B7h3 antibody-exatecan analog conjugates and medical uses thereof

Abstract

Provided are B7H3 antibody-Exatecan analog conjugates and medical uses thereof. Specifically, provided are B7H3 antibody-drug conjugates or pharmaceutically acceptable salts thereof, and uses thereof in the preparation of a medicament for treating a B7H3-mediated disease or disorder; especially in the preparation of an anti-cancer medicament.

Description

[0001] This application claims priority to Chinese patent application filed on March 25, 2020 (application number CN 202010218100.7). Technical Field

[0002] This disclosure relates to anti-B7H3 antibody-eczema analog conjugates and their pharmaceutical use. Further, this disclosure relates to anti-B7H3 antibody-eczema analog conjugates or pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising the aforementioned conjugates or pharmaceutically acceptable salts thereof, and their use in the preparation of medicaments for treating B7H3-mediated diseases or conditions; particularly in the preparation of anticancer medicaments. Background Technology

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

[0004] T cell-mediated immune responses play a crucial role in the body's anti-tumor process. T cell activation and proliferation require not only antigen signals recognized by the TCR but also a second signal provided by co-stimulatory molecules. The B7 family of molecules belongs to the co-stimulatory immunoglobulin superfamily. Increasing research indicates that this family of molecules plays an important regulatory role in both normal immune function and pathological conditions.

[0005] B7H3 is a member of the B7 family, belonging to type I transmembrane proteins. It contains an amino-terminal signal peptide, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and a cytoplasmic tail region containing approximately 45 amino acids (Tissue Antigens. 2007 Aug; 70(2):96-104). Currently, B7H3 mainly exists in two splice variants, B7H3a and B7H3b. The extracellular segment of B7H3a consists of two immunoglobulin domains, IgV and IgC, and is also known as 2IgB7H3; while the extracellular segment of B7H3b consists of four immunoglobulin domains, IgV-IgC-IgV-IgC, and is also known as 4IgB7H3.

[0006] B7H3 is not expressed or is expressed at very low levels in normal tissues and cells, but is highly expressed in various tumor tissues. B7H3 is closely related to tumor progression, patient survival, and prognosis. Clinically, it has been reported that B7H3 is overexpressed in many cancer types, especially in non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, and pancreatic cancer (Lung Cancer. 2009 Nov; 66(2):245-249; Clin Cancer Res. 2008 Aug 15; 14(16):5150-5157). In addition, there are also reports that in prostate cancer, the expression intensity of B7H3 is positively correlated with clinicopathological malignancy (such as tumor volume, extraprostatic invasion, or Gleason score) and is also associated with cancer progression (Cancer Res. 2007 Aug 15; 67(16):7893-7900). Similarly, in glioblastoma multiforme, B7H3 expression is negatively correlated with event-free survival; and in pancreatic cancer, B7H3 expression is associated with lymph node metastasis and pathological progression. Therefore, B7H3 is considered a novel tumor marker and a potential therapeutic target.

[0007] Currently, therapeutic strategies targeting B7H3 have been used in preclinical studies. For example, antibodies targeting B7H3 in mice can enhance invasive CD8-positive T cells within the tumor and inhibit tumor growth. Mod Pathol. 2010 Aug; 23(8):1104-1112). Furthermore, patent application WO2008 / 066691 shows that antibodies recognizing B7H3a exhibit in vivo antitumor activity against adenocarcinoma. In clinical studies, a murine B7H3 antibody was used in conjunction with radioactive I... 131 The conjugate drug can significantly inhibit the growth of neuroblastoma in patients (J Neufooocol 97(3):409-l 8(2010)). However, the projects currently under research are all humanized antibodies modified from murine antibodies. Humanized antibodies have the problem of relatively high immunogenicity during immunization, which is an unfavorable factor when used in humans.

[0008] Phage display technology involves fusing exogenous proteins or peptides with phage coat proteins, thereby expressing the exogenous protein on the surface of the phage. A phage antibody library is an antibody library established using a comprehensive set of techniques, combining phage display technology, PCR amplification technology, and protein expression technology.

[0009] The greatest advantage of phage antibody libraries is that they can produce fully human antibodies without in vivo immunization, mimicking the three processes of antibody production in vivo. In addition, phage antibody libraries also have the following advantages:

[0010] ① It achieves the unification of genotype and phenotype. In addition, the experimental method is simple and rapid. Traditional antibody production methods using hybridoma technology take several months, while antibody library technology only takes a few weeks.

[0011] ② It expresses a fully human antibody with a small molecular weight, mainly expressed in the form of active fragments Fab and scFv, which have significant advantages in tissue penetration compared with intact antibodies.

[0012] ③ Large screening capacity: Hybridoma technology screens from thousands of clones, while antibody library technology can select from millions or even billions of molecules, resulting in a wide variety of antibodies.

[0013] ④ It has a wide range of applications and adopts a prokaryotic expression system. Its advantages are more obvious when it is produced on a large scale (Curr Opin Biotechnol. 2002 Dec; 13(6):598-602; Immunotechnology, 2013, 48(13):63-73).

[0014] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antigen-binding fragments to biologically active cytotoxic agents via adaptor compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, and the high efficiency of cytotoxic substances, while avoiding the drawbacks of low efficacy of antibodies and excessive toxic side effects of cytotoxic agents. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce the impact on normal cells.

[0015] Currently, several ADC drugs are being used in clinical trials or research; for example, Kadcyla is an ADC drug formed by trastuzumab targeting Her2 and DM1. Meanwhile, there are also patent reports on antibodies and ADC drugs targeting B7H3, such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, and WO2016044383.

[0016] Several classes of small molecules with cytotoxicity are used in antibody-drug conjugates; one of these is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Reports on the application of the camptothecin derivative ethanotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) include WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107:1039-1046. However, further development of more effective ADC drugs is still needed. Summary of the Invention

[0017] To reduce the immunogenicity of antibody molecules, a more suitable B7H3 antibody-drug conjugate is provided. This disclosure provides an antibody-drug conjugate of the general formula Pc-LYD or a pharmaceutically acceptable salt thereof:

[0018]

[0019] in:

[0020] Y is selected from -O-(CR) a R b ) m -CR 1 R 2 -C(O)-、-O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0021] R a and R bThey may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl and heterocyclic groups;

[0022] R 1 Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0023] Or, R a and R 2 Together with the carbon atom attached to it, it forms a cycloalkyl or heterocyclic group;

[0024] m is an integer from 0 to 4; non-restrictive examples include m being selected from 0, 1, 2, 3, and 4;

[0025] n is between 1 and 10, and n is a decimal or an integer;

[0026] L represents the connector unit;

[0027] Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises:

[0028] The heavy chain variable region, being a variant of SEQ ID NO: 1, comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S, and A113T; preferably, the variant comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, and A113T; and

[0029] The light chain variable region is SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said variant comprises an amino acid substitution of S57G;

[0030] The amino acid numbering is based on the natural sequence numbering of the variable region sequence.

[0031] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises:

[0032] The heavy chain variable region is a variant of SEQ ID NO: 1, which contains an amino acid substitution of T16R; preferably, it contains amino acid substitutions of T16R and A113T.

[0033] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a light chain variable region as shown in sequence SEQ ID NO: 2.

[0034] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a variant of the light chain variable region of SEQ ID NO: 2, the variant comprising an amino acid substitution of S57G.

[0035] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the heavy chain variable region of the anti-B7H3 antibody or its antigen-binding fragment is a variant of SEQ ID NO: 1, the variant comprising amino acid substitutions selected from any of the following groups:

[0036] a, T16R, Y103F, and A113T;

[0037] b, T16R, I28T, Y103F, and A113T;

[0038] c, T16R and A113T; and

[0039] d, T16R, I28T and A113T.

[0040] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises any one of the following:

[0041] e. Heavy chain variable region, which contains amino acid substitutions for T16R, Y103F, and A113T, and

[0042] As shown in SEQ ID NO: 2, the light chain variable region;

[0043] f. Heavy chain variable region, which contains amino acid substitutions for T16R, I28T, Y103F, and A113T, and

[0044] As shown in SEQ ID NO: 2, the light chain variable region;

[0045] g. Heavy chain variable region, which contains amino acid substitutions for T16R, Y103F, and A113T, and

[0046] The light chain variable region contains amino acid substitutions for S57G;

[0047] h, the heavy chain variable region, which contains amino acid substitutions for T16R, I28T, Y103F, and A113T, and

[0048] The light chain variable region contains amino acid substitutions for S57G;

[0049] i. Heavy chain variable region, which contains amino acid substitutions for T16R and A113T, and

[0050] The light chain variable region contains amino acid substitutions for S57G;

[0051] j. Heavy chain variable region, which contains amino acid substitutions at T16R, I28T, and A113T, and

[0052] The light chain variable region contains amino acid substitutions for S57G.

[0053] This disclosure provides an antibody-drug conjugate of general formula Pc-LYD or a pharmaceutically acceptable salt thereof:

[0054]

[0055] in:

[0056] Y is selected from -O-(CR) a R b ) m -CR 1 R 2 -C(O)-、-O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0057] R a and R bThey may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl and heterocyclic groups;

[0058] R 1 Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0059] Or, R a and R 2 Together with the carbon atom attached to it, it forms a cycloalkyl or heterocyclic group;

[0060] m is an integer from 0 to 4; non-restrictive examples include m being selected from 0, 1, 2, 3, and 4;

[0061] n is between 1 and 10, and n is a decimal or an integer;

[0062] L represents the connector unit;

[0063] Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises:

[0064] The heavy chain variable region, which is a variant of the heavy chain variable region in SEQ ID NO: 1, comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S, and A113T; preferably, the variant comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, and A113T; and

[0065] The light chain variable region is the light chain variable region in SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said variant comprises an amino acid substitution of S57G;

[0066] The amino acid numbering is based on the natural sequence numbering of the variable region sequence.

[0067] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises:

[0068] The heavy chain variable region is a variant of the heavy chain variable region in SEQ ID NO: 1, said variant containing an amino acid substitution of T16R; preferably containing amino acid substitutions of T16R and A113T.

[0069] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a light chain variable region as shown in sequence SEQ ID NO: 2.

[0070] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a light chain variable variant of SEQ ID NO: 2, the light chain variable region comprising an amino acid substitution of S57G.

[0071] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the heavy chain variable region of the anti-B7H3 antibody or its antigen-binding fragment is a variant of the heavy chain variable region of SEQ ID NO: 1, the variant comprising amino acid substitutions selected from any of the following groups:

[0072] a, T16R, Y103F, and A113T;

[0073] b, T16R, I28T, Y103F, and A113T;

[0074] c, T16R and A113T; and

[0075] d, T16R, I28T and A113T.

[0076] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises any one of the following:

[0077] e. Heavy chain variable region, which contains amino acid substitutions for T16R, Y103F, and A113T, and

[0078] The light chain variable region in SEQ ID NO: 2;

[0079] f. Heavy chain variable region, which contains amino acid substitutions for T16R, I28T, Y103F, and A113T, and

[0080] The light chain variable region in SEQ ID NO: 2;

[0081] g. Heavy chain variable region, which contains amino acid substitutions for T16R, Y103F, and A113T, and

[0082] The light chain variable region contains amino acid substitutions for S57G;

[0083] h, the heavy chain variable region, which contains amino acid substitutions for T16R, I28T, Y103F, and A113T, and

[0084] The light chain variable region contains amino acid substitutions for S57G;

[0085] i. Heavy chain variable region, which contains amino acid substitutions for T16R and A113T, and

[0086] The light chain variable region contains amino acid substitutions for S57G;

[0087] j. Heavy chain variable region, which contains amino acid substitutions at T16R, I28T, and A113T, and

[0088] The light chain variable region contains amino acid substitutions for S57G.

[0089] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or its antigen-binding fragment is selected from any one of the following:

[0090] As shown in SEQ ID NO: 3, the heavy chain variable region, and

[0091] As shown in SEQ ID NO: 4, the light chain variable region;

[0092] As shown in SEQ ID NO: 5, the heavy chain variable region, and

[0093] As shown in SEQ ID NO: 6, the light chain variable region;

[0094] As shown in SEQ ID NO: 7, the heavy chain variable region, and

[0095] As shown in SEQ ID NO: 8, the light chain variable region;

[0096] As shown in SEQ ID NO: 9, the heavy chain variable region, and

[0097] As shown in SEQ ID NO: 10, the light chain variable region;

[0098] As shown in SEQ ID NO: 11, the heavy chain variable region, and

[0099] As shown in SEQ ID NO: 12, the light chain variable region;

[0100] As shown in SEQ ID NO: 13, the heavy chain variable region, and

[0101] The light chain variable region is shown in SEQ ID NO: 14.

[0102] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises an antibody constant region; the heavy chain constant region of the antibody constant region is derived from human IgG1, IgG2, IgG3 or IgG4, preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1; the light chain constant region of the antibody constant region is derived from human antibody κ or λ chains.

[0103] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the anti-B7H3 antibody comprises a subset selected from any of the following:

[0104] As shown in SEQ ID NO: 17, heavy chains, and

[0105] The light chain shown in SEQ ID NO: 18;

[0106] As shown in SEQ ID NO: 19, heavy chains, and

[0107] The light chain shown in SEQ ID NO: 20;

[0108] As shown in SEQ ID NO: 21, heavy chains, and

[0109] The light chain shown in SEQ ID NO: 22; and

[0110] As shown in SEQ ID NO: 23, heavy chains, and

[0111] The light chain shown in SEQ ID NO: 24.

[0112] In another embodiment, this disclosure provides an antibody-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), and disulfide-stabilized V region (dsFv).

[0113] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein n is 1 to 8, preferably 3-8, more preferably 3-7, and n is a decimal or an integer. As a non-restrictive example, n may be mentioned as 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 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, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or any of the aforementioned values; technicians are permitted to determine the precision after the decimal point as needed.

[0114] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0115] in:

[0116] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0117] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and C1 atoms. -6 alkyl;

[0118] R 1 Halogenated C 1-6 Alkyl or C 3-6 cycloalkyl;

[0119] R 2 Selected from hydrogen atoms, halogenated C 1-6 Alkyl and C 3-6 cycloalkyl;

[0120] Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl;

[0121] m is 0 or 1.

[0122] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding claims, wherein Y is selected from any of the following:

[0123]

[0124] The O end of Y is connected to the connector unit L.

[0125] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0126] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-; where W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight-chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0127] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0128] L 3It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (Cit), serine (S), glutamic acid (E) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0129] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6;

[0130] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0131] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0132] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0133] L 1 for s 1 It is an integer from 2 to 8 (specifically, 2, 3, 4, 5, 6, 7, or 8);

[0134] L 2 It is a chemical bond;

[0135] L 3 It is a tetrapeptide residue; preferably, L 3 It is a tetrapeptide residue of GGFG (SEQ ID NO: 33);

[0136] L 4 For -NR 5 (CR 6 R7 )t-,R 5 R 6 Or R 7 They may be the same or different, and each is independently a hydrogen atom or an alkyl group, with t being 1 or 2;

[0137] The L mentioned therein 1 The terminal is connected to the PC, L 4 The end is connected to Y.

[0138] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding claims, wherein -L- is:

[0139]

[0140] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding claims, wherein -LY- is optionally selected from any of the following:

[0141]

[0142] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, which is of the general formula (Pc-L a The antibody-drug conjugate or its pharmaceutically acceptable salt shown in (-YD) is:

[0143]

[0144] in,

[0145] Pc, n, m, R 1 R 2 As defined in the general formula (Pc-LYD);

[0146] W, L 2 L 3 R 5 R 6 and R 7 As defined in connector unit-L-;

[0147] Specifically,

[0148] Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises:

[0149] The heavy chain variable region, which is a variant of the heavy chain variable region in SEQ ID NO: 1, comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S, and A113T; preferably, the variant comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, and A113T; and

[0150] The light chain variable region is the light chain variable region in SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said variant comprises an amino acid substitution of S57G;

[0151] m is an integer from 0 to 4;

[0152] n is between 1 and 10, and n is a decimal or an integer;

[0153] R 1 Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0154] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight-chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0155] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0156] L 3It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0157] R 5 Selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

[0158] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0159] In another embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, which is of the general formula (Pc-L b The antibody-drug conjugate or its pharmaceutically acceptable salt shown in (-YD) is:

[0160]

[0161] in:

[0162] s 1 Integers between 2 and 8;

[0163] Pc, R 1 R 2 R 5 To R 7 m and n are as shown in the general formula (Pc-L) a Defined in -YD).

[0164] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the antibody-drug conjugate is selected from any of the following:

[0165]

[0166]

[0167] Where Pc and n are as in the general formula (Pc-L) a Defined in -YD).

[0168] In another embodiment, this disclosure provides an antibody-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is:

[0169]

[0170] in:

[0171] n is 1 to 8, preferably 3 to 8; more preferably 3 to 7, where n is a decimal or an integer;

[0172] h1702-DS-107 is an anti-B7H3 antibody, which contains a heavy chain with the sequence shown in SEQ ID NO: 21 and a light chain with the sequence shown in SEQ ID NO: 22.

[0173] In other embodiments, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein n is 1 to 8, preferably 3-8, more preferably 3-7, and n is a decimal or an integer.

[0174] On the other hand, one embodiment of this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region, which is a variant of the heavy chain variable region in SEQ ID NO: 1, said variant comprising one or more amino acid substitutions selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S, and A113T; preferably, said variant comprises one or more amino acid substitutions selected from T16R, Y103F, I28T, and A113T; and

[0175] The light chain variable region is the light chain variable region in SEQ ID NO: 2 or a variant thereof, the variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, the variant comprises an amino acid substitution of S57G.

[0176] In another embodiment, this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the anti-B7H3 antibody or the antigen-binding fragment thereof is a variant of the heavy chain variable region of SEQ ID NO: 1, the variant comprising amino acid substitutions selected from any of the following groups:

[0177] a, T16R, Y103F, and A113T;

[0178] b, T16R, I28T, Y103F, and A113T;

[0179] c, T16R and A113T; and

[0180] d, T16R, I28T and A113T.

[0181] In another embodiment, this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises:

[0182] e. Heavy chain variable region, which includes amino acid substitutions of T16R, Y103F and A113T, and light chain variable region in SEQ ID NO: 2;

[0183] f. Heavy chain variable region, which includes amino acid substitutions of T16R, I28T, Y103F and A113T, and light chain variable region in SEQ ID NO: 2;

[0184] g. Heavy chain variable region, which contains amino acid substitutions of T16R, Y103F and A113T, and light chain variable region, which contains amino acid substitutions of S57G.

[0185] h, heavy chain variable region, which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and light chain variable region, which contains amino acid substitutions of S57G;

[0186] i. Heavy chain variable region, which contains amino acid substitutions of T16R and A113T, and light chain variable region, which contains amino acid substitutions of S57G.

[0187] j. Heavy chain variable region containing amino acid substitutions of T16R, I28T, and A113T, and light chain variable region containing amino acid substitutions of S57G.

[0188] In another embodiment, this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof is selected from:

[0189] As shown in SEQ ID NO:3, the heavy chain variable region, and as shown in SEQ ID NO:4, the light chain variable region;

[0190] As shown in SEQ ID NO: 5, the heavy chain variable region, and as shown in SEQ ID NO: 6, the light chain variable region;

[0191] As shown in SEQ ID NO: 7, the heavy chain variable region; and as shown in SEQ ID NO: 8, the light chain variable region.

[0192] As shown in SEQ ID NO: 9, the heavy chain variable region, and as shown in SEQ ID NO: 10, the light chain variable region;

[0193] The heavy chain variable region as shown in SEQ ID NO: 11, and the light chain variable region as shown in SEQ ID NO: 12; and

[0194] The heavy chain variable region as shown in SEQ ID NO: 13, and the light chain variable region as shown in SEQ ID NO: 14.

[0195] In another embodiment, this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises an antibody constant region; the heavy chain constant region of the antibody constant region is derived from human IgG1, IgG2, IgG3 or IgG4, preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1, more preferably the heavy chain constant region as shown in SEQ ID NO: 15; the light chain constant region of the antibody constant region is derived from human antibody κ or λ chains; preferably the light chain constant region as shown in SEQ ID NO: 16.

[0196] In another embodiment, this disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody is selected from:

[0197] As shown in SEQ ID NO: 17, the heavy chain, and in SEQ ID NO: 18, the light chain;

[0198] As shown in SEQ ID NO: 19, the heavy chain, and in SEQ ID NO: 20, the light chain;

[0199] As shown in SEQ ID NO: 21, the heavy chain, and in SEQ ID NO: 22, the light chain; and

[0200] As shown in SEQ ID NO: 23, the heavy chain, and in SEQ ID NO: 24, the light chain.

[0201] On the other hand, one embodiment of this disclosure provides a nucleic acid molecule that encodes an anti-B7H3 antibody or an antigen-binding fragment thereof as described above.

[0202] On the other hand, one embodiment of this disclosure provides a host cell that contains nucleic acid molecules as described above.

[0203] On the other hand, one embodiment of this disclosure provides a method for preparing a product such as general formula Pc-L a The method for the compound shown in -YD includes the following steps:

[0204]

[0205] Pc' is the reduced Pc, and is consistent with the general formula (L aThe compound represented by -YD) undergoes a coupling reaction to yield the compound of general formula (Pc-L). a The compound shown in -YD); as an example, Pc' is a compound with a reactive group (such as a thiol), and the reducing agent is preferably TCEP, especially the disulfide bond on the antibody, to obtain the thiol;

[0206] in:

[0207] Pc is the anti-B7H3 antibody or its antigen-binding fragment as described above;

[0208] W, L 2 L 3 R 1 R 2 R 5 To R 7 m and n are defined as in the general formula Pc-La-YD.

[0209] On the other hand, one embodiment of this disclosure provides a method for preparing a product such as general formula Pc-L a The method for the compound shown in -YD includes the following steps:

[0210]

[0211] The anti-B7H3 antibody h1702-DS-107 was reduced to obtain h1702-DS-107', and h1702-DS-107' was coupled with the compound shown in formula 9-A to obtain the compound shown in general formula (h1702-DS-107-9-A); the reducing agent is preferably TCEP, and in particular, the disulfide bonds on the antibody are preferably reduced to obtain thiol groups;

[0212] n can be 1 to 8, preferably 3 to 8, and n can be a decimal or an integer;

[0213] 1702-DS-107 is an anti-B7H3 antibody, which contains a heavy chain with the sequence shown in SEQ ID NO:3 and a light chain with the sequence shown in SEQ ID NO:4.

[0214] On the other hand, this disclosure provides a pharmaceutical composition comprising:

[0215] -An antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or an anti-B7H3 antibody or an antigen-binding fragment thereof as described in any of the preceding claims, and

[0216] - One or more pharmaceutically acceptable excipients, diluents or carriers.

[0217] In some embodiments, the unit dose of the pharmaceutical composition contains 0.1 mg to 3000 mg or 1 mg to 1000 mg of the anti-B7H3 antibody, its antigen-binding fragment, or the antibody-drug conjugate as described above.

[0218] On the other hand, this disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or an anti-B7H3 antibody or an antigen-binding fragment thereof as described in any of the preceding claims, or a pharmaceutical composition comprising the thereof, as a medicament.

[0219] On the other hand, this disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or an anti-B7H3 antibody or an antigen-binding fragment thereof as described in any of the preceding claims, or a pharmaceutical composition comprising the thereof, in the preparation of a medicament for treating B7H3-mediated diseases or conditions or tumors, wherein the B7H3-mediated diseases or conditions are B7H3-high-expressing cancers, medium-expressing cancers, or low-expressing cancers.

[0220] On the other hand, this disclosure provides the use of an antibody-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof, or an anti-B7H3 antibody as described in any of the preceding claims or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the thereof, in the preparation of a medicament for treating or preventing tumors, wherein the tumors and cancers are preferably squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, etc. Lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma; more preferably, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0221] On the other hand, this disclosure further relates to a method for treating or preventing tumors or cancer, the method comprising administering to a subject in need a therapeutically effective or preventatively effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or an anti-B7H3 antibody or an antigen-binding fragment thereof as described in any of the preceding claims, or a pharmaceutical composition comprising the thereof.

[0222] In some embodiments, tumors or cancers suitable for treatment with the active compounds of this application are associated with high, medium, or low expression of B7H3. In some implementations, the tumors and cancers are selected from: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma.

[0223] In some specific implementations, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma.

[0224] In some specific implementations, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer.

[0225] In some specific implementation schemes, the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0226] On the other hand, this disclosure further provides the aforementioned anti-B7H3 antibody or its antibody-drug conjugate as a drug, preferably as a drug for treating cancer or tumors, and more preferably as a drug for treating B7H3-mediated cancers.

[0227] The active compound (e.g., the ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in this disclosure) can be formulated in a form suitable for administration via any appropriate route, preferably in a unit dose manner or in a manner that allows the subject to self-administer a single dose. The unit dose of the compounds or compositions disclosed herein can be in the form of tablets, capsules, sachets, bottled liquids, powders, granules, lozenges, suppositories, regenerated powders, or liquid formulations.

[0228] The dosage of the active compound or composition used in the treatment methods disclosed herein will generally vary depending on the severity of the disease, the subject's weight, and the relative efficacy of the compound. As a general guideline, a suitable unit dose may be from 0.1 mg to 1000 mg.

[0229] In addition to the active compound, the pharmaceutical compositions disclosed herein may contain one or more excipients selected from the following: fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1% to 99% by weight of the active compound.

[0230] The anti-B7H3 antibody and antibody-drug conjugate disclosed herein have reduced immunogenicity, higher tumor-suppressive efficacy and therapeutic activity, lower toxicity, better pharmacokinetic properties and drug-likeness (e.g., stability). Attached Figure Description

[0231] Figure 1 Different ADCs inhibited the proliferation of Detroit562 cell lines with different B7H3 expression levels.

[0232] Figure 2 The efficacy of different ADCs on Detroit562 xenografts in nude mice. Detailed Implementation

[0233] I. Terminology

[0234] Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

[0235] When a trade name is used in this disclosure, it is intended to include the formulation of the product under that trade name, the pharmaceutical product under that trade name, and the active pharmaceutical ingredient portion thereof.

[0236] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0237] The term "drug" refers to cytotoxic drugs, which are chemical molecules that strongly disrupt the normal growth of cells within the cell. In principle, cytotoxic drugs can kill cells at sufficient concentrations; however, due to their lack of specificity, while killing tumor cells, they can also induce apoptosis in normal cells, leading to serious side effects. This term also includes toxins (such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals), and radioactive isotopes (such as At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 (and radioactive isotopes of Lu), chemotherapy drugs, antibiotics, and nucleolysins.

[0238] The terms “L”, “linker unit”, “linker”, “connector unit”, or “connector fragment” refer to a chemical structural fragment or bond that is connected to a ligand (specifically an antibody or antigen-binding fragment) at one end and to a drug at the other end. It can also be connected to other linkers before being connected to a ligand or drug.

[0239] The linker (including extensions, spacers, and amino acid units) can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a “cleavable linker” that facilitates the release of drugs into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0240] The term "antibody-drug conjugate" (ADC) refers to an antibody linked to a drug via a linker unit. In this disclosure, "antibody-drug conjugate" refers to the linking of a monoclonal antibody or antigen-binding fragment to a biologically active toxic drug via a stable linker unit.

[0241] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0242] The term "antibody" refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins are classified into five classes, or isotypes, based on the amino acid composition and sequence of their heavy chain constant regions: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of 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 subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. 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.

[0243] The sequence of approximately 110 amino acids near the N-terminus of both the heavy and light chains of the full-length antibody varies considerably and is known as the variable region (Fv region); the amino acid sequence near the C-terminus is relatively stable and is known as the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in this disclosure conform to the known IMGT rules in terms of both number and position.

[0244] The term "fully human antibody," also known as "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, thus reducing immunogenicity and toxicity. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0245] The term "antigen-binding fragment" refers to one or more segments of an antibody that retain its ability to bind antigens. Fragments of a full-length antibody can be used to perform the antigen-binding function. Examples of binding fragments included in an "antigen-binding fragment" include:

[0246] (i)Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains;

[0247] (ii) F(ab')2 fragment, a divalent fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0248] (iii) Fd fragments composed of VH and CH1 domains;

[0249] (iv) The Fv fragment consisting of the VH and VL domains of the single arm of the antibody;

[0250] (v) Single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of VH domains; and

[0251] (vi) Separated complementarity-determining regions (CDRs); or

[0252] (vii) A combination of two or more separate CDRs connected by a connector.

[0253] Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a linker using recombination methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule, called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" in the term antibody.

[0254] Such antigen-binding fragments are obtained using conventional techniques known to those skilled in the art and are functionally screened in the same manner as for intact antibodies. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0255] Typically, Fab is a fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (e.g., cleaving amino acid residue 224 of the H chain), wherein the N-terminal portion of the H chain and the L chain are linked together by disulfide bonds.

[0256] Typically, F(ab')2 is obtained by digesting the portion below the disulfide bond in the hinge region of IgG with the enzyme pepsin. It is a fragment with a molecular weight of approximately 100,000, possesses antigen-binding activity, and contains two Fab regions connected at the hinge position.

[0257] Typically, Fab' is a fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0258] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryotic or eukaryotic organism for expression.

[0259] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or VH) of the antibody heavy chain and a variable domain (or VL) of the antibody light chain linked by a linker. 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 amino acid sequences or variants thereof, for example, using 1–4 repeating variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure 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.

[0260] The term "frame region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0261] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0262] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10...-7 M, for example, approximately less than 10 -8 M, 10 -9 M or 10 -10 Binds with M or less affinity (in KD).

[0263] The term "nucleic acid molecule" refers to either a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0264] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the expression vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the expression vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0265] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0266] The antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the N-terminal site of the Fc region. Positive clones are scaled up in a bioreactor medium to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antigen-binding fragment is detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0267] The term "peptide" refers to a compound fragment with a molecular weight between that of an amino acid and a protein, consisting of two or more amino acid molecules linked together by peptide bonds.

[0268] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, the alkyl group is a lower alkyl group containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point, and the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0269] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0270] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). Alkylene groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point, and the substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, and oxo.

[0271] The term "alkoxy" refers to -O- (alkyl) and -O- (cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0272] The term "cycloalkyl" refers to a cyclic hydrocarbon substituent that is saturated or partially unsaturated, either monocyclic or polycyclic, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (containing 3, 4, 5, 6, 7, or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, or bridged-ring cycloalkyl groups.

[0273] The term "heterocyclic group" refers to a cyclic hydrocarbon substituent that is saturated or partially unsaturated, consisting of a monocyclic or polycyclic ring containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The ring consists of heteroatoms (where m is an integer 0, 1, or 2), but excludes ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3, or 4 heteroatoms); more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (containing 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, or bridged-ring heterocyclic groups.

[0274] The term "spiroheterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which one or more ring atoms are selected from nitrogen, oxygen, or S(O). m The ring atoms are (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6 to 14 fused, more preferably 7 to 10 fused. Spirocyclic groups are classified according to the number of shared spiroatoms between rings: monospirocyclic, bispirocyclic, or multispirocyclic, preferably monospirocyclic or bispirocyclic. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic groups include:

[0275]

[0276] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m(where m is an integer 0, 1, or 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, they are 6 to 14 members, more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings). Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0277]

[0278] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m The heteroatom is a carbon atom (where m is an integer 0, 1, or 2). Preferably, it consists of 6 to 14 members, more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings). Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0279]

[0280] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0281] wait.

[0282] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0283] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered (6-, 7-, 8-, 9-, or 10-membered), such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0284]

[0285] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0286] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms (1, 2, 3, or 4 heteroatoms) and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered (5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl), more preferably 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0287]

[0288] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0289] The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenemethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be replaced by 1 to 3 substituents (one, two, or three substituents) selected from halogens, alkoxy groups, or nitro groups. The amino protecting group is preferably 9-fluorenemethoxycarbonyl.

[0290] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more halogens, wherein the alkyl group is as defined above.

[0291] The term “deuterated alkyl” refers to an alkyl group in which one or more deuterium atoms are replaced by hydrogen atoms, wherein the alkyl group is as defined above.

[0292] The term "hydroxyl group" refers to the -OH group.

[0293] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0294] The term "amino" refers to -NH2.

[0295] The term "nitro" refers to -NO2.

[0296] The term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0297] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0298] This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuterontetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0299] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0300] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one, two, or three hydrogen atoms that are independently substituted by a substituent. Substituents are only considered in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0301] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active compound and its biological activity.

[0302] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the antibody-drug conjugate of this disclosure, or a salt of the active compound described in this disclosure, which is safe and effective when used in a subject and has the intended biological activity. As an example, the antibody-drug conjugate of this disclosure contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0303] "Drug loading," also known as the drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range, for example, from about 1 to about 10 drugs per antibody; and in some embodiments, it is preferably selected from the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8, within the range of about 1 to about 8 drugs per antibody. Exemplarily, the drug loading can be an arithmetic mean obtained on the basis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range of drug loading.

[0304] In embodiments of this disclosure, the drug loading is expressed as n, and the drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC characterization.

[0305] The loading capacity of antibody-drug conjugates can be controlled using the following non-restrictive methods, including:

[0306] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0307] (2) Control the reaction time and temperature.

[0308] (3) Choose different reaction reagents.

[0309] Conventional preparation methods for pharmaceutical compositions are described in the Chinese Pharmacopoeia.

[0310] The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters a subject and its distribution within the body, control the rate of drug release, and deliver the drug to a target. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, polymeric surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferred examples being micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates possess the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0311] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active ingredient; it can also be called a pharmacological agent. Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0312] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation. Examples of absorbents include starch, lactose, inorganic salts of calcium, and microcrystalline cellulose.

[0313] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Water, Ringer's solution, and isotonic sodium chloride solution may be among the acceptable solvents and media used. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active compound is dissolved in an oil phase. For example, the active compound is dissolved in a mixture of soybean oil and lecithin, and then the oil solution is treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the subject by local large-volume injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound disclosed herein. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0314] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

[0315] II. Synthesis Method

[0316] To achieve the synthesis objective, the following synthesis technique was adopted:

[0317] A method for preparing antibody-drug conjugate h1702-DS-107 includes the following steps:

[0318]

[0319] The anti-B7H3 antibody h1702-DS-107 is reduced to obtain h1702-DS-107', and h1702-DS-107' is coupled with the compound shown in formula 9-A to obtain the compound shown in general formula (h1702-DS-107-9-A); the reducing agent is preferably TCEP, and in particular, the reducing agent is preferably the reducing agent for disulfide bonds on the antibody to obtain thiol groups; n is 1 to 8, preferably 3 to 8, and n is a decimal or an integer.

[0320] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will become apparent from the description and claims.

[0321] In the specification and claims, unless the context clearly indicates otherwise, the singular form includes the plural referent.

[0322] Unless otherwise defined, all technical and scientific terms used in this disclosure have the general meanings understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the specification are incorporated herein by reference.

[0323] The following embodiments are provided to more fully illustrate preferred embodiments of the present disclosure. These embodiments should not be construed in any way as limiting the scope of the present disclosure, which is defined by the claims.

[0324] I. Antibody Preparation

[0325] Example 1-1 Preparation, Expression and Purification of Antibodies

[0326] (I) Antibody Design

[0327] WO2019 / 024911 discloses the preparation of an anti-B7H3 antibody h1702-DS, the full text of which can be referenced. The specific sequence of antibody h1702-DS includes the heavy chain (IgG1) amino acid sequence (as described in sequence number 22 in WO2018 / 177393):

[0328]

[0329] And the light chain amino acid sequence (such as sequence number 26 as described in publication WO2018 / 177393):

[0330]

[0331] The above-mentioned anti-B7H3 antibody was mutated to obtain an antibody with lower immunogenicity. Its binding activity against B7H3 was tested (Test Example 1), and a new antibody was obtained. The corresponding light chain variable region and heavy chain variable region of the antibody are as follows:

[0332] Table 1. Mutants of anti-B7H3 antibodies and their sequences

[0333]

[0334]

[0335] The antibody variable region was then homologously recombinated with the constant region gene (CH1-FC / CL) fragment to construct the complete antibody VH-CH1-FC / VK-CL / VL-CL. The constant region sequence is as follows:

[0336] Heavy chain constant region:

[0337]

[0338]

[0339] Light chain constant region:

[0340]

[0341] The complete antibody was obtained, and the specific antibody sequence included, but was not limited to:

[0342] Antibody h1702-DS-105

[0343] Heavy chain amino acid sequence:

[0344]

[0345] Light chain amino acid sequence:

[0346]

[0347] Antibody h1702-DS-106

[0348] Heavy chain amino acid sequence:

[0349]

[0350] Light chain amino acid sequence:

[0351]

[0352]

[0353] Antibody h1702-DS-107

[0354] Heavy chain (IgG1) amino acid sequence:

[0355]

[0356] Light chain amino acid sequence:

[0357]

[0358] Antibody h1702-DS-108

[0359] Heavy chain amino acid sequence:

[0360]

[0361] Light chain amino acid sequence:

[0362]

[0363] The CDR sequences (IMGT numbering rules) in the light and heavy chains of the above-mentioned complete antibodies are shown in Table 2.

[0364] Table 2. CDR region sequences of each heavy chain and light chain

[0365]

[0366] (II) Expression and purification of fully human antibodies

[0367] Plasmids expressing the light and heavy chains of the antibody were transfected into HEK293E cells. After 6 days, the supernatant was collected, centrifuged at high speed to remove impurities, and purified using a Protein A column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was further purified using PBS-equilibrated Superdex 200 (GE) gel chromatography to remove aggregates. The monomer peaks were collected and aliquoted for later use.

[0368] Examples 1-2: Preparation of relevant detection cell lines and antibodies

[0369] (I) Cell lines overexpressing B7H3

[0370] This disclosure uses recombinant cell lines overexpressing B7H3 (CT26 / B7H3, where CT26 is derived from the Chinese Academy of Sciences Cell Bank, TCM37) or tumor cells (A498) to detect the binding ability of the disclosed antibody to the B7H3 antigen.

[0371] Human B7H3 full-length amino acid sequence: B7H3 (SEQ ID NO: 31):

[0372]

[0373] Note:

[0374] The double-lined portion represents the signal peptide (Signal peptide: 1–28);

[0375] The underlined portion represents the extracellular domain of B7H3 (29-466), where 29-139 is the Ig-like V-type 1 domain, 145–238 is the Ig-like C2-type 1 domain; 243-357 is the Ig-like V-type 2 domain, and 363–456 is the Ig-like C2-type 2 domain.

[0376] The dotted-lined area represents the transmembrane domain (467-487);

[0377] The italicized portion is the intracellular region (Cytoplasmic domain: 488-534).

[0378] (II) Full-length amino acid sequence of monkey B7H3

[0379]

[0380] Note:

[0381] The double-lined portion represents the signal peptide (Signal peptide: 1–28);

[0382] The underlined portion represents the extracellular domain of B7H3 (29-466), where 29-139 is the Ig-like V-type 1 domain, 145–238 is the Ig-like C2-type 1 domain; 243-357 is the Ig-like V-type 2 domain, and 363–456 is the Ig-like C2-type 2 domain.

[0383] The dotted-dash section represents the transmembrane domain (467-487).

[0384] The italicized portion is the intracellular domain (Cytoplasmic domain: 488-534).

[0385] II. Preparation of Compounds

[0386] Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

[0387] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). Chemical shifts were expressed as 10⁻⁶. -6 (ppm) is given as the unit.

[0388] MS measurements were performed using a Finnigan LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

[0389] The UPLC determination was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

[0390] HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0391] The UV-HPLC determination was performed using a Thermo nanodrop2000 UV spectrophotometer.

[0392] proliferation inhibition rate and IC 50 The values ​​were determined using a Phera StarFS microplate reader (BMG GmbH, Germany).

[0393] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC have a diameter of 0.15 mm to 0.2 mm, while those used for TLC separation and purification of products have a diameter of 0.4 mm to 0.5 mm.

[0394] Column chromatography typically uses 200 to 300 mesh silica gel from Yantai Huanghai as a carrier.

[0395] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0396] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere refers to a reaction flask connected to an approximately 1L volume argon or nitrogen gas balloon.

[0397] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0398] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0399] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0400] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0401] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0402] Unless otherwise specified in the examples, the reaction temperature is room temperature. Room temperature is the optimal reaction temperature, ranging from 20°C to 30°C.

[0403] Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g of KH2PO4, 8.56g of K2HPO4·3H2O, 5.85g of NaCl, and 1.5g of EDTA and place them in a bottle. Make up the volume to 2L, sonicate to dissolve completely, and shake well.

[0404] The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

[0405] Some of the compounds disclosed herein were characterized by Q-TOF LC / MS. The Q-TOF LC / MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

[0406] The YD drug portion of this disclosure of antibody-drug conjugates is referenced in PCT / CN2019 / 107873. The full text, including related compound synthesis and test examples, is cited in this patent. The non-limiting example synthesis is cited below:

[0407] Example 2-1

[0408] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0409]

[0410] Add 1 mL of N,N-dimethylformamide to eczema sulfonate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopropylformic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (3.8 mg, 13.7 μmol) to the reaction solution in sequence. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%).

[0411] MS m / z(ESI): 520.2 [M+1]

[0412] 1 H NMR (400MHz, CDCl3): δ7.90-7.84(m, 1H), 7.80-7.68(m, 1H), 5.80-5.70(m, 1H), 5.62-5.54(m, 2H), 5.44-5.32(m, 2H), 5.28-5.10(m, 2H) , 3.40-3.15(m, 3H), 2.44(s, 3H), 2.23(t, 1H), 2.06-1.75(m, 2H), 1.68-1.56(m, 1H), 1.22-1.18(m, 2H), 1.04-0.98(m, 2H), 0.89(t, 3H).

[0413] Example 2-2

[0414] (S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide 2-A

[0415] (R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide 2-B

[0416]

[0417] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (4 mg, 7.53 μmol), purge three times with argon, cool to 0-5 °C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared using the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) sequentially to the reaction solution. After the additions are complete, stir the reaction mixture at 0-5 °C for 1 hour. Remove the ice-water bath, heat to 30 °C, and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridgePrep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

[0418] MS m / z(ESI):534.0[M+1].

[0419] Single-configuration compound 2-B (shorter retention time):

[0420] UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0421] 1H NMR (400MHz, DMSO-d6): δ8.37(d, 1H), 7.76(d, 1H), 7.30(s, 1H), 6.51(s, 1H) ,5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H) , 3.19-3.13(m, 1H), 2.38(s, 3H), 2.20-2.14(m, 1H), 1.98(q, 2H), 1.87-1.83( m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H).

[0422] Compound 2-A with a single configuration (longer retention time):

[0423] UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0424] 1 H NMR (400MHz, DMSO-d6): δ8.35(d, 1H), 7.78(d, 1H), 7.31(s, 1H), 6.52(s, 1H ), 5.58-5.53(m, 1H), 5.42(s, 2H), 5.37(d, 1H), 5.32(t, 1H), 3.62(t, 1H), 3. 20-3.15(m, 2H), 2.40(s, 3H), 2.25-2.16(m, 1H), 1.98(q, 2H), 1.87-1.82(m , 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

[0425] Example 2-3

[0426] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-A

[0427] (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

[0428]

[0429] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (5.0 mg, 9.41 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath, heat to 30 °C, and stir for 8 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (1.5mg, 1.5mg).

[0430] MS m / z(ESI): 561.9 [M+1].

[0431] Single-configuration compounds (shorter retention time):

[0432] UPLC analysis: retention time 1.11 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0433] 1H NMR (400MHz, DMSO-d6): δ8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H) , 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4.66- 4.57(m, 1H), 3.18-3.12(m, 1H), 2.40(s, 3H), 2.26-2.20(m, 1H), 2.16-2.08( m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

[0434] Single-configuration compounds (longer retention time):

[0435] UPLC analysis: retention time 1.19 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0436] 1 H NMR (400MHz, DMSO-d6): δ8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1 H), 4.66-4.57(m, 1H), 3.18-3.12(m, 1H), 2.40(s, 3H), 2.22-2.14(m, 1H ), 2.04-1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

[0437] Examples 2-4

[0438] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 4

[0439]

[0440] Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared using the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 4 (2.5 mg, yield: 80.9%).

[0441] MS m / z(ESI): 548.0 [M+1].

[0442] 1 H NMR (400MHz, CDCl3): δ7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H), 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2.08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

[0443] Examples 2-5

[0444] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

[0445]

[0446] Add 1 mL of N,N-dimethylformamide to 1b (2.0 mg, 3.76 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared using the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2 mg, 7.24 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 5 (1.0 mg, yield: 50%).

[0447] MS m / z(ESI): 533.9 [M+1].

[0448] 1 H NMR (400MHz, CDCl3): δ8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55-6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3.30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35 -2.28 (m, 1H), 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

[0449] Examples 2-6

[0450] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

[0451]

[0452] Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0–5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared according to the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 6 (2.1 mg, yield: 67.9%).

[0453] MS m / z(ESI): 548.0 [M+1].

[0454] 1 H NMR (400MHz, DMSO-d6): δ7.85-7.62 (m, 1H), 6.88 (br, 1H), 5.87-5.48 (m, 2H), 5.47-5.33 (m, 1H), 5.31-5.06 (m, 1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

[0455] Examples 2-7

[0456] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclobutane-1-carboxamide 7

[0457]

[0458] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplier's reagent), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, and purify the residue by thin-layer chromatography using developing solvent system B to give title product 7 (2.5 mg, yield: 83.1%).

[0459] MS m / z(ESI):534.0[M+1].

[0460] 1 H NMR (400MHz, DMSO-d6): δ8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H) , 6.12(s, 1H), 5.59-5.51(m, 1H), 5.41(s, 2H), 5.20-5.01(m, 2H), 3.27-3.17( m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 (m, 1H) , 2.03-1.94(m, 2H), 1.92-1.78(m, 4H), 1.50-1.42(m, 1H), 0.90-0.83(m, 4H).

[0461] Examples 2-8

[0462] 1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

[0463]

[0464] Step 1: 1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid benzyl ester 8c

[0465] 1-Hydroxycyclopropane-1-carboxylic acid benzyl ester 8a (104 mg, 0.54 mmol; prepared by the method disclosed in patent application "US2005 / 20645") and 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in patent application "CN105829346A") were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, the temperature was lowered to 0-5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, the mixture was heated to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of 1,4-dioxane, and 0.6 mL of water was added. Sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the title product 8c (100 mg, yield: 73.6%).

[0466] MS m / z(ESI):501.0[M+1].

[0467] Step 2: 1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d

[0468] Dissolve 8c (50 mg, 0.10 mmol) in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), add palladium on carbon (25 mg, 10%), purge three times with hydrogen, and stir at room temperature for 1 hour. Filter the reaction solution with diatomaceous earth, wash the filter cake with tetrahydrofuran, concentrate the filtrate to give the title product 8d (41 mg, yield: 100%).

[0469] MS m / z(ESI):411.0[M+1].

[0470] Step 3: (9H-fluorene-9-yl)methyl(2-(((1-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

[0471] Add 1b (7 mg, 0.013 mmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add 0.5 mL of 8d (7 mg, 0.017 mmol) in N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (7 mg, 0.026 mmol), and stir in an ice bath for 35 minutes. Add 10 mL of water, extract with ethyl acetate (5 mL × 3), wash the organic phase with saturated sodium chloride solution (10 mL), dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by thin-layer chromatography with developing solvent system B to give the title product 8e (8.5 mg, yield 78.0%).

[0472] MS m / z(ESI):828.0[M+1].

[0473] Step 4: 1-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8f

[0474] 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, and 0.1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred. The upper n-hexane layer was decanted. This process was repeated three times. The crude product 8f (2.9 mg) was concentrated under reduced pressure. The product was used directly in the next reaction without purification.

[0475] MS m / z(ESI): 606.0 [M+1].

[0476] Step 5: 1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

[0477] Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. 0.3 mL of N,N-dimethylformamide solution containing 8 g (2.7 mg, 5.80 μmol, prepared using the method disclosed in patent application "EP2907824") of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrolo-1-yl)hexamylamino)acetamido)acetamido)-3-phenylpropionic acid was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol). The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was heated to room temperature and stirred for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).

[0478] MS m / z(ESI):1060.0[M+1].

[0479] 1 H NMR (400MHz, DMSO-d6): δ9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m, 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07(m, 4H), 6.98(s, 1H), 6.50(s, 1H), 5.61(q, 1H), 5.40(s, 2H), 5.32(t, 1H), 5.12(q, 2H), 4.62(t, 1H) , 4.52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47 (m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37 -2.23(m, 4H), 2.12-1.93(m, 4H), 1.90-1.74(m, 2H), 1.52-1.38(m, 4H), 1.33-1.11(m, 5H), 0.91-0.81(m, 4H).

[0480] Examples 2-9

[0481] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-A

[0482] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

[0483]

[0484]

[0485] Step 1: 2-Cyclopropyl-2-hydroxyacetic acid benzyl ester 9a

[0486] 2a (1.3 g, 11.2 mmol; prepared by the method disclosed in patent application "WO2013 / 106717") was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent system C to give the title product 9a (2 g, yield: 86.9%).

[0487] Step 2: 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid benzyl ester 9b

[0488] Add 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) to the reaction flask, add 4 mL of tetrahydrofuran, purge three times with argon, cool to 0-5 °C in an ice-water bath, add potassium tert-butoxide (109 mg, 0.98 mmol), remove the ice bath, raise to room temperature and stir for 40 minutes, add 10 mL of ice water, and extract with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). Combine the organic phases and concentrate. Dissolve the residue in 4 mL of dioxane, add 2 mL of water, add sodium bicarbonate (49.2 mg, 0.586 mmol) and fluorene methyl chloroformate (126 mg, 0.49 mmol), and stir at room temperature for 2 hours. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 9b (48 mg, yield: 19%).

[0489] MS m / z(ESI): 515.0 [M+1].

[0490] Step 3: 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid 9c

[0491] 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to obtain the crude product 9c (13 mg). This product was used directly in the next reaction without further purification.

[0492] MS m / z(ESI):424.9[M+1].

[0493] Step 4: (9H-fluorene-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

[0494] Add 1b (10 mg, 18.8 μmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add crude product 9c (13 mg, 30.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (16.9 mg, 61.2 μmol), and stir in an ice bath for 40 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by thin-layer chromatography using developing solvent system B to give the title product 9d (19 mg, yield: 73.6%).

[0495] MS m / z(ESI):842.1[M+1].

[0496] Step 5: 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide 9e

[0497] 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 9e (17 mg). The product was used directly in the next reaction without purification.

[0498] MS m / z(ESI): 638.0 [M+18].

[0499] Step 6: N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide 9-A

[0500] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

[0501] Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (21.2 mg, 44.8 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

[0502] MS m / z(ESI):1074.4[M+1].

[0503] Compound 9-A with a single configuration (shorter retention time):

[0504] UPLC analysis: retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0505] 1H NMR (400MHz, DMSO-d6): δ8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H) , 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1 H), 4.54-4.40(m, 2H), 3.76-3.64(m, 4H), 3.62-3.48(m, 2H), 3.20-3.07(m, 2H), 3.04-2.94(m, 1H), 2.80-2.62(m, 1H), 2.45-2.30(m, 3H), 2.25-2.15(m, 2H), 2.15-2.04(m, 2H), 1.93-1.78(m, 2H), 1.52-1.39(m, 3H), 1.34-1.12(m, 5H), 0.87(t, 3H), 0.64-0.38(m, 4H).

[0506] Single-configuration compound 9-B (longer retention time):

[0507] UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0508] 1H NMR (400MHz, DMSO-d6): δ8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31(s, 1H), 7.26-7.13(m, 3H), 6.99(s, 1H), 6.55-6.48(m, 1H), 5.60- 5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60- 4.40(m, 2H), 3.76-3.58(m, 4H), 3.58-3.48(m, 1H), 3.20-3.10(m, 2H) , 3.08-2.97(m, 2H), 2.80-2.72(m, 2H), 2.45-2.30(m, 3H), 2.25-2.13( m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52- 1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

[0509] III. Preparation of ADC

[0510] ADC drug loading analysis

[0511] Experimental Objectives and Principles

[0512] The ADC load was determined using ultraviolet spectrophotometry (UV-Vis). Instrument: Thermo Nanodrop 2000 UV-Vis spectrophotometer. The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbance values ​​of the drug and the monoclonal antibody at that wavelength.

[0513] Experimental methods

[0514] After placing cuvettes containing sodium succinate buffer solution into the reference absorption cell and the sample absorption cell, respectively, and subtracting the solvent blank, cuvettes containing the test solution were placed into the sample absorption cell, and absorbance was measured at 280 nm and 370 nm.

[0515] Result calculation:

[0516] A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug Equation (1)

[0517] εDrug-280 The drug has an average molar extinction coefficient of 5100 at 280 nm.

[0518] C Drug Drug concentration;

[0519] ε mab-280 The average molar extinction coefficient of the monoclonal antibody at 280 nm is 214,600.

[0520] C mab : Concentration of monoclonal antibody;

[0521] b: The optical path length is 1cm.

[0522] Similarly, the equation for the total absorbance of the sample at 370 nm can be obtained:

[0523] A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug Equation (2)

[0524] ε Drug-370 The drug has an average molar extinction coefficient of 19000 at 370 nm.

[0525] C Drug Drug concentration;

[0526] ε mab-370 The monoclonal antibody has an extinction coefficient of 0 at 370 nm.

[0527] C mab : Concentration of monoclonal antibody;

[0528] b: The optical path length is 1cm.

[0529] The drug loading in the ADC can be calculated by combining equations (1) and (2) with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

[0530] Drug load = C Drug / C mab .

[0531] Example 3: Preparation of Antibody-Drug Conjugation

[0532] (I) Preparation of antibody-drug conjugates with h1702-DS-107-9-A with different DAR values:

[0533]

[0534] The preparation process of the ADC conjugate h1702-DS-107-9-A is as follows: Humanized antibody (h1702-DS-107) was placed in 0.05M PBS buffer solution at pH 6.5 (antibody concentration 10 mg / mL). A 10mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (Innochem, CAS: 51805-45-9, Cat#B45573) was added, and the mixture was placed in a 37℃ constant temperature shaking incubator for 3 hours. The reaction solution was then cooled to 25℃ in an ice bath.

[0535] Compound 9-A was dissolved in dimethyl sulfoxide and added to the above reaction solution. The mixture was placed on a shaker at room temperature and reacted for 3 hours before the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05M PBS buffered aqueous solution at pH 6.5 containing 0.001M EDTA) to obtain the target antibody-drug conjugate.

[0536] By adjusting the ratio of antibody to drug, the scale of reaction, and other conditions, antibody-drug conjugates with different DAR values ​​(n) can be obtained. The preferred DAR value is 1-8, more preferably 3-8, and most preferably 3-7.

[0537] The specific preparation method for obtaining the ADC compound h1702-DS-107-9-A is as follows:

[0538] Example 3-1 ADC-1 (DAR = 6.92)

[0539] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 383 μL, 3830 nmol) was added to the PBS buffered aqueous solution of antibody h1702-DS-107 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 10.7 mL, 723 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0540] Compound 9-A (12.2 mg, 11358 nmol) was dissolved in 600 μl DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.33 mg / mL, 70 mL) of the title product ADC-1, which was stored at 4 °C.

[0541] UV-Vis calculated average: n = 6.92.

[0542] Example 3-2 ADC-2 (DAR = 4.75)

[0543] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 20.6 μL, 206 nmol) was added to the PBS buffered aqueous solution of antibody h1702-DS-107 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.99 mL, 66.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0544] Compound 9-A (1.07 mg, 996 nmol) was dissolved in 28.5 μl DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.57 mg / mL, 12.9 mL) of the title product ADC-2, which was stored at 4 °C.

[0545] UV-Vis calculated average: n = 4.75.

[0546] Example 3-3 ADC-3 (DAR = 3.09)

[0547] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 12.0 μL, 120 nmol) was added to the PBS buffered aqueous solution of antibody h1702-DS-107 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.99 mL, 66.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0548] Compound 9-A (0.71 mg, 661 nmol) was dissolved in 18.9 μl DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.54 mg / mL, 13.9 mL) of the title product ADC-3, which was stored at 4 °C.

[0549] UV-Vis calculated average: n = 3.09.

[0550] (II) Preparation of reference antibody-drug conjugate with h1702-DS-9-A with different DAR values:

[0551]

[0552] Example 3-4 ADC-4 (DAR = 6.87)

[0553] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 6.20 mL, 62.0 μmol) was added to the PBS buffered aqueous solution of antibody h1702-DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 180 mL, 12.16 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0554] Compound 9-A (195.9 mg, 182.4 nmol) was dissolved in a mixed solution of acetonitrile (3.6 mL) and DMSO (1.8 mL), and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified successively by ultrafiltration membrane exchange buffer using 50 mM pH 6.5 PBS buffer (containing 4% v / v acetonitrile and 2% v / v DMSO) and 10 mM pH 5.3 succinate buffer to remove small molecules. Sucrose was added to a concentration of 60 mg / mL, and Tween-20 was added to a concentration of 0.2 mg / mL. After lyophilization, the lyophilized powder sample of the title product ADC-4 (20 mg / vial) was obtained and stored at 4 °C.

[0555] UV-Vis calculated average: n = 6.87.

[0556] Example 3-5 ADC-5 (DAR=4.80)

[0557] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 25.1 μL, 251 nmol) was added to the PBS buffered aqueous solution of antibody h1702-DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.20 mL, 81.1 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0558] Compound 9-A (1.30 mg, 1210 nmol) was dissolved in 34.7 μl DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.68 mg / mL, 13.6 mL) of the title product ADC-5, which was stored at 4 °C.

[0559] UV-Vis calculated average: n = 4.80.

[0560] Example 3-6 ADC-6 (DAR = 2.97)

[0561] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 14.6 μL, 146 nmol) was added to the PBS buffered aqueous solution of antibody h1702-DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.20 mL, 81.1 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0562] Compound 9-A (0.87 mg, 931 nmol) was dissolved in 23.2 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.73 mg / mL, 12.2 mL) of the title product ADC-6, which was stored at 4 °C.

[0563] UV-Vis calculated average: n = 2.97.

[0564] Reference example

[0565] This disclosure incorporates the entire contents of application WO2020063676. Reference is made to PCT / CN2019 / 107873, pages 104-105, regarding the preparation of ADC-28 and ADC-29.

[0566] Wherein, ADC-28 is an exemplary product of the general formula FADC-26, with the following structure:

[0567]

[0568] UV-Vis calculated average: n = 7.46.

[0569] ADC-29 is an exemplary product of the general formula FADC-25, and its structure is as follows:

[0570]

[0571] UV-Vis calculated average: n = 7.24.

[0572] The h1702-DS has a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2.

[0573] The compound 20 (prepared according to Example 20, page 88 of PCT / CN2019 / 107873) has the structure shown in the following formula.

[0574]

[0575] Biological evaluation

[0576] In vitro bioactivity evaluation

[0577] Test Example 1: Expression of the h1702-DS mutant and detection of its B7H3 binding activity

[0578] To eliminate potential T cell epitopes of h1702-DS, a series of mutants of h1702-DS were designed, expressed, and purified. The binding of the purified mutants to the CT26-B7H3 cell line was detected by FACS.

[0579] The results showed that the disclosed mutant h1702-DS-107 exhibited a significant binding signal with the CT26-B7H3 cell line. Subsequently, after serial dilution of the mutant, the EC50 binding of its mutant with the CT26-B7H3 cell line was further examined. 50 As shown in Table 3.

[0580] Table 3. Expression levels of each h1702-DS mutant and EC50 binding to the CT26-B7H3 cell line 50

[0581]

[0582]

[0583] To further verify the binding of the mutant to B7H3 on the surface of tumor cell lines and monkey B7H3, the binding of the mutant to A498 cell lines and CHOK1-cynoB7H3 was further examined.

[0584] The results, as shown in Table 4, indicate that these mutants can bind to the A498 cell line and CHOK1-cynoB7H3.

[0585] Table 4. Binding of mutants to B7H3 on the surface of tumor cell lines and monkey B7H3

[0586]

[0587] Test Example 2: In vitro cell proliferation experiment of ADC

[0588] Test Example 2-1: Experiment on the Inhibition of Tumor Cell Line Proliferation by ADC

[0589] This experiment detects intracellular ATP levels and, based on IC50... 50 The size of the ADC disclosed herein was used to evaluate its inhibitory effect on cell proliferation.

[0590] The tumor cells to be tested included: Calu-6 cells (ATCC, Catalog #). HTB-56 TM Detroit 562 cells (ATCC, Catalog #) CCL-138 TM ), and CHO-K1 (ATCC, Catalog# CCL-61 TM ).

[0591] The ADC samples to be tested were serially diluted 3-fold with PBS or DMSO to nine concentrations (each sample starting at 500 nM). The samples were added to culture plates and incubated for 6 days (37°C, 5% CO2). Detection was performed using CellTiter-Glo reagent (Promega, G7571), and the chemiluminescence signal values ​​were read in a Victor3 display. Data were processed using GraphPad software. The measured IC50 values ​​were... 50 The values ​​are shown in Table 5.

[0592] The results showed that the ADCs corresponding to h1702-DS-107 and h1702-DS antibodies had similar inhibitory effects on the proliferation of various tumor cells when the DAR values ​​were similar, and the inhibitory effect was positively correlated with the DAR value. The larger the DAR value, the more obvious the inhibitory effect.

[0593] Table 5. Inhibitory effects of different ADCs on cell proliferation

[0594]

[0595] Test Example 2-2: The inhibitory effect of ADC on the proliferation of tumor cell lines was positively correlated with the expression level of B7H3.

[0596] To further verify whether the inhibitory effect of ADC on cell proliferation in this disclosure is positively correlated with the expression level of B7H3, B7H3 overexpression and B7H3 knockout were performed on the Detriot562 tumor cell line (Wildtype Detriot562) respectively (to obtain Detriot562). B7H3- / - Cell lines). For the Detroit562 cell line overexpressing B7H3, B7H3 expression levels were determined to be different from those of the Detriot562 cell line. B7H3中 ) and B7H3 overexpressing cell line (Detriot562) B7H3高 ),like Figure 1 As shown (the antibody used for FACS identification is from Sino Biology, catalog number: 11188-MM06-A).

[0597] The method for the cell proliferation inhibition assay is described in Test Example 2-1. The results are shown in Table 6. The ADC-1 corresponding to antibody h1702-DS-107 and the ADC-4 corresponding to h1702-DS have similar inhibitory effects on the proliferation of each Detroit562 cell line. Moreover, the inhibitory effect of the two ADCs on the proliferation of Detroit562 cells is significantly positively correlated with the expression level of B7H3. The higher the expression level of B7H3, the more obvious the proliferation inhibition effect.

[0598] Table 6. Inhibition of Detroit 562 cell line proliferation by ADC at different B7H3 expression levels

[0599]

[0600] In vivo bioactivity evaluation

[0601] Test Example 3: Evaluation of the efficacy of ADC in Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases

[0602] 1. Testing Method:

[0603] Female BALB / c-nude nude mice, 6-7 weeks old, were subcutaneously inoculated with Detroit 562 cells, a metastatic cell line from human pharyngeal carcinoma pleural effusion. On day 10 post-inoculation, animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections were administered once weekly for a total of 3 times, with two doses: 1 MPk and 3 MPk, or a single 10 MPk injection. Observation continued until day 28, with tumor volume and body weight measured 2-3 times weekly and data recorded. Tumor volume (V) was calculated using the following formula:

[0604] V = 1 / 2 × a × b²

[0605] Where a and b represent length and width, respectively.

[0606] Relative volume (RTV) = VT / V0

[0607] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0608] V0 and VT represent the tumor volume at the beginning and end of the experiment, respectively. CRTV and TRTV represent the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.

[0609] 2. Test subjects:

[0610] ADC-1;

[0611] ADC-4;

[0612] Control group (PBS).

[0613] 3. The antitumor effects of antibody ADCs are shown in Table 7 and... Figure 2 As shown:

[0614] The tumor inhibition rates of the tested ADCs were as follows: ADC-1 1 mg / kg (1 mpk) achieved a tumor inhibition rate of 50.68%; ADC-1 3 mg / kg (3 mpk) achieved a tumor inhibition rate of 78.21% (P<0.05); ADC-1 10 mg / kg (10 mpk) achieved a tumor inhibition rate of 59.7% after a single dose (P<0.05); ADC-4 1 mg / kg (1 mpk) achieved a tumor inhibition rate of 55.91%; ADC-4 3 mg / kg (3 mpk) achieved a tumor inhibition rate of 72.47% (P<0.05); and ADC-4 10 mg / kg (10 mpk) achieved a tumor inhibition rate of 86.37% after a single dose (P<0.001).

[0615] Animals in all groups maintained normal body weight during the administration process, indicating that ADC had no obvious toxic side effects.

[0616] Table 7. Efficacy of antibody administration on Detroit 562 xenografts in tumor-bearing nude mice (D28)

[0617]

[0618] vs. control group: *p<0.05; **p<0.001.

[0619] Test Example 4: T1 / 2 Evaluation of SD Rats

[0620] Four SD rats (half male and half female) were purchased from JessJet Laboratory Animal Co., Ltd. They were kept under 12 / 12-hour light / dark cycles, maintained at a constant temperature of 24±3℃ and humidity of 50-60%, and allowed free access to food and water. On the day of the experiment, the SD rats were injected intravenously with the test drug ADC at a dose of 3 mg / kg and an injection volume of 5 mL / kg.

[0621] Blood was collected at the following time points: 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days after drug administration. 300 μL of blood was collected from the fundus vein of rats each time. The collected blood samples were left at room temperature for half an hour until agglutination, and then centrifuged at 1000 × g for 15 minutes at 4°C. The supernatant was collected and immediately stored at -80°C.

[0622] The concentrations of B7H3 antibody and ADC in serum were detected by ELISA. The p-values ​​of intact ADC and total antibody (ADC-conjugated antibody and free antibody in serum) were detected separately. The detection methods were antitoxin-coated antibody or B7H3 antigen-coated antibody to detect B7H3 antibody (Anti-Human IgG (HRP) mouse-epreadsorbed, abcam, ab97175) in serum.

[0623] The results are shown in Table 8, indicating that the half-lives of ADC-1 and ADC-4 in both the total antibody and the whole ADC are very close.

[0624] Table 8. T in SD rats with B7H3 antibody ADC 1 / 2

[0625] Rat half-life (3 mpk) ADC-4 ADC-1 Total antibodies 9.19±1.69 9.47±0.30 Complete ADC 8.14±1.23 8.72±0.24

[0626] Reference Test Case: Evaluation of the efficacy of ADC on Detroit 562 human pharyngeal carcinoma pleural effusion metastatic cells in nude mice (WO2020063673, Test Case 8)

[0627] I. Experimental Objective

[0628] This experiment used BALB / c-nude nude mice as test animals to evaluate the efficacy of ADC compounds on Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

[0629] II. Test Drugs and Materials

[0630] 1. Test drug

[0631] ADC-29 (3 mg / kg);

[0632] ADC-28 (3 mg / kg);

[0633] Negative control ADC (3 mg / kg): a ligand toxin conjugate formed by conjugating a non-B7H3 target antibody with compound 20.

[0634] 2. Preparation method: All preparations are made by diluting with PBS.

[0635] 3. Experimental animals

[0636] BALB / c-nude nude mice: purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0637] III. Test Methods

[0638] The experiment used female BALB / c-nude nude mice, 6-7 weeks old, which were subcutaneously inoculated with Detroit 562 cells (ATCC, Catalog #) that were metastatic human pharyngeal carcinoma cells in pleural effusion. CCL-138 TM On the tenth day after cell inoculation, animals were randomly divided into groups (D0), with 8 animals in each group. Intraperitoneal injection of the drug was initiated once a week for a total of 3 times. Tumor volume and body weight were measured 2-3 times per week, and the data were recorded. The formula for calculating tumor volume (V) is:

[0639] V = 1 / 2 × a × b 2

[0640] Where a and b represent length and width, respectively.

[0641] Relative volume (RTV) = V T / V0

[0642] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[0643] Among them, V0, V T The figures represent the tumor volume at the beginning and end of the experiment, respectively. C RTV T RTV The figures represent the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment.

[0644] IV. Test Results

[0645] The drugs were administered intraperitoneally once a week for a total of three times. By day 28, the tumor inhibition rates of the tested ADCs were as follows: ADC-29 3 mg / kg (3 MPk) achieved a tumor inhibition rate of 72.27% (P<0.001); ADC-28 3 mg / kg (3 MPk) achieved a tumor inhibition rate of 56.2% (P<0.001). ADC-29 showed stronger antitumor efficacy than ADC-28 in both cases.

[0646] During the administration process, the weight of animals in all groups remained normal, indicating that the ADC had no obvious toxic side effects. The test results are shown in Table 9. The tested antibody effectively inhibited the growth of Detroit 562 xenografts in tumor-bearing nude mice in a dose-dependent manner.

[0647] Table 9. Efficacy of antibody administration on Detroit 562 xenografts in tumor-bearing nude mice (D28)

[0648] sequence list <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Hengrui Medicine Co., Ltd. <120> B7H3 antibody-ecetane analogue conjugate and its pharmaceutical uses <130> 721023CPCT <150> CN202010218100.7 <151> 2020-03-25 <160> 33 <170> SIPOSequenceListing 1.0 <210> 1 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702-DS heavy chain sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 2 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223>\(h1702 - DS\) light chain sequence <400> 2 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys 210 215 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑79 HV <400> 3 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑79 LV <400> 4 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <221> DOMAIN <223> h1702‑DS‑84 HV <400> 5 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 110 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <221> DOMAIN <223> h1702‑DS‑84 LV <400> 6 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑105 HV <400> 7 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702-DS-105 LV <400> 8 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN<00,01882><223> h1702‑DS‑106 HV <400> 9 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg [[ID=4,6]]1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 10 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702-DS-106 LV <400> 10 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 11 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑107 HV <400> 11 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 12 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑107 LV <400> 12 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 13 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑108 HV <400> 13 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> h1702‑DS‑108 LV <400> 14 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 15 <211> 330 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Heavy chain constant region <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser1] 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 16 <211> 1 05 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Light chain constant region <400> 16 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys 100 105 <210> 17 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702‑DS‑105 HC <400> 17 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys 210 215 <210> 19 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702-DS-106 HC <400> 19 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 20 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702‑DS‑106 LC <400> 20 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 15`0 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys 210 215 <210> 21 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702-DS-107 HC <400> `21 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 22 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702‑DS‑107 LC <400> 22 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys 210 215 <210> 23 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> h1702‑DS‑108 HC <400> 23 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 24 <211> 215 <212> PRT [[ID=2S]]<213> Artificial Sequence <220> <221> CHAIN <223> h1702-DS-108 LC <400> 24 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Gly Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys 210 215 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR1 <400> 25 Gly Phe Ile Phe Ser Ser Ser Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400> 26 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR3 <400> 27 Ala Arg Ser Ala Arg Leu Phe Ala Ser Phe Asp Tyr 1 5 10 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 <400> 28 Ser Gly Ser Val Ser Thr Ser His Tyr 1 5 <210> 29 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR2 <400> 29 Asn Thr Asn 1 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR3 <400> 30 Ala Ile His Val Asp Arg Asp Ile Trp Val 1 5 10 <210> 31 <211> 534<0​​​​​​​​​​​​​​Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln 225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln 305 310 315 320 Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330 335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val 340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu 355 360 365 Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser 370 375 380 Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln 385 390 395 400 Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415 Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425 430 Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp 435 440 445 Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro 450 455 460 Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu 465 470 475 480 Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys 485 490 495 Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly 500 505 510 Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp 515 520 525 Asp Gly Gln Glu Ile Ala 530 <210> 32 <211> 534 <212> PRT <213> Macaca fascicularis <400> 32 Met Leu His Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr 65 70 75 80 Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Leu Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Ala Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Ile Thr Ile Thr Pro Gln 225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Leu Asp Leu Leu Ala Gln 305 310 315 320 Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330 335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val 340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu 355 360 365 Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser 370 375 380 Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln 385 390 395 400 Gly Ala Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415 Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425 430 Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp 435 440 445 Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro 450 455 460 Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Val Ala Leu 465 470 475 480 Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys 485 490 495 Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly 500 505 510 Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp 515 520 525 Asp Gly Gln Glu Leu Ala 530 <210> 33 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (1)..(4) <223> Tetrapeptide linker <400> 33 Gly Gly Phe Gly<了 1

Claims

1. An anti-B7H3 antibody or its antigen-binding fragment, comprising: As shown in SEQ ID NO:3, the heavy chain variable region, and as shown in SEQ ID NO:4, the light chain variable region; As shown in SEQ ID NO: 5, the heavy chain variable region, and as shown in SEQ ID NO: 6, the light chain variable region; As shown in SEQ ID NO: 7, the heavy chain variable region; and as shown in SEQ ID NO: 8, the light chain variable region. As shown in SEQ ID NO: 9, the heavy chain variable region, and as shown in SEQ ID NO: 10, the light chain variable region; The heavy chain variable region as shown in SEQ ID NO: 11, and the light chain variable region as shown in SEQ ID NO: 12; and The heavy chain variable region as shown in SEQ ID NO: 13, and the light chain variable region as shown in SEQ ID NO:

14.

2. The anti-B7H3 antibody or its antigen-binding fragment according to claim 1, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region; The heavy chain constant region is derived from human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is derived from human antibody κ or λ chains.

3. The anti-B7H3 antibody or its antigen-binding fragment according to claim 2, wherein the heavy chain constant region is as shown in SEQ ID NO: 15 and the light chain constant region is as shown in SEQ ID NO:

16.

4. The anti-B7H3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the anti-B7H3 antibody is selected from any one of the following groups: As shown in SEQ ID NO: 17, the heavy chain, and in SEQ ID NO: 18, the light chain; As shown in SEQ ID NO: 19, the heavy chain, and in SEQ ID NO: 20, the light chain; As shown in SEQ ID NO: 21, the heavy chain, and in SEQ ID NO: 22, the light chain; and As shown in SEQ ID NO: 23, the heavy chain, and in SEQ ID NO: 24, the light chain.

5. A nucleic acid molecule encoding an anti-B7H3 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 4.

6. A host cell comprising the nucleic acid molecule as described in claim 5.

7. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is of the general formula (Pc-LYD): in: n is between 1 and 10, and n is a decimal or an integer; Pc is the anti-B7H3 antibody or its antigen-binding fragment as described in any one of claims 1 to 4; Wherein, -Y- is selected from: , , , , , and , Among them, the O end of -Y- is connected to the connector unit -L-; Where -L- stands for: 。 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein n is 1 to 8, and n is a decimal or an integer.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein n is 3 to 7, and n is a decimal or an integer.

10. The antibody-drug conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein -LY- is selected from any of the following: 、 and 。 11. The antibody-drug conjugate according to any one of claims 7 to 10, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from any one of the following: ， and ； in, Pc and n are as defined in claim 7.

12. The antibody-drug conjugate according to claim 11, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is: in: n is between 1 and 8, where n is a decimal or an integer; h1702-DS-107 is an anti-B7H3 antibody that contains a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO:

22.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12, wherein n is 3 to 7, and n is a decimal or an integer.

14. A pharmaceutical composition comprising: an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 13, or an anti-B7H3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, and one or more pharmaceutically acceptable carriers.

15. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 13, the anti-B7H3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating and / or preventing tumors and cancer. in, The tumors and cancers mentioned are selected from: head and neck cancer, brain cancer, glioma, neuroblastoma, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, leukemia, lymphoma, bone cancer, myeloma, myelodysplastic syndrome, and urothelial carcinoma.

16. The use according to claim 15, wherein The head and neck cancer mentioned is selected from: squamous cell carcinoma of the head and neck; The neuroblastoma mentioned is selected from: glioblastoma multiforme; The renal cell carcinoma was selected from: clear cell renal cell carcinoma; The skin cancer mentioned was selected from: melanoma; The bone cancers mentioned are selected from: chondrosarcoma and Ewing's sarcoma; The myeloma is selected from: multiple myeloma; The lymphomas mentioned are selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma. The lung cancers mentioned are selected from: non-small cell lung cancer and small cell lung cancer; The leukemia mentioned is selected from: lymphocytic leukemia and myeloid leukemia.

17. The use according to claim 16, wherein The lymphocytic leukemia mentioned is selected from: acute lymphoblastic leukemia and chronic lymphocytic leukemia; The myeloid leukemia mentioned is selected from: acute myeloid leukemia and chronic myeloid leukemia.

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