specific implementations, the R 1 R 2 The atoms connected to them together form

[0102] In some specific implementations, R 4 Selected from H.

[0103] In some specific implementations, R 5 It is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl.

[0104] In some specific implementations, R 5 Selected from H.

[0105] In some specific implementations, R 6 Selected from H.

[0106] In some specific implementations, R 7 Selected from cyclopropyl.

[0107] In some specific embodiments, the compound represented by formula (DI) is selected from one of the following compounds:

[0108] In some specific implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt, wherein the linker unit L is selected from... Its a-terminus is covalently linked to the antibody unit Pc, and its b-terminus is covalently linked to the cytotoxic drug D, wherein:

[0109] R b1 R b2 Each is independently selected from H, halogen, CN, C1-C6 alkyl or C3-C6 cycloalkyl, or R b1 R b2 The carbon atoms connected to them together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, which is optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic group;

[0110] m1 is selected from integers 2 to 8;

[0111] L a Selected from Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl groups, m2 is selected from integers 1 to 8, R b4 R b5 Each is independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl,

[0112] L b The peptide residues are selected from 1 to 8 amino acids, and the peptide residues are optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic groups.

[0113] In some specific implementations, R b1 R b2 All are selected from H or R b1 R b2 The carbon atoms connected to them together form 4-7 membered heterocyclic groups.

[0114] In some specific implementations, structural units Selected from

[0115] In some specific implementations, L a Selected from chemical bonds or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl, R b4 R b5 One is selected from H, and the other is selected from

[0116] In some specific implementations, L a Selected from Its * end and L b connect.

[0117] In some specific implementations, the L b It consists of Gly-Gly-Phe-Gly tetrapeptide residues.

[0118] In some specific implementations, m1 is an integer from 2 to 6.

[0119] In some specific implementations, m1 is 2, 3, 4 or 5.

[0120] In some specific implementations, the connecting subunit L is

[0121] Its a-end is covalently linked to the antibody unit Pc, and its b-end is covalently linked to the drug unit D.

[0122] In some specific implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt, wherein n is selected from a real number from 1 to 16, for example n is selected from a real number from 2 to 12, for example n is selected from a real number from 4 to 10, for example n is selected from a real number from 3 to 9, for example n is selected from a real number from 4 to 8, for example n is selected from a real number from 6 to 8.

[0123] In some specific implementations, n is selected from real numbers from 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5 7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0124] In some specific implementations, n is selected from real numbers from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0125] In some specific implementations, the general formula of this disclosure is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt is selected from the following compounds or their pharmaceutically acceptable salts:

[0126] Where Pc and n are as defined in any of the preceding definitions.

[0127] In a seventh aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned multispecific antibody or its antigen-binding fragment, nucleic acid fragment, carrier, host cell, product prepared by the aforementioned methods, or antibody-drug conjugate or its pharmaceutically acceptable salt; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0128] In an eighth aspect, this disclosure provides the use of the aforementioned multispecific antibodies or antigen-binding fragments thereof, nucleic acid fragments, vectors, host cells, products prepared by the aforementioned methods, or antibody-drug conjugates or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of tumors; wherein the tumors are selected from solid tumors, hematologic malignancies, or cancers that invasively express B7H3 or DLL3;

[0129] Preferably, the tumor is selected from small cell lung cancer, pancreatic cancer, and other solid tumors.

[0130] In an eighth aspect, this disclosure provides a method for preventing and / or treating tumors, comprising administering to a patient in need an effective amount of the aforementioned multispecific antibody or antigen-binding fragment thereof, nucleic acid fragment, vector, host cell, product prepared by the aforementioned method, or antibody-drug conjugate or a pharmaceutically acceptable salt thereof; wherein the tumor is selected from solid tumors, hematologic malignancies, or cancers that infiltrate and express B7H3 or DLL3;

[0131] Preferably, the tumor is selected from small cell lung cancer, pancreatic cancer, and other solid tumors.

[0132] In a ninth aspect, this disclosure provides methods for the prevention and / or treatment of tumors using the aforementioned multispecific antibodies or their antigen-binding fragments, nucleic acid fragments, vectors, host cells, products prepared by the aforementioned methods, or antibody-drug conjugates or their pharmaceutically acceptable salts; wherein the tumors are selected from solid tumors, hematologic malignancies, or cancers that invasively express B7H3 or DLL3;

[0133] Preferably, the tumor is selected from small cell lung cancer, pancreatic cancer, and other solid tumors.

[0134] In a tenth aspect, this disclosure provides a method for preventing and / or treating tumors, comprising administering to a patient in need an effective amount of a multispecific antibody or an antigen-binding fragment thereof, which includes a first antigen-binding domain specifically binding to B7H3 and a second antigen-binding domain specifically binding to DLL3.

[0135] In some specific embodiments, the first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions:

[0136] Heavy chain CDR1, such as SEQ ID NO.27, 48, 69;

[0137] Heavy chain CDR2 such as SEQ ID NO.28, 49, 70;

[0138] Heavy chain CDR3 such as SEQ ID NO.29, 50, 71;

[0139] Light chain CDR1, such as SEQ ID NO.39, 60, 81;

[0140] Light chain CDR2 such as SEQ ID NO.40, 61, 82;

[0141] Light chain CDR3, such as SEQ ID NO.41, 62, 83.

[0142] In some specific embodiments, the second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions:

[0143] Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78;

[0144] Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79;

[0145] Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80;

[0146] Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87;

[0147] Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88;

[0148] Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89.

[0149] In some specific embodiments, the VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 21; the VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; Compared to NO.23 or 25, the amino acid sequence has 90%, 95%, 96%, 97%, 98%, 99% or higher homology.

[0150] In some specific embodiments, the multispecific antibody or its antigen-binding fragment is further conjugated with a cytotoxic drug D; optionally, the cytotoxic drug D is selected from one of the following compounds.

[0151] In some specific embodiments, the cytotoxic drug D is linked to the multispecific antibody or its antigen-binding fragment via a linker unit L; optionally, the linker unit L is... Its a-end is covalently linked to the antibody, and its b-end is covalently linked to the drug D. Attached Figure Description

[0152] Figures 1A-1C are schematic diagrams of the dual-resistance structure.

[0153] Figure 2A shows the binding activity of the penicillin antibody, its conjugated ADC, the control antibody, and the control ADC to human DLL3-his protein detected by ELISA.

[0154] Figure 2B shows the binding activity of the penicillin antibody, its conjugated ADC, the control antibody, and the control ADC to monkey DLL3-his protein detected by ELISA.

[0155] Figure 2C shows the ELISA detection of the binding activity of the penicillin antibody, its conjugated ADC, the control antibody, and the control ADC to human B7H3-his protein.

[0156] Figure 2D shows the binding activity of the penicillin antibody, its conjugated ADC, the control antibody, and the control ADC to monkey B7H3-his protein detected by ELISA.

[0157] Figures 3A-3B show the binding activity of the bispecific antibody, its conjugated ADC, control antibody, and control ADC to human tumor cells NCI-H82 and NCI-H510A cells as detected by FACS.

[0158] Figure 3C shows the non-specific binding of the double antibody, its conjugated ADC, the control antibody, and the control ADC to the negative MDA-MB-453 cells detected by FACS.

[0159] Figure 4A shows the evaluation of the killing activity of the bispecific antibody ADC molecule and the control ADC against the NCI-H146 tumor cell line.

[0160] Figure 4B shows the evaluation of the killing activity of the bispecific antibody ADC molecule and the control ADC against the NCI-H526 tumor cell line.

[0161] Figure 4C shows the evaluation of the killing activity of the bispecific antibody ADC molecule and the control ADC against the tumor cell line QGP-1.

[0162] Figure 5A shows the tumor growth curves of human small cell lung cancer NCI-H82 ADC candidate molecules and control ADC under the regulation of human small cell lung cancer NCI-H82.

[0163] Figure 5B shows the curves of weight change in NCI-H82 tumor-bearing mice with human small cell lung cancer regulated by ADC candidate molecules and control ADC.

[0164] Figure 6A shows the growth curves of NCI-H2171 small cell lung cancer tumors regulated by ADC candidate molecules and control ADC.

[0165] Figure 6B shows the curves of weight change in NCI-H2171 tumor-bearing mice regulated by ADC candidate molecules and control ADC.

[0166] Terminology Definitions and Explanations

[0167] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0168] Furthermore, unless otherwise stated herein, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0169] The terms “comprising,” “including,” and “having” are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for solutions “composed of.” For example, “a composition comprising A and B” should be understood to include compositions consisting of A and B, as well as compositions containing other components besides A and B, both falling within the scope of the aforementioned “a composition.”

[0170] The term “and / or” as used herein includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0171] The term "B7H3" in this article refers to a member of the B7-CD28 superfamily, expressed on antigen-presenting cells. B7-H3 binds to T cells, but the B7-H3 counter-receptor on the surface of these T cells has not been fully characterized. The human B7H3 protein is 534 amino acids long and primarily contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC). Although initially thought to contain only two Ig domains (IgV-IgC), a four-immunoglobulin extracellular domain variant ("4Ig-B7-H3") has been identified and found to be the more common human form. The native murine form (2Ig) and the human 4Ig form show similar functions. The 4Ig-B7-H3 molecule inhibits NK cell-mediated lysis of cancer cells. B7H3 mRNA expression has been found in heart, kidney, testis, lung, liver, pancreas, prostate, colon, and osteoblasts.

[0172] The term "Delta-Like Ligand 3 (DLL3)" in this article refers to a single-pass transmembrane protein attached to the cell surface, belonging to the Notch ligand family. The human DLL3 gene is located on chromosome 19q13, with an open reading frame length of approximately 1800 bp. The human DLL3 protein consists of 619 amino acids, and its complete structure includes one DSL domain, one intracellular domain, and six epidermal growth factor-like domains. The DSL gene sequence at the N-terminus of the extracellular domain is highly conserved within the ligand family and is the essential functional domain for binding to the Notch receptor. The intracellular domain of DLL3 is relatively short, and its function is unclear. Studies have found that DLL3 is highly expressed in SCLC and other neuroendocrine tumors, but rarely expressed in normal tissues. Activation of DLL3 can exert pro-cancer or anti-cancer effects. DLL3 is widely expressed in human cancers, including small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary adenoma, endometrioma, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer, and esophageal cancer.

[0173] The term "specific binding" in this article refers to the ability of antigen-binding molecules (such as antibodies) to specifically bind to antigens and substantially the same antigens with high affinity, but not to unrelated antigens with high affinity. Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. For example, for antibodies, high affinity typically refers to a KD of 1 × 10⁻⁶. -7 M or lower, approximately 1×10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, 1×10-11 M or lower or 1×10 -12 M or lower KD. KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see the method for obtaining the KD value shown in Example 7 of this document.

[0174] The term "antigen-binding molecule" is used in the broadest sense in this document to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen-binding molecules include, but are not limited to, antibodies or antibody mimics. "Antibody mimic" refers to an organic compound or binding domain that can specifically bind to an antigen but is independent of the antibody structure. Exemplarily, antibody mimics include, but are not limited to, affibody, affitin, affilin, designed ankylosing spondylamine repeat (DARPin), aptamers, or Kunitz-type domain peptides.

[0175] The term “antibody” in this article, used in its broadest sense, refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain, thereby enabling specific binding to an antigen. The term “antibody” in this article encompasses a wide range of forms and structures, provided they exhibit the desired antigen-binding activity. The term “antibody” in this article includes alternative protein scaffolds or artificial scaffolds with transplanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (containing mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for use in transplanting CDRs, including but not limited to tendinins, fibronectins, peptide aptamers, etc.

[0176] The term "antibody" in this article includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin family composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0177] The term "antibody" in this article includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanocamels (Lama guanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0178] As used herein, the term "heavy chain antibody" refers to an antibody that lacks the light chain of a conventional antibody. This term specifically includes, but is not limited to, homodimeric antibodies containing a VH antigen-binding domain and constant CH2 and CH3 domains in the absence of a CH1 domain.

[0179] As used in this article, the term "nanobody" refers to a naturally occurring heavy chain antibody in camels that lacks the light chain. Cloning its variable region yields a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0180] The terms "nanobody" and "single domain antibody" (sdAb) used in this article have the same meaning and are used interchangeably. They refer to the cloning of the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, a naturally occurring heavy chain antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0181] Further descriptions of "heavy chain antibodies" and "nanobodies" can be found in: Hamers-Casterman et al., Nature. 1993; 363; 446-8; Muyldermans' review article (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO97 / 49805, WO 01 / 21817, WO WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and other prior art mentioned in these applications.

[0182] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0183] The term "multispecific" in this article refers to having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0184] The term "valence" in this article refers to the presence of a specified number of binding sites in an antibody / antigen binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" represent the presence of one, two, four, and six binding sites in an antibody / antigen binding molecule, respectively.

[0185] In this document, "antigen-binding fragment" and "antibody fragment" are used interchangeably. They do not possess the complete structure of a full antibody, but only contain a portion or a local variant of the full antibody, which has the ability to bind antigens. "Antigen-binding fragment" or "antibody fragment" in this document includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabody, and single-domain antibodies.

[0186] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing variable domains for both the heavy and light chains, as well as a constant domain for the light chain and a first constant domain (CH1) for the heavy chain. Thus, the term "Fab fragment" in this paper refers to the light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the Fab' fragment in which the cysteine ​​residues in the constant domain carry a free thiol group. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0187] The term "Fd" in this paper refers to an antibody composed of VH and CH1 domains. The term "Fv" refers to an antibody fragment composed of a single-arm VL and VH domain. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to antibodies. However, even a variable region (such as an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site.

[0188] The term "scFv" (single-chain variable fragment) in this paper refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 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. In some cases, a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).

[0189] The term "diabody" in this article refers to a single polypeptide chain in which the VH and VL domains are expressed, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0190] The term "naked antibody" in this article refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" in this article refers to an antibody that is conjugated to a therapeutic agent or tracer.

[0191] The term "humanized antibody" in this article refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0192] The term "fully human antibody" in this document refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" in this document does not include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0193] The term "variable region" in this article refers to the region in the antibody heavy or light chain involved in enabling antibody binding to antigens. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably in this article. They typically refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form a precise complementarity with the antigen epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" and "FR region" are used interchangeably, referring to the amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. A typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0194] For further description of CDR, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). The term “CDR” in this paper may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tools used may include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abysis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0195] The term "Kabat numbering system" in this article usually refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0196] The term "IMGT numbering system" in this article generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0197] The term “Chothia numbering system” in this article usually refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

[0198] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. A "heavy chain constant region" contains at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0199] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0200] The term "Fc" in this document refers to the C-terminal portion of an antibody obtained by papain hydrolysis of a complete antibody, and is used to define the C-terminal region of an antibody heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its C-terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447. Typically, it contains the CH3 and CH2 domains of the antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. The "CH2 region" of the human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 region. The CH2 domain in this article can be the natural CH2 region or a variant CH2 region. The "CH3 region" includes the residues at the C-terminus of the CH2 region in the Fc region (i.e., amino acid residues from approximately position 341 to approximately position 447 of IgG). The CH3 region in this article can be the natural CH3 region or a variant CH3 region. Fc variants are generated from these variant CH3 regions. Inter-Fc variants can form space-filling effects, electrostatic conduction, hydrogen bonding, hydrophobic interactions, etc., which contribute to the formation of stable heterodimeric proteins.

[0201] The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "Knob-into-Hole" form proposed by Cater et al.; the formation of Fc-containing heterodimers using electrostatic steering by Amgen engineers (US 20100286374 A1); the heterodimer form (SEEDbodies) formed through IgG / Ig chain exchange proposed by Jonathan H. Davis et al.; bispecific molecules formed using Genmab's DuoBody platform technology; heterodimer protein forms formed by Xencor engineers through a combination of structural calculations and Fc amino acid mutations, integrating different modes of action; the Fc modification method based on charge networks from Suzhou Corning Jerry (CN201110459100.7) to obtain heterodimer protein forms; and other genetic engineering methods based on Fc amino acid changes or functional modifications to achieve the formation of heterodimer functional proteins. The Knob / Hole structure on the Fc variant fragments described in this application refers to the mutation of each of the two Fc fragments, which can then bind in a "knob-into-hole" manner. Preferably, the "knob-into-hole" model of Cater et al. is used to modify the Fc region through site mutations, so that the resulting first and second Fc variants can bind together in a "knob-into-hole" manner to form a heterodimer. Selecting specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the scope of those skilled in the art. Preferably, the Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are used, more preferably the Fc region of human antibody IgG1. One of the first or second Fc variants is randomly selected to undergo a knockb mutation (knob chain), and the other to undergo a hole mutation (hole chain). In some specific embodiments, the CH3 region of the knockb chain contains the T366W mutation, and the hole chain contains the T366S, L368A, and / or Y407V mutations, resulting in the Fc variant. (The above content is incorporated herein by reference in its entirety.) In some embodiments, the first Fc variant may be Fc1 (SEQ ID NO. 90), and the second Fc variant may be Fc2 (SEQ ID NO. 91).

[0202] SEQ ID NO.90:

[0203] SEQ ID NO.91:

[0204] The term "conservative amino acid" in this document generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group constitute substitutions of conserved amino acids:

[0205] For example, the following six groups are examples of amino acids that are considered to have conserved substitutions for each other:

[0206] 1) Alanine (A), Serine (S), Threonine (T);

[0207] 2) Aspartic acid (D), glutamic acid (E);

[0208] 3) Asparagine (N), glutamine (Q);

[0209] 4) Arginine (R), Lysine (K), Histidine (H);

[0210] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0211] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0212] The term "identity" in this document can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0213] Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the common positions shared by the sequences.

[0214] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at www.gcg.com) in the GAP program integrated into the GCG software package can be used to determine the percentage of identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. As another example, the GAP program in the GCG software package (available at www.gcg.com) can be used to determine the percentage of identity between two nucleotide sequences using an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0215] The PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0216] Additionally or alternatively, the nucleic acid and protein sequences described in this invention can be further used as "query sequences" to perform searches against public databases to, for example, identify sequences of other family members or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of this invention. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of this invention. For comparison purposes, vacancy-based alignment results can be obtained using vacancy BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When using the BLAST and empty BLAST procedures, the default parameters for the corresponding procedures (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0217] The term "nucleic acid" in this document includes any compound and / or substance comprising a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1). “Isolated” nucleic acids herein refer to nucleic acid molecules that have been separated from components of their native environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are present outside the chromosome or at a chromosomal location different from their native chromosomal location.

[0218] The term "vector" in this paper refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors can direct the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0219] The term "host cell" in this article refers to a cell in which foreign nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0220] In this article, "n is a real number from 1 to 16" means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0221] In this article Indicates the connection site.

[0222] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0223] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0224] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0225] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0226] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0227] C in this article m -C n , refers to having an integer number of carbon atoms in the range mn.

[0228] The term "alkyl" refers to the general formula CnH. 2n+1 The alkyl group can be straight-chain or branched. The term "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0229] The “C1-C6 alkyl” mentioned in this article may further include “C1-C3 alkyl”.

[0230] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0231] The term "heterocyclic group" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridged ring group containing 1-5 heteroatoms or heteroatom groups (i.e., atomic groups containing heteroatoms). These "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, containing 1-3 independently selected heteroatoms or heteroatom groups as described above. The term "5-6 membered heterocyclic group" refers to a heterocyclic group with 5 or 6 ring atoms, and whose ring atoms contain 1-3 independent heteroatoms or heterogroups selected from those described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrocyclobutane and oxacyclobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. "4-7 membered heterocyclic group" can encompass the ranges of "4-7 membered heterocyclic alkyl," "5-6 membered heterocyclic group," and "5-6 membered heterocyclic alkyl."

[0232] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0233] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0234] The term "effective dose" refers to the amount of a therapeutic agent, when administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0235] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that receive treatment for a disease or condition.

[0236] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0237] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0238] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0239] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the disclosed compounds to an organism.

[0240] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0241] The term "cancer" in this document refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The term "tumor" or "tumor" in this document refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used in this document.

[0242] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0243] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0244] Certain isotope-labeled compounds of this disclosure (e.g., using...) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0245] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0246] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0247] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0248] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety. Detailed Implementation

[0249] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0250] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0251] Example 1: Preparation and purification methods of recombinant protein and control antibody

[0252] 1.1 Design, Expression and Purification of Recombinant Proteins

[0253] Construction of B7H3-4Ig recombinant protein: Using human B7H3-4Ig protein (UniProt: Q5ZPR3-1) as a template sequence, a tagged fusion protein was designed, cloned into the pTT5 vector (UniProt, VT2202), and the B7H3-4Ig plasmid was constructed. Similarly, construction of B7H3-2Ig recombinant protein: Using human B7H3-2Ig protein (UniProt: Q5ZPR3-2) as a template sequence, a tagged fusion protein was designed, cloned into the pTT5 vector, and the B7H3-2Ig plasmid was constructed. The antigen and detection protein of this invention were transiently expressed in Expi 293F cells (Gibco, A14527). The preparation method of the cynomolgus monkey B7H3 recombinant protein is the same as that of the human recombinant protein. The cynomolgus monkey B7H3 sequence is from UniProt: A0A2K5U2B3. The specific sequence information of the recombinant protein is shown in Table 1.

[0254] Construction of recombinant DLL3 protein: The amino acid sequence containing the truncated extracellular region encoding the human DLL3 protein (UniProt: Q9NYJ7) was cloned into a His-tagged pTT5 vector (Ubibio, VT2202). Plasmids were prepared according to the plasmid extraction kit method and transiently transfected into Expi 293F cells (Gibco, A14527). Cell components were removed by centrifugation, and the culture supernatant was obtained. The protein in the cell culture supernatant was purified using a Ni affinity chromatography column (purchased from GE Healthcare). Equilibration was first performed with 3-5 column volumes of equilibration buffer (PBS, pH 7.4), and then the clarified culture supernatant was loaded onto the Ni affinity chromatography column at a flow rate of 5 mL / min. After loading, the protein A column was washed with equilibration buffer, the volume of which was 3-5 times the column bed volume of the Ni affinity chromatography column. Gradient elution was performed with 0-500 mM imidazole, and the elution status was monitored using a nucleic acid protein detector (A280 UV absorption peak). The eluted protein was collected and dialyzed into PBS phosphate buffer at 4°C using a dialysis card (Thermo Scientific). The protein was then aseptically filtered through a 0.22 μm filter (Millipore) and aseptically stored to obtain the antigen and detection protein of this invention. The preparation method of the truncated extracellular region protein of cynomolgus monkey DLL3 is the same as that of the human recombinant protein. The cynomolgus monkey DLL3 sequence is derived from Uniprot ID: A0A2K5WSR4, and the specific sequence information of the recombinant protein is shown in Table 1.

[0255] Nickel column purification of recombinant proteins: Cell expression supernatant samples were centrifuged at high speed to remove impurities. The nickel column was equilibrated with 20 mM PBS + 500 mM NaCl solution and washed with 2-5 column volumes. The culture supernatant was loaded onto a Ni affinity chromatography column (GE Healthcare), and the UV absorbance (A280 nm) was monitored using a UV detector. The column was washed with equilibration buffer until the A280 reading returned to baseline. Then, a gradient elution was performed using equilibration buffers containing 10 mM, 20 mM, 40 mM, 90 mM, 250 mM, and 500 mM imidazole. Each elution peak was collected, and the fraction containing the target protein was identified based on the SDS-PAGE gel image. The collected elution product containing the target protein was concentrated and further purified using Superdex 200 (GE) gel chromatography with PBS as the mobile phase to remove aggregates and other protein peaks. The elution peak of the target product was collected. The obtained protein was identified by electrophoresis, peptide mapping, and LC-MS before being aliquoted and used. The proteins purified using this method include human B7H3 4Ig-His, human B7H3 2Ig-His, monkey B7H3-His, human DLL3-His, and monkey DLL3-His.

[0256] Table 1 Recombinant protein sequences

[0257] 1.2 Design and expression of control antibodies

[0258] The control antibodies used in this invention are all derived from published patents. The DS7300 antibody is derived from the published patent CN103687945B, and the YL212 antibody is derived from the published patent WO2024012523. Unless otherwise specified, both the DS7300 and YL212 control antibodies are recombinantly expressed using the human IgG1+κ subtype.

[0259] The expression and purification process of the control antibody was as follows: The antibody sequence gene was synthesized and cloned into the expression vector pTT5, then transiently transfected into Expi293F cells (purchased from Gibco, A14527). After culturing at 37°C for 7 days on a shaker, the cell supernatant was collected for Protein A antibody purification. The Protein A affinity column was washed with 3-5 column volumes of 0.1M NaOH, followed by 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes of 1×PBS (pH 7.4) buffer. The cell supernatant was loaded at a low flow rate for binding, controlling the flow rate to maintain a retention time of approximately 1 min or longer. After binding, the column was washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returned to baseline. Samples were eluted using a 50 mM citrate / sodium citrate buffer (pH 3.0-3.5). Elution peaks were collected based on UV detection. The eluted products were temporarily stored by rapidly adjusting the pH to 5-6 with 1 M Tris-HCl (pH 8.0). Solution replacement of the eluted products could be performed using methods well-known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube followed by solution replacement to the desired buffer system, or desalting using size exclusion columns such as G-25, or removing aggregate components from the eluted products using a high-resolution size exclusion column such as Superdex 200 to improve sample purity. The resulting control antibodies were named DS7300-hIgG1 and YL212-hIgG1. Specific antibody sequence information is shown in Table 2.

[0260] Table 2. Control antibody sequence listing

[0261] Example 2: Design and Construction of Bispecific Antibodies

[0262] Bispecific antibody molecules targeting B7H3×DLL3 were constructed using the sequences of anti-DLL3 humanized antibodies mAb01, mAb02, and mAb03 and the sequence of anti-B7H3 humanized antibody SCR10437. The specific structures of the bispecific antibodies are shown in Figures 1A-1C. To reduce homology mismatch, the antibody molecules adopted an asymmetric structure, with KIH mutations introduced into the Fc regions of both heavy chains. Simultaneously, to reduce light chain mismatch, VH / VL interchange mutations were introduced (see Figure 1 for details). The constructed bispecific antibodies were named Bis01, Bis02, and Bis03, and the amino acid sequences of each chain are shown in Table 3 below. SCR10437 is derived from patent CN202410110647.3, mAb01 and mAb02 are derived from patent PCT / CN2024 / 121296, and mAb03 is derived from patent PCT / CN2024 / 111678. For details of their VH, VL and CDR sequence information, please refer to Table 4-5.

[0263] Table 3. Amino acid sequence listing of bispecific antibodies

[0264] Table 4. Variable region sequences of bispecific antibodies

[0265] Table 5. CDR analysis results of bispecific antibodies

[0266] Example 3: Expression and purification of bispecific antibodies

[0267] 3.1 Transfection and expression of double antibiotic plasmid

[0268] The light and heavy chain nucleotide sequences encoding Bis01, Bis02, and Bis03 were cloned into the pTT5 vector. The plasmid and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. This mixture was then added to Expi293 cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai AOPMai, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0269] 3.2 Purification of Bispecific Antibodies

[0270] 3.2.1 Purification method of Bis01

[0271] After collecting the culture supernatant, the protein was purified using AKTAPure via Protein A affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. The obtained antibodies were quantitatively and qualitatively analyzed using UV spectrophotometry, SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification method is as follows: Protein A column (MabSelect SuRe, purchased from Cytiva) was used for capture. The Protein A column was equilibrated with 3–5 column volumes of equilibration buffer (1X PBS phosphate buffer, pH 7.4), and then the clear culture supernatant was loaded at a flow rate of 7 mL / min. After loading, the Protein A column was washed with 3–5 column volumes of equilibration buffer. The Protein A column was then washed with 3–5 column volumes of wash buffer (mAb Wash). After washing, the Protein A column was washed with 3–5 column volumes of equilibration buffer. The protein bound to the Protein A column was eluted with elution buffer (0.05 M acetate buffer, pH 3.5). Collect the eluted protein and neutralize to pH 5-6 with 1M pH 8.0 Tris-HCl. Adjust the pH of the protein A eluted sample to 5.5 using 1M acetic acid and ultrapure water. Treat the cation exchange column (HiTrap Capto SPImpRes) with 3-5 column volumes of 0.5M NaOH, followed by 3-5 column volumes of equilibration buffer (50mM acetic acid-sodium acetate, pH 5.5). Then load the adjusted sample onto the cation exchange column at a flow rate of 5 mL / min. After loading, wash with 3-5 column volumes of equilibration buffer (50mM acetic acid-sodium acetate, pH 5.5). Elute the sample on the column using a buffer gradient from pump A (50mM acetic acid-sodium acetate, pH 5.5) to pump B (50mM acetic acid-sodium acetate, 1M NaCl, pH 5.5), and collect the target protein. Subsequently, purification was performed using a hydrophobic chromatography column (BorgLone, EzLoad 16 / 10Octyl 4FF). An absorptive buffer (50 mM acetate-sodium acetate, pH 5.5, 1.5 M (NH4)2SO4) and buffer B (50 mM acetate-sodium acetate, pH 5.5) were prepared; the sample eluted from SP was transferred to the absorptive buffer for later use. The hydrophobic chromatography column (BorgLone, EzLoad 16 / 10Octyl 4FF) was treated with 3–5 column volumes of 0.5 M NaOH and then equilibrated with the absorptive buffer. The sample after buffer replacement was then loaded at a rate of 5 mL / min. After loading, a gradient of buffers A and B was used to elute the sample from the column. The target protein was collected, concentrated, dialyzed, and the buffer was transferred to 559 buffer (10 mM acetate-sodium acetate, 9% sucrose, pH 5.5). The bispecific antibody, which meets the purity requirements of the drug, is obtained by aseptic filtration using a 0.22μm filter and aseptic storage.

[0272] 3.2.2 Purification methods for BisO2 and BisO3

[0273] After collecting the culture supernatant, the protein was purified using AKTAPure via Protein A affinity chromatography and ion exchange chromatography. The obtained antibody was then quantitatively and qualitatively analyzed using UV spectrophotometry, SDS-PAGE, SEC-HPLC, CE-SDS, and LC-MS. The specific purification methods are as follows.

[0274] Protein A: Equilibrate the Protein A column with 3–5 column volumes of equilibration buffer (1X PBS phosphate buffer, pH 7.4), then load the clear culture supernatant at a flow rate of 7 mL / min. After loading, wash the Protein A column with 3–5 column volumes of equilibration buffer. Then wash the Protein A column with 3–5 column volumes of wash buffer (mAb Wash). After washing, wash the Protein A column with 3–5 column volumes of equilibration buffer. Elute the protein bound to the Protein A column with elution buffer (0.05 M acetate buffer, pH 3.5). Collect the eluted protein and neutralize to pH 5–6 with Tris buffer.

[0275] Ion exchange: The sample eluted from Protein A was adjusted to pH 5.5 using 1M acetic acid and ultrapure water. The cation exchange column (HiTrap Capto SPImpRes) was treated with 3–5 column volumes of 0.5M NaOH, followed by 3–5 column volumes of equilibration buffer (50mM acetic acid-sodium acetate, pH 5.5). The adjusted sample (pH 5.5) was then loaded onto the cation exchange column at a flow rate of 5 mL / min. After loading, the column was washed with equilibration buffer (50mM acetic acid-sodium acetate, pH 5.5) for 3–5 column volumes. A buffer gradient was used between pump A (50mM acetic acid-sodium acetate, pH 5.5) and pump B (50mM acetic acid-sodium acetate, 1M NaCl, pH 5.5) to elute the sample. The target protein was collected, yielding the bispecific antibody meeting the purity requirements of the drug.

[0276] 3.3 Purity detection of bispecific antibodies

[0277] 3.3.1 SEC-HPLC analysis

[0278] SEC-HPLC was used to analyze the test samples, characterize the molecular size uniformity of the bispecific antibodies, and determine their purity. The HPLC system used was an Agilent 1260, with a TSKgel G3000SWXL column from Tosoh Bioscience. The mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropanol (v / v 9:1) (batch number: 20220616101). The detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein was diluted 10-fold with DI water, the sample loading was 50 μg, and the analysis time was 40 minutes. The chromatograms of the SEC-HPLC data were analyzed using the manual integration method, and the protein purity was calculated using the area normalization method. The main peak was considered the monomer, the peaks before the main peak were called aggregates, and the peaks after the main peak were called fragments. The purity of the obtained bispecific antibodies was greater than 95%, meeting the drug-grade standards.

[0279] 3.3.2 CE-SDS Analysis

[0280] The non-reducing CE-SDS method was used to analyze the test samples, determine the purity of the bispecific antibodies, and characterize the size uniformity of the test samples. The capillary electrophoresis system used in this method was an AB Sciex PA800Plus, the detector was a PDA, the detection wavelength was 220 nm, the effective capillary detection length was 20 cm, the protein separation voltage was 15.0 kV, and the NR-CE SDS detection time was 40 min. The sample volume was 100 μg. For the NR-CE-SDS method, SDS Sample Buffer (20 mM PB, 5 mM citric acid, 1% SDS, pH 6.5) was added to the protein sample, dissolved to a volume of 94 μL, followed by 5 μL of 100 mmol / L NEM and 1 μL of 10 KD marker. The sample was incubated at 70 °C for 10 min to obtain the NR-CE-SDS test sample. The chromatograms of the NR-CE SDS data were analyzed using the manual integration method, and the protein purity was calculated using the area normalization method. The purity of the obtained bispecific antibodies was greater than 95%, which meets the standards for finished drugs.

[0281] Example 4: Preparation method of antibody-drug conjugate

[0282] 4.1 Synthesis of drug-linker L3-1

[0283] Synthesis route:

[0284] Step 1: Synthesis of tert-butyl 3-(2-cyclopentathio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionate (intermediate 3-2)

[0285] At room temperature, cyclopentylthiol (367 mg) and 2 M sodium hydroxide aqueous solution (1.65 mL) were added sequentially to a solution of compound 3-1 (1.066 g) dissolved in DCM (10 mL). The resulting mixture was stirred at room temperature for 30 min. The mixture was then separated by column chromatography (EA / PE, 0-50%) to obtain the crude title compound, which was used directly in the next reaction.

[0286] Step 2: Synthesis of tert-butyl 3-(2'-(cyclopentylthio)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-3)

[0287] Intermediate 3-2 (200 mg) was dissolved in N,N-dimethylformamide (2 mL) at 0 °C under a nitrogen atmosphere. NaH (127.15 mg, 60% effective content) and 1-iodo-2-(2-iodoethoxy)ethane (518.01 mg) were added. The reaction mixture was stirred at 0 °C for 1 h under nitrogen protection. Ice water (2 mL) was then added to the reaction mixture, followed by extraction with ethyl acetate (1 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (ethyl acetate / petroleum ether, 0-60%) to give the title compound (110 mg).

[0288] MS m / z (ESI): 448.2 [M+H] + .

[0289] Step 3: Synthesis of tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-4)

[0290] Intermediate 3-3 (60 mg) was dissolved in anhydrous dichloromethane (1 mL) at 0 °C, and m-chloroperoxybenzoic acid (108.86 mg, 85% purity) was added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure and separated by column chromatography (ethyl acetate / petroleum ether, 0-55%) to give the title compound (9.8 mg).

[0291] MS m / z (ESI): 480.3 [M+H] + .

[0292] 1H NMR (400MHz, Chloroform-d) δ = 9.40 (s, 1H), 4.35-4.18 (m, 1H), 4.00 (m, 2H), 3.86-3.72 (m, 6H), 2.68 (t, J= 6.3Hz,2H),2.31-2.23(m,2H),2.18(m,2H),2.06(m,2H),1.93-1.81(m,2H),1.77-1.65(m,4H),1.45(s,9H)

[0293] Step 4: Synthesis of 3-(2'-(cyclopentanyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionic acid (intermediate 3-5)

[0294] Intermediate 3-4 (680 mg) was dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (0.68 mL) was added to the reaction solution. The reaction solution was stirred at 25 °C for 1 hour. After the reaction was complete, the solvent was removed by direct concentration under reduced pressure. The residue was purified by rapid silica gel column chromatography. 12g The title compound (200 mg) was obtained by rapid silica column chromatography with a gradient of 0–53% tetrahydrofuran / petroleum ether at a flow rate of 40 mL / min.

[0295] MS m / z (ESI): 423.8 [M+H] +

[0296] Step 5: Synthesis of (S)-5-allyloxycarbonyl-1-(9H-fluorene-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxo-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatritetradecane-34-carboxylic acid (intermediates 3-6)

[0297] This compound was synthesized using a peptide solid-phase synthesis method, as follows:

[0298] 1) Dichloromethane was added to a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g) under nitrogen protection;

[0299] 2) Add diisopropylethylamine (DIEA) dropwise and mix and stir for 2 hours;

[0300] 3) Add methanol (13 mL) and mix and stir for 0.5 h;

[0301] 4) Filter and wash three times with DMF;

[0302] 5) Add 20% piperidine / DMF solution and react for 30 min;

[0303] 6) Filter and wash five times with DMF;

[0304] 7) Add the substances listed in the "Ingredients" column of the table below and mix for 30 seconds, then add the substances listed in the "Reagents" column of the table below. The reaction is carried out under nitrogen bubbling conditions for 1 hour, where Fmoc-Sar-OH represents N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine, and Fmoc-Asp-OAll-OH represents (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutyric acid;

[0305] 8) Repeat steps 4-7 to complete the desired peptide synthesis.

[0306] Note:

[0307] After solid-phase preparation, a 20% dichloromethane solution of 1,1,1,3,3,3-hexafluoroisopropanol was added to the resulting mixture and the mixture was stirred for 1.5 h. The mixture was then filtered, the filtrate was collected and concentrated, and the resulting mixture was purified by preparative liquid chromatography (A: 0.075% aqueous trifluoroacetic acid, B: acetonitrile) to give the title compound (2.15 g).

[0308] MS m / z (ESI): 1035.6 [M+H] + .

[0309] Step 6: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid allyl ester (intermediate 3-7)

[0310] Intermediate 3-6 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), and HATU (728.95 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 3 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (800 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0311] MS m / z (ESI): 1034.6 [M+H] + .

[0312] Step 7: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid (intermediates 3-8)

[0313] Intermediate 3-7 (800 mg), tetraphenylphosphine palladium (201.84 mg), and 1,3-dimethylbarbituric acid (108.71 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 4 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (700 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0314] MS m / z (ESI): 994.9 [M+H] + .

[0315] Step 8: Synthesis of resin-supported (2S,10S,19S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediates 3-9)

[0316] Intermediate 3-8 (700 mg) was added to DMF (10 mL), followed by intermediate L1-25 (total weight of resin and substrate 400 mg, content approximately 0.18 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was purged with nitrogen three times and then reacted at 25 °C with shaking for 1 h. After the reaction was complete, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (420 mg).

[0317] MS m / z (ESI): 1461.4 [M+Na]+ .

[0318] Step 9: Synthesis of resin-supported (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediate 3-10)

[0319] Intermediate 3-9 (420 mg) was dissolved in DMF (5 mL), and piperidine (1.25 mL) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (400 mg).

[0320] MS m / z (ESI): 1217.4 [M+H] + .

[0321] Step 10: Resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34 Synthesis of 37,40,43,46-Nonmethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-Decadecyloxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-Tetraazaoctadecane-1-carboxylic acid (intermediate 3-11)

[0322] Intermediate 3-10 (880 mg) and intermediate 3-5 (393.14 mg) were dissolved in N,N-dimethylformamide (15 mL), and O-benzotriazole-tetramethylurea hexafluorophosphate (453.65 mg) and N,N-diisopropylethylamine (313.82 μL) were added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 hour. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (920 mg).

[0323] Step 11: (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34,37 Synthesis of 40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid-1-carboxylic acid (intermediate 3-12)

[0324] Intermediate 3-11 (920 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25 °C for 0.5 hr. After the reaction was complete, the reaction solution was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (65 mg).

[0325] Step 12: (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosane)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxopentano[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxa-2,7,10,13,16-pentazaoctadecane-18-yl)-2-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyridino[4,3-d]pyrimidinyl]-6'(7'H)-yl)propionylamino)-N 4 Synthesis of methylbutyramide (drug linker L3-1)

[0326] Intermediate 3-12 (65 mg), intermediate 2-11 (22.10 mg, preparation method see Example 9.2 below), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.36 mg), pyridine (9.51 mg), and 1-hydroxybenzotriazole (10.82 mg) were dissolved in N,N-dimethylformamide (3 mL), purged with nitrogen three times, and the reaction solution was stirred for 2 hours at 25°C under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 21%-51%, 11 min) to obtain the title compound (35 mg).

[0327] 1 H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.69-8.55 (m, 2H), 8.30-8.21 (m, 2H), 8.01 (m, 2H), 7.79 (s, 1H), 7.52 (s, 1H), 7.39-7.27 (m, 1H), 7. 26-7.21(m,5H),7.15(s,1H),7.11-7.00(m,1H),6.49(s,1H),5.43(d,J=5.8Hz,4H),4.86-4.70(m,2H),4.70-4.63(m,1H),4.62-4.53(m,1 H),4.52-4.44(m,1H),4.40-4.17(m,10H),4.12-3.89(m,9H),3.86-3.76(m,5H),3.75-3.65(m,9H),3.63-3.52(m,2H),3.51-3.43(m,2H) ,3.00-2.68(m,32H),2.07-1.92(m,6H),1.91-1.80(m,2H),1.71-1.59(m,6H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.27(m,4H)

[0328] MS m / z(ESI): 1014.8 [(M+2) / 2] + .

[0329] 4.2 Antibody-Drug Linker Coupling Reaction

[0330] The three candidate antibodies obtained in Example 3, along with the positive control antibodies mAb02, SCR10437, and the negative control antibody anit-FITC-hIgG1, were dialyzed to PBS buffer and then 8-25 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added. The reaction solution was mixed and reduced at 4°C for 16-18 h or at 37°C for 2-3 h. 15-27 molar equivalents of the drug-linker compound L3-1 (dissolved in DMSO) were added to the reaction system, and the reaction solution was mixed and coupled at 4°C for 6 h or at 25°C for 3 h. Unreacted free small molecule toxins were removed using Capto SPImpRes cation exchangers (purchased from Cytiva). Finally, the solution was dialyzed to 559 buffer (10 mM acetate-sodium acetate, 9% sucrose, pH 5.5). The purity and DAR values ​​of the ADC products were analyzed using SEC and LC-MS, and the content of free small molecules was analyzed using RP-HPLC. The obtained ADCs were named ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, and ADC-ISO-1.

[0331] 4.3 Coupling reaction of positive controls DS7300 and YL212

[0332] After dialysis of the naked DS7300 antibody with PBS buffer, 3-5 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added, and the reaction solution was mixed and reduced at 4°C for 16 h. 5% DMSO was added to the reduced antibody solution, followed by 10 molar equivalents of the drug-linker compound (MC-GGFG-DXD, MCE, HY-13631E) dissolved in DMSO. The reaction solution was mixed and coupled at 4°C for 6 h. Unreacted free small molecule toxins were removed using Capto SpimpRes cation exchangers (purchased from Cytiva), and finally, the solution was dialyzed and replaced with 559 buffer (10 mM acetate-sodium acetate, 9% sucrose, pH 5.5). The purity and DAR value of the ADC product were analyzed by SEC and LC-MS, and the content of free small molecules was analyzed by RP-HPLC. The obtained ADC was DS7300-Dxd.

[0333] The naked YL212 antibody solution was replaced with PBS buffer, and 8-25 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added. The reaction solution was mixed and reduced at 4°C for 16 h. 5% DMSO was added to the reduced antibody solution, followed by 10 molar equivalents of the drug-linker compound (DL-01formic, MCE, HY-155870A) dissolved in DMSO. The reaction solution was mixed and coupled at 25°C for 3 h. After the reaction, unreacted free small molecule toxins were removed using Capto SpimpRes cation exchangers (purchased from Cytiva). Finally, the solution was dialyzed and the buffer was replaced with 559 buffer (10 mM acetate-sodium acetate, 9% sucrose, pH 5.5). The purity and DAR value of the ADC product were analyzed by SEC and LC-MS. The obtained ADC was YL212-DL01.

[0334] 4.4 ADC Sample Purity Analysis and DAR Value Determination

[0335] SEC Purity Analysis: The SEC-HPLC method was used to analyze the protein samples, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein. The HPLC system used was an Agilent 1260, the column was a TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase was 200 mM phosphate buffer, pH 7.0, the detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0336] DAR value determination: The DAR value of ADC molecules was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-sugar modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37℃ for 1 h to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected into a Waters ACQUITY Protein BEH size-exclusion column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min, and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were: spray voltage 3.8 kV, capillary heating temperature 300℃, sheath gas flow rate 35 arb, and precursor ion scan range 800-3000. Finally, the mass spectrometry data was analyzed using the Biopharma Finder software. 4.1 The Respect algorithm was used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component, thereby calculating the DAR value of the ADC sample to be tested. The coupling purity and DAR value detection data for each sample are shown in Table 6.

[0337] Table 6. Preparation, DAR value, and SEC purity of antibody-drug conjugates

[0338] Example 5: Affinity determination of ADC

[0339] The binding strength of the ADC to the antigen was detected using the Protein A capture method on a BIAcore 8K instrument. First, Protein A was immobilized onto a CM4 chip (GE, BR-1005-34) using an amino-coupling method. Following the instructions of the Amine Coupling Kit (GE, BR100633), HBS-EP + pH 7.4 was used as the mobile phase. After mixing NHS and EDC, the chip was activated for approximately 600 seconds. Protein A was then diluted to 50 μg / mL with 10 mM sodium acetate at pH 4.5 and injected for 600 seconds. Finally, the remaining activation sites were blocked with ethanolamine. Then, a multi-cycle kinetic method was used to determine the affinity between the ADC and the antigen. In each cycle, the ADC to be tested was first captured using a Protein A chip, and then a single concentration of antigen protein was injected. The binding and dissociation processes of the ADC and antigen protein were recorded. Finally, the chip was regenerated using Glycine pH 1.5. The mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P2O), the flow rate was 30 μL / min, the regeneration time was 30 s, and the detection temperature was 25 °C. Finally, the data were analyzed according to a 1:1 binding model to fit the antibody-antigen binding kinetic parameters, including the binding rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.

[0340] The binding rates (Ka), dissociation rates (Kd), and binding affinity (KD) of the ADC and control antibodies with B7H3 and DLL3 proteins are shown in Tables 7 and 8.

[0341] Table 7. SPR (Biacore) assay for affinity between ADC and DLL3 protein.

[0342] Table 8. SPR (Biacore) assay for affinity between ADC and B7H3 protein.

[0343] Example 6: Identification of the binding activity of the antibody and its conjugated ADC

[0344] 6.1 ELISA detection of the binding of antibodies and their conjugated ADCs to DLL3 and B7H3 proteins

[0345] Human DLL3-his and cyno DLL3-his proteins were diluted to a final concentration of 0.5 μg / mL with PBS, and then 50 μl was added to each well of a 96-well ELISA plate (9018, Corning). The plates were incubated overnight at 4°C. The next day, the plates were washed twice with PBST, and blocking buffer [PBS + 2% (w / v) BSA] was added, followed by blocking at room temperature for 2 hours. The blocking buffer was discarded, and 50 μl of the antibody and its conjugated ADC (100 nM starting concentration, serially diluted 10-fold), along with positive and negative control antibodies and their conjugated ADCs, were added to each well. After incubation at 37°C for 1 hour, the plates were washed three times with PBST [PBS + 0.05% Tween 20]. Secondary antibody Peroxidase Affini Pure Goat Anti-Human IgG, Fcγfragment specific (purchased from Jackson Immuno, catalog number: 109-035-098) was added, and the plates were incubated at 37°C for 1 hour, followed by washing five times with PBST. Add 50 μl of TMB substrate to each well, incubate at room temperature for 3 minutes, then add 50 μl of stop solution (1.0 M HCl) to each well. Read the OD values ​​using an ELISA plate reader (PowerWaveHT, Biotec). 450nm The binding activity of the antibodies to human DLL3-his and cyno DLL3-his proteins is shown in Figures 2A-2B. The results showed that the negative control anti-FITC-hIgG1 (from the literature J Biol Chem. 1990 Jan 5; 265(1):133-8) did not bind to DLL3. The bispecific antibodies and their conjugated ADCs could effectively bind to human DLL3-his and cyno DLL3-his proteins, and the binding ability was comparable to that of the control antibody YL212 naked antibody and YL212-DL01.

[0346] Human B7H3-his and cyno B7H3-his proteins were diluted with PBS to a final concentration of 2 μg / mL, and then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of the antibodies to human B7H3-his and cyno B7H3-his proteins was analyzed using the same detection method as in Example 6.1. The results showed that the bispecific antibody and its conjugated ADC could effectively bind to B7H3 protein, and the binding ability was comparable to that of the control DS7300-Dxd (Figures 2C-2D).

[0347] 6.2 Flow cytometry (FACS) assay to detect the binding of antibodies and their conjugated ADCs to tumor cells

[0348] The expression of DLL3 and B7H3 on tumor cells NCI-H82, NCI-H510A, and MDA-MB-453 were detected by FACS, and the results are shown in Table 9. The binding activity of each antibiotic and ADC to cells was detected as follows: NCI-H82, NCI-H510A, and MDA-MB-453 cells were cultured in T-175 flasks until the logarithmic growth phase. The suspended cells were collected, mixed by pipetting, and counted. After cell counting, the cells were centrifuged, and the cell pellet was resuspended in FACS buffer (PBS + 2% (w / v) FBS) to a final volume of 2 × 10⁻⁶. 6 -4×10 6 Add 50 μl of cells per well to a 96-well FACS plate, centrifuge, discard the supernatant, add 50 μl of the antibody sample (200 nM as the starting concentration, serially diluted 5-fold) per well, mix well, and incubate at 4°C for 1 hour. Wash three times with PBS buffer by centrifugation, and add 50 μl of Alexa antibody to each well. 647AffiniPure Goat Anti-Human IgG, Fcγfragment-specific labeled secondary antibody (purchased from Jackson Immuno, catalog number: 109-605-098) was incubated at 4°C for 40 minutes. After washing twice with PBS buffer by centrifugation, the cells were resuspended in 100 μl PBS and the results were detected and analyzed using FACS (FACS Canto™, purchased from BD). The data were then analyzed using software (GraphPad Prism 10) for data fitting and EC50 calculation. The results are shown in Figures 3A-3C and Table 10. The bispecific antibody and its conjugate ADC specifically bound to DLL3×B7H3 double-positive tumor cells. On NCI-H82 and NCI-H510A double-positive cells, the binding ability of the bispecific antibody and its conjugate ADC was superior to the control monoclonal antibody (Figures 3A-3B). Neither the bispecific antibody nor its conjugate ADC bound MDA-MB-453 cells, showing no non-specific binding (Figure 3C).

[0349] Table 9. Expression of DLL3 and B7H3 on the surface of tumor cells.

[0350] Table 10. Data on antibody binding to tumor cells

[0351] Example 7: In vitro proliferation inhibition test of ADC on tumor cells

[0352] The NCI-H146 (catalog number HTB-173) and NCI-H526 (catalog number CRL-5811) cells used in this experiment were purchased from ATCC. The complete culture medium was RPMI-1640 medium containing 10% FBS. QGP-1 (catalog number JCRB0183) cells were purchased from JCRB. All ADCs to be tested were derived from Example 4. ADC-1, ADC-2 and ADC-3 were candidate molecules, ADC-4, YL212-DL01, DS7300-Dxd and ADC-5 were control molecules, and ADC-ISO-1 was a candidate molecule control isotype.

[0353] After adjusting NCI-H146, NCI-H526, and QGP-1 cells to the logarithmic growth phase, the cells were collected, resuspended, and counted. The cells were then seeded into 384-well plates (Corning, catalog number 3765) at the following density: NCI-H146 cells 1.5 × 10⁶. 3 1.0 × 10⁶ cells / 40 μL, NCl-H526 cells 3 0.5 × 10⁵ cells / 40 μL, QGP-1 cells 3 Cells / 40 μL were incubated overnight at 37°C in a 5% CO2 incubator. The next day, ADC was serially diluted with complete culture medium according to the experimental design to an initial concentration of 100 nM or 500 nM, followed by 5-fold serial dilutions to a total of 10 concentrations. 20 μL of each diluted ADC was transferred to the corresponding well of the plate. The cell culture plate was incubated at 37°C in a 5% CO2 incubator for 7 days; subsequently, 25 μL of ADC was added to each well. The Luminescent Cell Viability Detection Kit (purchased from vkey-bio, catalog number A2010003N, usage instructions follow the product manual) was used with an Envision microplate reader (purchased from PerkinElmer, model Envision 2105) to detect fluorescence signal values, thereby reflecting the inhibitory effect of the sample on cell proliferation.

[0354] Data Analysis: Positive and negative control groups were set up as 0% and 100% cell killing controls, respectively. The positive control group received no test drug, but all other procedures were the same as the experimental group. The negative control group received no cells, only the same volume of culture medium, and all other procedures were the same as the experimental group. The cell proliferation inhibition rate was calculated using the formula: Inhibition rate = ((Positive control - Sample well reading) / (Positive control - Negative well reading)) × 100%. This formula was used to obtain the inhibition rate of the corresponding drug at each concentration. The maximum inhibition rate was represented by Emax. The IC50 was calculated using GraphPad Prism software. 50 .

[0355] Experimental results: Under the experimental conditions, the candidate molecules ADC-1, ADC-2 and ADC-3 showed significantly better IC50 killing effects on NCI-H146, NCI-H526 and QGP-1 cells than ADC-4, YL212-DL01, DS7300-Dxd and the control ADC-5. The results are shown in Table 12 and Figures 4A-4C.

[0356] Table 11 Expression of DLL3 and B7H3 on the surface of tumor cells

[0357] Table 12 Inhibitory effect of ADCs on endogenous tumor cell proliferation

[0358] Example 8: In vivo efficacy evaluation of ADC on tumor cells

[0359] In this embodiment, human small cell lung cancer cells NCI-H82 and NCI-H2171, which are double-positive for tumor cells B7H3 and DLL3, were selected to establish an in vivo mouse model (Balb / c-nude, female, 6-8 weeks old, Beijing Vitonda Laboratory Animal Technology Co., Ltd.) and these models were used to evaluate the anti-tumor efficacy of the candidate molecules in vivo.

[0360] 8.1 ADC candidate molecules inhibit tumor growth in NCI-H82 human small cell lung cancer cells in tumor-bearing mice

[0361] The seeding time of human small cell lung cancer cells NCI-H82 was designated as day 0 of this experiment. On day 0, human small cell lung cancer cells NCI-H82 (American Cell Culture Collection Center) that had been expanded to the required number and were in the logarithmic growth phase (approximately 80% confluence; the culture medium was replaced with fresh medium the day before seeding) were collected and seeded. First, the cell suspension was collected into 50 mL centrifuge tubes, centrifuged at 300 g for 7 min, and an appropriate amount of serum-free RPMI-1640 medium (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Sigma, #R8758-500 mL) was used to resuspend the cells. 500 μL of the cell suspension was then used to count the cells in a cell counter (Beckman Coulter, SIC-TP-573). Finally, based on the cell count results, the cell density was adjusted to 100 × 10⁶ cells / mL using serum-free medium. 6 Cells / mL were placed on ice and transferred through a transfer window to the SPF animal room for inoculation and modeling. Before inoculation, the above cell suspension was mixed with Matrigel (Corning Biotech, #356234) in an equal ratio, and 100 μL of the above cell mixture was subcutaneously injected into the right axilla of each mouse.

[0362] After inoculation with human small cell lung cancer cells NCI-H82, tumor growth was monitored. When the mean overall tumor volume was 100-150 mmHg... 3 Around 100 mice with suitable tumor volume were randomly divided into groups of 6. Each group received the corresponding drug via tail vein administration according to the protocol, and the tumor volume and mouse weight changes were measured. The specific administration protocol is shown in Table 13. PBS was the negative control group, ADC-ISO-1 was the isotype control, ADC-1, ADC-2, and ADC-3 were ADC candidate molecules, and ADC-4 and DS7300-Dxd were ADC control molecules. The ADC candidate molecules were administered at the same unit weight as the isotype control.

[0363] The tumor inhibition rate is calculated as follows: Tumor inhibition rate (%) = [1 - (Vt(treatment group) - V0(treatment group)) / (Vt(negative control group) - V0(negative control group))] × 100%, where V0 is the average tumor volume at the time of grouping, and Vt is the average tumor volume at a certain measurement after treatment. The relative change in mouse body weight is calculated as: RCBW(%) = (BWi – BW0) / BW0 × 100, where BWi is the body weight at a certain tumor measurement after administration, and BW0 is the body weight at the time of the first administration.

[0364] The results are shown in Figures 5A and 5B and Table 14. On day 14 after administration, all tested drug groups showed good efficacy, with ADC-2 showing the best efficacy, followed by ADC-3 and ADC-1. The efficacy of ADC-1, ADC-2, and ADC-3 was significantly better than that of ADC-4 and DS7300-Dxd. During the monitoring period, the relative change rate of mouse body weight in each administration group was not significantly different from that in the negative control PBS group, suggesting that the tumor-bearing mice tolerated the test substances well.

[0365] Table 13. Drug Efficacy and Dosing Regimens of ADC Candidate Molecules in Human Small Cell Lung Cancer Cell NCI-H82 Tumor-Bearing Mice

[0366] Table 14. Efficacy of ADC candidate molecules in NCI-H82 tumor-bearing mice with human small cell lung cancer cells. Notes: a. Mean ± SD b. Comparisons among the 8 groups in this experiment were calculated using one-way ANOVA and then analyzed using Tukey's method. The p-values ​​on day 14 are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0367] 8.2 ADC candidate molecules inhibit the growth of human small cell lung cancer NCI-H2171 tumors.

[0368] Day 0 of the experiment was designated as the inoculation time of human small cell lung cancer cells NCI-H2171. On day 0, human small cell lung cancer cells NCI-H2171 (American Cell Culture Collection Center) that had been expanded to the required number and were in the logarithmic growth phase (approximately 80% confluence; the culture medium was replaced with fresh medium the day before inoculation) were collected and inoculated. First, the cell suspension was collected into 50 mL centrifuge tubes, centrifuged at 300 g for 7 min, and an appropriate amount of serum-free RPMI-1640 medium (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Sigma, #R8758-500 mL) was used to resuspend the cells. 500 μL of the cell suspension was then counted using a cell counter (Beckman Coulter, SIC-TP-573). Finally, based on the cell count results, the cell density was adjusted to 200 × 10⁶ cells / mL using serum-free medium. 6 Cells / mL were placed on ice and transferred through a transfer window to the SPF animal room for inoculation and modeling. Before inoculation, the above cell suspension was mixed with Matrigel (Corning Biotech, #356234) in an equal ratio, and 100 μL of the above cell mixture was subcutaneously injected into the right axilla of each mouse.

[0369] After inoculation with human small cell lung cancer cells NCI-H2171, tumor growth was monitored. When the mean overall tumor volume was 100-150 mmHg... 3 Around 100 mice with suitable tumor volume were randomly divided into groups of 6. Each group received the corresponding drug via tail vein administration according to the protocol, and the tumor volume and mouse weight changes were measured. The specific administration protocol is shown in Table 15. PBS was the negative control group, ADC-ISO-1 was the isotype control, ADC-1, ADC-2, and ADC-3 were ADC candidate molecules, and ADC-4, DS7300-DXd, and ADC-5 were ADC control molecules. The ADC candidate molecules were administered at the same unit weight as the isotype control.

[0370] The tumor inhibition rate is calculated as follows: Tumor inhibition rate (%) = [1 - (Vt(treatment group) - V0(treatment group)) / (Vt(negative control group) - V0(negative control group))] × 100%, where V0 is the average tumor volume at the time of grouping, and Vt is the average tumor volume at a certain measurement after treatment. The relative change in mouse body weight is calculated as: RCBW(%) = (BWi – BW0) / BW0 × 100, where BWi is the body weight at a certain tumor measurement after administration, and BW0 is the body weight at the time of the first administration.

[0371] The results are shown in Figures 6A and 6B and Table 16. On day 20 after administration, all tested drug groups showed good efficacy. Among them, ADC-2 and ADC-1 had comparable efficacy, both better than ADC-3. ADC-2 and ADC-1 were significantly more effective than ADC-4, DS7300-Dxd, and ADC-5. ADC-3 was significantly more effective than DS7300-Dxd and ADC-5, and better than ADC-4. During the monitoring period, the relative change rate of mouse body weight in each treatment group was not significantly different from that in the negative control PBS group, indicating that the tumor-bearing mice tolerated the test substances well.

[0372] Table 15. Drug efficacy and administration regimens of ADC candidate molecules in human small cell lung cancer NCI-H2171 tumor-bearing mice.

[0373] Table 16. Efficacy of ADC candidate molecules in human small cell lung cancer NCI-H2171 tumor-bearing mice. Notes: a. Mean ± SD b. The comparisons between the 8 groups in this experiment were calculated using one-way ANOVA and then analyzed using Tukey's method. The p-values ​​on day 20 are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Claims

A multispecific antibody or an antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to B7H3 and a second antigen-binding domain that specifically binds to DLL3. According to claim 1, the multispecific antibody or its antigen-binding fragment thereof, wherein, The first antigen-binding domain and the second antigen-binding domain are each independently selected from antibodies, antibody fragments, F(ab')2, Fab', Fab, Fv, scFv, nanobodies, or VHH. The multispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The multispecific antibody or its antigen-binding fragment comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain. According to claim 3, the multispecific antibody or its antigen-binding fragment thereof, wherein, (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1; (2) The first light chain includes the following structure: VL B7H3 -CL; (3) The second heavy chain includes the following structure: VL DLL3 -CH1-Fc2; (4) The second light chain includes the following structure: VH DLL3 -CL; Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 These are the heavy chain variable region and light chain variable region that specifically bind to DLL3, respectively; Fc1 and Fc2 are each the Fc region of any antibody. The multispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The multispecific antibody or its antigen-binding fragment comprises three polypeptide chains: a first heavy chain, a first light chain, and a second heavy chain. According to claim 5, the multispecific antibody or its antigen-binding fragment thereof, wherein, (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1; (2) The first light chain includes the following structure: VL B7H3 -CL; (3) The second heavy chain includes the following structure: VL DLL3 -(G4S)3-VH DLL3 -GGG-Fc2; or VHH DLL3 -GGG-Fc2; Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 They specifically bind to the heavy chain variable region and light chain variable region of DLL3, respectively, and form scFv; VHH DLL3 These are nanobodies that specifically bind to DLL3; Fc1 and Fc2 are the Fc regions of any antibody. The multispecific antibody or its antigen-binding fragment according to claim 4 or 6, wherein, The scFv that specifically binds to DLL3 introduces a CC mutation; Fc1 and Fc2 are different, with Fc1 being a knock-Fc and Fc2 being a hole-Fc; preferably, the knock-Fc contains a T366W mutation, and / or the hole-Fc contains a T366S, L368A, and / or Y407V mutation; preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4. The multispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The first antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO.20; and light chain LCDRs of the VL domain as shown in SEQ ID NO.

21. According to claim 8, the multispecific antibody or its antigen-binding fragment thereof, wherein, The first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions: Heavy chain CDR1, such as SEQ ID NO.27, 48, 69; Heavy chain CDR2 such as SEQ ID NO.28, 49, 70; Heavy chain CDR3 such as SEQ ID NO.29, 50, 71; Light chain CDR1, such as SEQ ID NO.39, 60, 81; Light chain CDR2 such as SEQ ID NO.40, 61, 82; Light chain CDR3, such as SEQ ID NO.41, 62, 83. The multispecific antibody or its antigen-binding fragment according to claim 8 or 9, wherein, The VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO.

21. The multispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The second antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 22, 24 or 26; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 23 or 25; Preferably, the second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably, it has conserved amino acid substitutions: Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78; Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79; Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80; Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87; Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88; Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89. According to claim 11, the multispecific antibody or its antigen-binding fragment thereof, wherein, The VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 23 or 25. The multispecific antibody or its antigen-binding fragment according to any one of claims 1-12, wherein, The first heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 12; the first light chain comprises an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 13; The second heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 14, 16, 17 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 14, 16, 17; the second light chain comprises an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO.

15. The multispecific antibody or its antigen-binding fragment according to any one of claims 1-13, wherein, The multispecific antibody or its antigen-binding fragment is (1) Chimeric antibodies or fragments thereof; (2) Humanized antibodies or fragments thereof; or, (3) Fully human antibodies or fragments thereof. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein, The multispecific antibody or its antigen-binding fragment is trivalent, tetravalent, pentavalent, or hexavalent; preferably, the multispecific antibody or its antigen-binding fragment is bivalent. The multispecific antibody or its antigen-binding fragment according to any one of claims 1-15, wherein, The multispecific antibody or its antigen-binding fragment is further conjugated with a cytotoxic drug, which is selected from chemotherapeutic drugs or antibiotics; optionally, the cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents, or DNA topoisomerase inhibitors, wherein the tubulin inhibitors include dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins, the DNA damaging agents include PBD drugs, and the DNA topoisomerase inhibitors include camptothecin drugs. According to claim 16, the multispecific antibody or its antigen-binding fragment thereof, wherein, The cytotoxic drug is linked to the multispecific antibody or its antigen-binding fragment via a linker. An isolated nucleic acid fragment, wherein, The nucleic acid fragment encodes the multispecific antibody or its antigen-binding fragment as described in any one of claims 1-17. A carrier, in which, The vector comprises the nucleic acid fragment of claim 18. A host cell, characterized in that, The host cell comprises the vector of claim 19; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line). A method for preparing the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1-17, characterized in that, The method includes culturing the cells of claim 20 and isolating the multispecific antibodies or antigen-binding fragments thereof expressed by the cells. An antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt thereof, Pc-(L-D) n (A) in, D is a cytotoxic drug; L is the connecting subunit; Pc is a multispecific antibody or its antigen-binding fragment, which contains a first antigen-binding domain that specifically binds to B7H3 and a second antigen-binding domain that specifically binds to DLL3. Furthermore, n is a real number from 1 to 16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 22, wherein, The first antigen-binding domain and the second antigen-binding domain are each independently selected from antibodies, antibody fragments, F(ab')2, Fab', Fab, Fv, scFv, nanobodies, or VHH. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 22 or 23, wherein, The multispecific antibody or its antigen-binding fragment comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 24, wherein, (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1; (2) The first light chain includes the following structure: VL B7H3 -CL; (3) The second heavy chain includes the following structure: VL DLL3 -CH1-Fc2; (4) The second light chain includes the following structure: VH DLL3 -CL; Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 These are the heavy chain variable region and light chain variable region that specifically bind to DLL3, respectively; Fc1 and Fc2 are each the Fc region of any antibody. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 22 or 23, wherein, The multispecific antibody or its antigen-binding fragment comprises three polypeptide chains: a first heavy chain, a first light chain, and a second heavy chain. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 26, wherein, (1) The first heavy chain includes the following structure: VH B7H3 -CH1-Fc1; (2) The first light chain includes the following structure: VL B7H3 -CL; (3) The second heavy chain includes the following structure: VL DLL3 -(G4S)3-VH DLL3 -GGG-Fc2; or VHH DLL3 -GGG-Fc2; Among them, VH B7H3 and VL B7H3 These are the heavy chain variable region and light chain variable region that specifically bind to B7H3, respectively. DLL3 and VL DLL3 They specifically bind to the heavy chain variable region and light chain variable region of DLL3, respectively, and form scFv; VHH DLL3 These are nanobodies that specifically bind to DLL3; Fc1 and Fc2 are each the Fc region of any antibody. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 25 or 27, wherein, The scFv that specifically binds to DLL3 introduces a CC mutation; Fc1 and Fc2 are different, with Fc1 being a knock-Fc and Fc2 being a hole-Fc; preferably, the knock-Fc contains a T366W mutation, and / or the hole-Fc contains a T366S, L368A, and / or Y407V mutation; preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 22 or 23, wherein, The first antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO.20; and light chain LCDRs of the VL domain as shown in SEQ ID NO.21; Preferably, the first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably, it has conserved amino acid substitutions: Heavy chain CDR1, such as SEQ ID NO.27, 48, 69; Heavy chain CDR2 such as SEQ ID NO.28, 49, 70; Heavy chain CDR3 such as SEQ ID NO.29, 50, 71; Light chain CDR1, such as SEQ ID NO.39, 60, 81; Light chain CDR2 such as SEQ ID NO.40, 61, 82; Light chain CDR3, such as SEQ ID NO.41, 62, 83. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, wherein, The VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO.

21. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 22 or 23, wherein, The second antigen-binding domain comprises heavy chain CDRs of the VH domain as shown in SEQ ID NO. 22, 24 or 26; and light chain LCDRs of the VL domain as shown in SEQ ID NO. 23 or 25; Preferably, the second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably, it has conserved amino acid substitutions: Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78; Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79; Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80; Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87; Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88; Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 31, wherein, The VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 23 or 25. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 29-32, wherein, The multispecific antibody or its antigen-binding fragment comprises three polypeptide chains: a first heavy chain, a first light chain, and a second heavy chain; or the multispecific antibody or its antigen-binding fragment comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain. The first heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 12; the first light chain comprises an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 13; The second heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 14, 16, 17 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 14, 16, 17; the second light chain comprises an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 22-33, wherein, The cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics; optionally, the cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents, or DNA topoisomerase inhibitors, wherein the tubulin inhibitors include dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins; the DNA damaging agents include PBD drugs; and the DNA topoisomerase inhibitors include... Including camptothecin-based drugs. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 34, wherein, The cytotoxic drug D is selected from compounds of formula (DI). in, R 1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more D atoms; R 4 Selected from H or C1-C3 alkyl groups; R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups; R 6 Selected from H or C1-C3 alkyl groups; R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl; Optionally, the R 1 R 2 The atoms connected to them together form Optionally, R 4 Selected from H; Optionally, R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups; Optionally, R 5 Selected from H; Optionally, R 6 Selected from H; Optionally, R 7 Selected from cyclopropyl. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 35, wherein, The compound represented by formula (DI) is selected from one of the following compounds: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 22-36, wherein, The connecting subunit L is selected from Its a-terminus is covalently linked to the antibody unit Pc, and its b-terminus is covalently linked to the cytotoxic drug D, wherein: R b1 R b2 Each is independently selected from H, halogen, CN, C1-C6 alkyl or C3-C6 cycloalkyl, or R b1 R b2 The carbon atoms connected to them together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, which is optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic group; m1 is selected from integers 2 to 8; L a Selected from chemical bonds or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl groups, m2 is selected from integers 1 to 8, R b4 R b5 Each is independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl, L b The peptide residues are selected from 1 to 8 amino acids, and the peptide residues are optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic groups; Optionally, R b1 R b2 All are selected from H or R b1 R b2 The carbon atoms connected to them together form 4-7 membered heterocyclic groups; Optionally, structural unit Selected from Optionally, L a Selected from chemical bonds or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl, R b4 R b5 One is selected from H, and the other is selected from Optionally, L a Selected from Its * end and L b connect; Optionally, the L b It consists of Gly-Gly-Phe-Gly tetrapeptide residues; Optionally, m1 is an integer from 2 to 6; Optionally, m1 can be 2, 3, 4 or 5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 37, wherein, The connecting subunit L is Its a-end is covalently linked to the antibody unit Pc, and its b-end is covalently linked to the drug unit D. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 22-38, wherein, n is selected from real numbers from 1 to 16, for example, n is selected from real numbers from 2 to 12, for example, n is selected from real numbers from 4 to 10, for example, n is selected from real numbers from 3 to 9, for example, n is selected from real numbers from 4 to 8, for example, n is selected from real numbers from 6 to 8; optionally, n is selected from real numbers from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0. The antibody-drug conjugate according to any one of claims 22-39, or a pharmaceutically acceptable salt thereof, is selected from the following compounds or pharmaceutically acceptable salts thereof: Where Pc and n are as defined in any of the preceding definitions. A pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof of any one of claims 1-17, a nucleic acid fragment of claim 18, a carrier of claim 19, a host cell of claim 20, a product prepared by the method of claim 21, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 22-40; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant. The use of the multispecific antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid fragment of claim 18, the vector of claim 19, the host cell of claim 20, the product prepared by the method of claim 21, or the use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 22-40 in the preparation of a medicament for the prevention and / or treatment of tumors; wherein the tumor is selected from solid tumors, hematologic malignancies, or cancers that invasively express B7H3 or DLL3; Preferably, the tumor is selected from small cell lung cancer, pancreatic cancer, and other solid tumors. A method for preventing and / or treating tumors, comprising administering to a patient in need an effective amount of a multispecific antibody or antigen-binding fragment thereof of any one of claims 1-17, a nucleic acid fragment of claim 18, a vector of claim 19, a host cell of claim 20, a product prepared by the method of claim 21, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 22-40; wherein the tumor is selected from solid tumors, hematologic malignancies, or cancers that invasively express B7H3 or DLL3; preferably, the cancer is selected from small cell lung cancer, pancreatic cancer, and other solid tumors. The multispecific antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid fragment of claim 18, the vector of claim 19, the host cell of claim 20, the product prepared by the method of claim 21, or the antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 22-40 for the prevention and / or treatment of tumors; wherein the tumor is selected from solid tumors, hematologic malignancies, or cancers that invasively express B7H3 or DLL3; Preferably, the tumor is selected from small cell lung cancer, pancreatic cancer, and other solid tumors. A method for preventing and / or treating tumors includes administering an effective amount of a multispecific antibody or an antigen-binding fragment thereof to a patient in need, the multispecific antibody comprising a first antigen-binding domain specifically binding to B7H3 and a second antigen-binding domain specifically binding to DLL3. The method according to claim 45, wherein, The first antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions: Heavy chain CDR1, such as SEQ ID NO.27, 48, 69; Heavy chain CDR2 such as SEQ ID NO.28, 49, 70; Heavy chain CDR3 such as SEQ ID NO.29, 50, 71; Light chain CDR1, such as SEQ ID NO.39, 60, 81; Light chain CDR2 such as SEQ ID NO.40, 61, 82; Light chain CDR3, such as SEQ ID NO.41, 62, 83. The method according to claim 45 or 46, wherein, The second antigen-binding domain comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conserved amino acid substitutions: Heavy chain CDR1 such as SEQ ID NO. 30, 33, 36, 51, 54, 57, 72, 75, 78; Heavy chain CDR2 such as SEQ ID NO. 31, 34, 37, 52, 55, 58, 73, 76, 79; Heavy chain CDR3 such as SEQ ID NO. 32, 35, 38, 53, 56, 59, 74, 77, 80; Light chain CDR1 such as SEQ ID NO. 42, 45, 63, 66, 84, 87; Light chain CDR2 such as SEQ ID NO. 43, 46, 64, 67, 85, 88; Light chain CDR3 such as SEQ ID NO.44, 47, 65, 68, 86, 89. The method according to any one of claims 45-47, wherein, The VH of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; the VL of the first antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 21; the VH of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 22, 24 or 26 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 22, 24 or 26; the VL of the second antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO. 23 or 25 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher homology with SEQ ID NO. 20; Compared to NO.23 or 25, the amino acid sequence has 90%, 95%, 96%, 97%, 98%, 99% or higher homology. The method according to any one of claims 45-48, wherein, The multispecific antibody or its antigen-binding fragment is further conjugated with a cytotoxic drug D; optionally, the cytotoxic drug D is selected from one of the following compounds. The method according to claim 49, wherein, The cytotoxic drug D is linked to the multispecific antibody or its antigen-binding fragment via a linker unit L; optionally, the linker unit L is... Its a-end is covalently linked to the antibody, and its b-end is covalently linked to the drug D.

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