Antibody-drug conjugate targeting ceacam5 and use thereof

Abstract

Provided are an antibody-drug conjugate, wherein the antibody is an antibody or an antigen-binding fragment thereof binding to CEACAM5; and a pharmaceutical composition comprising the antibody-drug conjugate. Provided is use of the antibody-drug conjugate and the pharmaceutical composition comprising the antibody-drug conjugate in preventing and / or treating a disease.

Description

Antibody drug conjugates against ceacam5 and uses thereof

[0001] This international patent application claims priority to Chinese patent application No. 202410636784.0, filed May 21, 2024, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention relates to antibody drug conjugates against CEACAM5 and uses thereof, in particular in the treatment of cancer. BACKGROUND

[0003] 1. INTRODUCTION

[0004] Antibody-drug conjugates (ADCs) are a class of highly targeted anticancer therapeutics that combine the high specificity of monoclonal antibodies with the potent cytotoxicity of chemotherapeutic drugs. ADC drugs can deliver chemotherapeutic drugs directly to cancer cells by targeting tumor markers, thereby maximizing the killing effect of drugs on tumor cells while reducing damage to normal cells. ADC drugs specifically bind to antigens through monoclonal antibodies, accurately positioning toxin molecules to tumor cells. ADCs then enter tumor cells through endocytosis, transported to lysosomes by endosomes in tumor cells. Under the action of low pH and hydrolytic enzymes inside the lysosome, ADC releases toxin molecules into tumor cells, thereby achieving precise killing of tumor cells.

[0005] 2. Structure and function of CEACAM5 protein

[0006] CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5), also known as Carcinoembryonic antigen (CEA), is a membrane protein widely used for tumor marker monitoring. CEACAM5 belongs to the immunoglobulin superfamily, mainly expressed on gastrointestinal cells and on cancer cells of various cancers, such as colorectal cancer, breast cancer and lung cancer, etc. Structurally, CEACAM5 is composed of a large N-terminal ectoplasmic region, one or more immunoglobulin-like domains, a transmembrane region and a short intracellular region. The extracellular region is composed of multiple repeated immunoglobulin-like (Ig-like) domains, with abundant glycosylation sites. According to structural similarity, the extracellular immunoglobulin-like domain can be divided into N, A, B domains, and CEACAM5 has seven domains, N, A1-A3, B1-B3. This unique structure enables CEACAM5 to participate in intercellular adhesion processes and regulate various biological functions such as cell proliferation, differentiation and apoptosis. Researchers David M. Goldenberg et al. used antibodies (MN-3 / MN-14 / MN-15) against different CEACAM5 domains to evaluate the effects of antibodies on the biological functions of a series of tumor cells, including cell adhesion to extracellular matrix (ECM), cell migration, cell invasion ability, etc. It was found that binding to the N and A1B1 domains of CEACAM5 can effectively inhibit tumor cell invasion and metastasis (Rosalyn D et al., Cancer Res (2005) 65(19):8809-8817.). CEACAM5 can also bind to CD8α through the N domain, and glycosylation can affect the strength of binding to CD8α. Through binding to CD8α, CEACAM5 can activate CD8 + T cells and inhibit CD4 + T cell proliferation (G Roda et al. Mucosal Immunology, Volume 7, Issue 3, 2014, Pages 615-624).

[0007] 3. Application of CEACAM5 in tumor treatment

[0008] Due to the high expression of CEACAM5 on most tumor cells and its low expression in normal tissues, it has become a hot candidate for tumor therapy targets. Especially in the treatment of colorectal cancer, ADC drugs targeting CEACAM5 have shown great potential. Several CEACAM5-based ADCs have entered clinical trials, some of which have shown considerable efficacy and manageable safety. Researchers have developed different antibody and drug linking technologies to maximize the efficacy of ADCs. Labetuzumab (also known as hMN-14) developed by Immunomedics has carried out development attempts for colorectal cancer indications by coupling SN-38. However, due to poor efficacy (1.1% PR, 48.8% SD, mPFS 3.6 months, mOS 6.9 months), Immunomedics stopped its development in colorectal cancer indications (Cancer. JCO 35, 3338-3346 (2017).). Nevertheless, the exploration of colorectal cancer indications using the CEACAM5 target continues, and Merck announced an ADC drug (M9140) with exatecan as a toxin payload in July 2022 and carried out phase I clinical development (NCT05464030). In November 2023, Sanofi announced a collaboration with Seagen to develop an ADC drug with a TOP isomerase I inhibitor as a payload for phase I clinical development, with plans to enroll 410 patients (NCT06131840).

[0009] 4. Limitations of the Prior Art

[0010] The main factors affecting the efficacy of ADC drugs are the specific targeting of the monoclonal antibody to the antigen protein and the stable release of the toxin molecule. Although the ADC technology is extremely attractive in theory, it still faces many challenges in practical application. First, the stability of the connection between the antibody and the drug needs to be precisely controlled to ensure that the drug will not be released before reaching the tumor, thereby reducing side effects. Second, it is necessary to ensure that the ADC can effectively penetrate the tumor tissue, reach the tumor cells, and not bind to other normal tissues to avoid toxicity to normal tissues. The immunoglobulin superfamily to which CEACAM5 belongs has multiple homologous membrane proteins, among which CEACAM1 (unipro ID: P13688), CEACAM6 (unipro ID: P40199), CEACAM7 (unipro ID: Q14002) and CEACAM8 (unipro ID: P31997) have high homology with CEACAM5 (unipro ID: P06731), with sequence homology as high as 62%, 79%, 60% and 71%, respectively. Researchers Laura A Strickland et al. found that CEACAM6 is expressed in human neutrophils and granulocyte precursor cells, and ADC targeting CEACAM6 causes dose-dependent neutropenia in non-human primates (Laura A Strickland at al. J Pathol 2009; 218:380-390). Therefore, the ADC antibody targeting CEACAM5 should avoid cross-reaction with CEACAM1 / 6 / 7 / 8 and other homologous proteins to avoid normal tissue toxicity, which undoubtedly poses a challenge to antibody screening. Second, there is no optimal payload selection for existing ADC drugs. The ADC drug SAR-408701 developed by Sanofi Company with maytansinoid DM4 as payload failed to achieve the primary endpoint of PFS in the phase III clinical trial for NSQ NSCLC patients (NCT04154956). The ADC drug IMMU130 developed by Immunomedics Company with SN-38 as payload also failed to show sufficient efficacy in the phase I / II clinical trial for colorectal cancer patients. At present, although there are several ADC drugs with TOP isomerase inhibitors as payload in the development stage, they have not shown clear clinical efficacy.

[0011] 5. Object of the present application

[0012] In view of the deficiencies of the prior art, the present application aims to propose a novel ADC drug targeting CEACAM5 membrane protein. The drug will have improved connection stability, optimized drug release dynamics, and better tumor tissue specificity. Through these innovations, the ADC of the present application is expected to provide higher anti-tumor activity under the premise of ensuring safety. SUMMARY

[0013] The present application aims to propose a novel antibody drug conjugate (ADC) targeting CEACAM5 membrane protein. The antibody of the present application can specifically bind to human CEACAM5 and has high affinity, good intracellular endocytosis activity, etc., and the antibody drug conjugate prepared therefrom can effectively kill tumor cells and inhibit tumor growth in mice.

[0014] Correspondingly, in one aspect, the present application provides an antibody drug conjugate of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof: T— [L— D] y (I)

[0015] wherein,

[0016] T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds to CEACAM5;

[0017] L is a linker unit;

[0018] D is a biologically active fragment;

[0019] y is selected from an integer or decimal number from 0.1 to 20.

[0020] In another aspect, the present application provides a pharmaceutical composition comprising the antibody drug conjugate of the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0021] In another aspect, the present application provides a method for preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject the antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof.

[0022] In another aspect, the antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof, for use in preventing and / or treating a disease.

[0023] In another aspect, the present application provides the use of an antibody drug conjugate of the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof, in the manufacture of a medicament for the prevention and / or treatment of a disease.

[0024] In a preferred embodiment, the disease is a cancer, for example a cancer associated with CEACAM5 expression;

[0025] Preferably, the disease is selected from the group consisting of gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, thyroid cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial carcinoma;

[0026] More preferably, the disease is selected from the group consisting of pancreatic cancer, colorectal cancer, gastric cancer, lung cancer and breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1. Affinity evaluation of anti-CEACAM5 humanized antibodies.

[0028] Figure 2. Endocytosis activity experiment evaluation of anti-CEACAM5 humanized antibodies DT3C.

[0029] Figure 3. Binding activity evaluation of anti-CEACAM5 humanized antibodies to CEACAM5-expressing tumor cells.

[0030] Figure 4. Immunogenicity evaluation of anti-CEACAM5 humanized antibodies to human PBMC cells.

[0031] Figure 5. PK parameter evaluation of anti-CEACAM5 humanized antibodies in FcRn humanized mouse model.

[0032] Figure 6. IC50 of anti-CEACAM5 humanized antibody ADCs (ADC-2, ADC-3, ADC-5 and ADC-17) for killing activity to CEACAM5-expressing tumor cells.

[0033] Figure 7. IC50 of anti-CEACAM5 humanized antibody ADCs (ADC-6, ADC-7, ADC-9, ADC-10 to ADC-13, ADC-16 and ADC-17) for killing activity to CEACAM5-expressing tumor cells.

[0034] Figure 8. Evaluation of bystander cytotoxicity of anti-CEACAM5 humanized antibody ADC: Figure 8A, evaluation of bystander effect of antibody conjugated with MMAE toxin; Figure 8B, evaluation of bystander effect of antibody conjugated with TOP I inhibitor toxin.

[0035] Figure 9. In vivo efficacy evaluation of anti-CEACAM5 humanized antibody ADC on pancreatic cancer organoid PDOX model.

[0036] Figure 10. Body weight change of anti-CEACAM5 humanized antibody ADC on pancreatic cancer organoid PDOX model mice.

[0037] Figure 11. In vivo efficacy evaluation of anti-CEACAM5 humanized antibody ADC on gastric cancer CDX model.

[0038] Figure 12. Body weight change of anti-CEACAM5 humanized antibody ADC on gastric cancer CDX model mice.

[0039] Figure 13. In vivo efficacy evaluation of anti-CEACAM5 humanized antibody ADC on non-small cell lung cancer PDX model mice.

[0040] Figure 14. Body weight change of anti-CEACAM5 humanized antibody ADC on non-small cell lung cancer PDX model mice. DETAILED DESCRIPTION

[0041] The above features and advantages of the present application, and additional features and advantages thereof, will be more clearly understood from consideration of the following detailed description when taken in conjunction with the accompanying drawings.

[0042] The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and serve the purpose of general understanding of the present application. The embodiments should not be construed as limiting the scope of the present application. Identical or similar elements and elements having identical or similar functions are denoted by the same reference numerals throughout the specification.

[0043] In the present application, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by a person skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided below.

[0044] Definitions

[0045] Chemical Definitions

[0046] The definitions of specific functional groups and chemical terms are described in more detail below.

[0047] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.

[0048] "C 1-10 alkyl" refers to a straight chain or branched chain saturated hydrocarbon group having from 1 to 10 carbon atoms. In some embodiments, C 1-8 alkyl, C 1-6 alkyl, and C 1-4 alkyl are preferred. Examples of C 1-6 alkyl include: methyl (Ci), ethyl (C2), n-propyl (C3), i-propyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), i-butyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). The term "C 1-6 alkyl" also includes heteroalkyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkyl group can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0049] "C 2-10 alkenyl" refers to a straight chain or branched chain hydrocarbon group having from 2 to 10 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-6 alkenyl is preferred. C 2-6Examples of alkenyl groups include ethenyl (C2), 1- propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 Alkenyl also includes heteroalkenyl groups wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0050] "C 2-10 Alkynyl refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-6 Alkynyl is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 Alkynyl also includes heteroalkynyl groups wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkynyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0051] "Halo" or "halogen" refers to fluoro (F), chloro (CI), bromo (Br), and iodo (I).

[0052] "C 1-6 Haloalkyl refers to "C 1-6 Alkyl" groups substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, more preferred C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro- 1,1-dimethyl-ethyl, and the like. A haloalkyl group can be substituted at any available attachment point, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0053] "C 1-6 Alkoxy refers to the group -OR, where R is C 1-6 Alkyl, as defined above. C 1-4 Alkoxy is preferred.

[0054] "C 1-10 "Alkylene" refers to a divalent group formed by removing a C 1-10 from an alkyl group, and can be substituted or unsubstituted. In some embodiments, C 1-8 alkylene, C 1-6 alkylene, C 1-4 alkylene, and C 1-2 alkylene are preferred. Unsubstituted alkylene groups include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, e.g., alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- 2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0055] "C 2-10 "Alokenylene" refers to a divalent group formed by removing a C 2-10 from an alkenyl group, and can be substituted or unsubstituted. In some embodiments, C 2-8 alkenylene, C 4-6 alkenylene, C 1-4 alkenylene, C 2-4 alkenylene, and C 2-10 alkenylene are preferred.

[0056] "C 2-10 "Alynylene" refers to a divalent group formed by removing a C 2-8 from an alkynyl group, and can be substituted or unsubstituted. In some embodiments, C 4-6 alkynylene, C 1-4 alkynylene, C 2-4 alkynylene, and C 3-10 alkynylene are preferred.

[0057] "C "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C5-7 cycloalkyl, C 3-7 cycloalkyl and C 3-5 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the connecting point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may optionally be substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0058] "3-10 membered heterocyclyl" refers to a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 4-9 membered heterocyclyl groups are preferred, which are 4- to 9-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 5-8 membered heterocyclyl groups are preferred, which are 5- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl groups are preferred, which are 3- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; 3-7 membered heterocyclyl groups are preferred, which are 3- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 2 ring heteroatoms; 4-7 membered heterocyclyl groups are preferred, which are 4- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 4-6 membered heterocyclyl groups are preferred, which are 4- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; and 5-6 membered heterocyclyl groups are preferred, which are 5- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the cycloalkyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, dioxanyl.Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, hexahydrotriazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0059] "C 6-10 Aryl" means a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p-electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. 10 Aryl" means a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p-electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0060] "5-10-membered heteroaryl" refers to a 4n+2 aromatic ring system of a 5-10-membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms (e.g., having 6, 10, or 14 shared π electrons arranged in a ring), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-9-membered heteroaryl is preferred, which is a 4n+2 aromatic ring system of a 5-9-membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 4n+2 aromatic ring systems of 5-6 membered monocyclic or bicyclic rings having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirmonoheptatrienyl, oxadiazinyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophene, isobenzothiophene, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxolinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0061] “C 3-10 "Hypercyclic alkyl", "3-10 membered heterocyclic", "C" 6-10 "Alphane" and "5-10 methyl alphane" refer to compounds that have had "C" removed from their names.3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" are as previously described. 3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" are as previously described.

[0062] "Carbonyl", whether used alone or as part of a larger moiety, e.g., amino carbonyl, refers to -C(O)-.

[0063] "Oxy" refers to =O.

[0064] "Thio" refers to =S.

[0065] Alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups defined herein are optionally substituted.

[0066] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa, -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa)2, -BR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0067] or two geminal hydrogens on a carbon atom are replaced with a group =0, =S, =NN(R bb )2, =NNR bb C(=0)R aa , =NNR bb C(=0)OR aa , =NNR bb S(=0)2R aa , =NR bb , or =NOR cc ;

[0068] each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0069] each R bb is independently selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=0)R aa , -C(=0)N(R cc )2, -C02R aa , -S02R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -S02N(R cc )2, -S02R cc , -S02OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=0)SR cc , -C(=S)SR cc , -P(=0)2R aa , -P(=0)(R aa)2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0070] R cc each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0071] R dd each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(Rff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0072] Each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0073] Each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0074] Each R gg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1-6 alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocyclyl, C5-C 10 heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0075] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SRcc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups attached to a nitrogen atom are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd , R aa , R bb , R cc , and R dd are as described above.

[0076] Other Definitions

[0077] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques employed herein are those that are within the purview of the relevant art and are consistent with the practices of the relevant art. Also, to better understand the present application, the following definitions and explanations of the relevant terms are provided.

[0078] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and so forth.

[0079] The terms "comprising", "comprise" and "comprised of", as well as "including", "include" and "included of", "having", "has", "have" or variants thereof, are not

[0080] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0081] The heavy chain of an immunoglobulin can be divided into three functional regions: an Fd region, a hinge region, and an Fc region (fragment crystallizable). The Fd region comprises the VH and CH1 domains and, together with the light chain, forms the Fab (antigen binding fragment). The Fc fragment is responsible for the effector functions of the immunoglobulin, including, for example, complement binding and binding to the cognate Fc receptors of effector cells. The hinge region, found in the IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. The hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0082] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions based on structure and function: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes the amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the fragment flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexible allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0083] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is comprised of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that are primarily responsible for specific antigenic epitope binding. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL domain and the VH domain each comprise, from amino-terminus to carboxyl-terminus, the following framework regions (FRs) and CDRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0084] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure can use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined according to the Kabat definition.

[0085] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to either lambda (l) chains or kappa (K) chains.

[0086] As used herein, the term "antibody" shall be construed in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bispecific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, and should not be construed as requiring production by any particular method.

[0088] The term "bispecific antibody" is understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be construed in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0089] The term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0090] The term "humanized antibody" generally refers to an antibody in which portions of the amino acid sequence outside of the CDR regions of a non-human antibody (e.g., murine antibody) are replaced with corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids can also be allowed, as long as they still retain the ability of the antibody to bind to a particular antigen. A humanized antibody can optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody that includes sequences derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced by CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate that have the desired properties, affinities and / or capabilities. In some cases, FR region residues of a human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, a humanized antibody can comprise amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0091] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0092] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment consisting of the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); (ix) a Nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, the term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) with dual variable domain (D3) antibodies, which form a pair of antigen binding regions with a complementary light chain polypeptide, as well as modifications of any of the foregoing structures.

[0093] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments screened in the same manner as for whole antibodies.

[0094] As used herein, the term "binds" or "binds specifically to" refers to a nonrandom, binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0095] Avidity is a measure of the strength of binding between an antibody and the relevant antigen. Avidity involves both the affinity between the antigenic determinant and the antigen binding site of an antibody and the number of relevant binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10-8 M to 10 -12 M, and / or has a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D represents non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as different variations thereof known in the art.

[0096] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein or proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually comprise at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are thus the specific targets of an antibody or antigen-binding fragment thereof.

[0097] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0098] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions of an amino acid residue for another amino acid residue having a similar chemical structure that has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0099] Such conservative substitutions are preferably substitutions of one amino acid residue for another in groups (a) to (e) below: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0100] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0101] As used herein, unless otherwise noted, a bivalent structure can be attached to the remainder of the compound in either direction, left to right or right to left. In one embodiment, the bivalent structure is preferably attached to the remainder of the compound in the left to right orientation.

[0102] The term "amino acid residue" means to include any natural or synthetic amino acid residue, not limited to the amino acid residues in the group consisting of the 20 naturally occurring amino acids, wherein the residue refers to the moiety remaining after the loss of water from an amino acid linked by a peptide bond. The 20 naturally occurring amino acid residues are selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

[0103] The term "antibody conjugate" generally refers to an antibody linked to another chemical moiety. The chemical moiety can be a cytotoxic drug, an immunostimulatory molecule, and a detectable label. The drug can be, for example, a microtubulin inhibitor, an antibiotic, a DNA synthesis inhibitor, a topoisomerase inhibitor, an RNA polymerase II inhibitor, and an RNA spliceosome inhibitor. The terms "antibody-drug conjugate," "antibody conjugate," and "ADC" can be used interchangeably.

[0104] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof, and / or such carrier or excipient is approved or can be used in a pharmaceutical composition intended for parenteral administration to humans.

[0105] As used herein, the terms "treat," "treatment," "therapy," and the like, refer to the application of a pharmaceutical agent or performance of a procedure for the purpose of effecting an outcome. The outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptoms thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptoms of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as reduction of symptoms; alleviation of disease symptoms or making the disease condition more tolerable to the patient; slowing in rate of disease progression or degeneration; or improving quality of life. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including physician-based measures. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0106] As used herein, the term "effective amount" refers to an amount of a pharmaceutical agent administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0107] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment, or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0108] "stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0109] "chiral" is a molecule which does not superimpose on its mirror image; while "achiral" refers to a molecule which is superimposable on its mirror image.

[0110] "enantiomers" refer to two isomers of a compound which are nonsuperimposable mirror images of one another.

[0111] "diastereomers" refer to two or more stereoisomers of a compound which are not mirror images of one another and which have different physical properties. Mixtures of diastereomers can be separated by high resolution analytical techniques such as electrophoresis and chromatography, e.g., HPLC.

[0112] The stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0113] Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound; (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. One specific stereoisomer is the enantiomeric form, and mixtures of such isomers are called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, and such a mixture results when the reactions or processes involved in the manufacture of the compound are stereorandom.

[0114] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present application can exist in the form of a racemic or enantiomeric enrichment, e.g., (R)-, (S)-, or (R,S)-configurational forms. In certain embodiments, each asymmetric atom has at least a 50% enantiomeric excess in the (R)- or (S)- configuration, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess, or at least a 99% enantiomeric excess.

[0115] Depending on the choice of starting materials and methods, the compounds of the present application can be present in the form of one or more of possible isomers, such as racemates and mixtures of diastereomers, depending on the number of asymmetric carbon atoms (see, e.g., WO 91 / 06283). The optically active forms of (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compounds contain a double bond, the substituents can be in the E or the Z configuration; if the compounds contain a disubstituted cycloalkyl, the substituents on the cycloalkyl can be in the cis- or trans-configuration.

[0116] Any mixture of stereoisomers can be separated into their individual isomers by conventional techniques, such as HPLC or fractional crystallization, and any enantiomers can be converted into each other following conventional procedures.

[0117] Any resulting end products or intermediates of racemates can be resolved into the optical antipodes by methods well known to those skilled in the art, such as, for example, by separation of the resulting diastereomeric salts thereof. The racemic products can also be separated by chiral chromatography, such as, for example, high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, the enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0118] The present application includes tautomers, which are isomers of functional groups resulting from the movement of a certain atom in a molecule between two positions. Compounds exist in different tautomeric forms, one said compound is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

[0119] The compounds of the present application can include one or more asymmetric centers and can thus occur as various stereoisomeric forms, such as enantiomeric and / or diastereomeric forms. For example, the compounds of the present application can be individual enantiomers, diastereomers or geometric isomers (such as cis- and trans-isomers), or can be mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be separated by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomer can be prepared by asymmetric synthesis.

[0120] Those skilled in the art will appreciate that organic compounds can exist in solvated forms, in which the compound is complexed or coordinated with a solvent, usually water, but also including less polar solvents such as methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. These solvated forms are equivalent to unsolvated forms. The term'solvate' is used throughout the application to describe a molecular complex of a compound of the application with one or more solvent molecules. Non-limiting examples of solvents that can be used to form solvates include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. The term 'hydrate' is used when the solvent is water. The compounds of the application can be prepared as solvates and can be isolated as solvates. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated into the crystal lattice of the solid state of the compound. 'Solvate' includes both solution-phase solvates and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0121] The term'solvate' is used throughout the application to describe a molecular complex of a compound of the application with one or more solvent molecules. Non-limiting examples of solvents that can be used to form solvates include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. The term 'hydrate' is used when the solvent is water. The compounds of the application can be prepared as solvates and can be isolated as solvates. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated into the crystal lattice of the solid state of the compound. 'Solvate' includes both solution-phase solvates and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0122] The term 'hydrate' is used when the solvent is water. In general, the ratio of water molecules to compound molecules in a hydrate of a compound is determined. Thus, a hydrate of a compound can be represented, for example, by the general formula R x H2O, where R is the compound and x is a number greater than zero. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than zero and less than 1, for example, a hemihydrate (R 0.5 H2O)), and polyhydrates (x is a number greater than 1, for example, dihydrates (R 2 H2O) and hexahydrates (R 6 H2O)).

[0123] The compounds of the present application can be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the present application can exist in one or more crystalline forms. Accordingly, the present application includes within its scope all amorphous or crystalline forms of the compounds of the present application. The term "polymorph" refers to crystalline forms of a compound (or salts, hydrates or solvates thereof) that have the same elemental composition but different crystal packing arrangements. All polymorphs of a compound have the same elemental makeup. Different polymorphs of a compound often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. The recrystallization solvent, the rate of crystallization, storage temperature, and other factors can cause one crystalline form to dominate over another. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0124] The present application also includes isotopically-labelled compounds (isotopic variants) which are identical to those recited in Formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be suitably substituted into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, and chlorine, for example 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl. The present application also embraces compounds of the present application that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present application. Certain isotopically-labelled compounds of the present application, for example those into which radioactive isotopes such as 3 H and 14 C) are incorporated can be useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Isotopically labelled compounds of Formula (I) of the present application and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and / or in the examples and

[0125] As described herein, the compounds of the present application can optionally be substituted with one or more substituents, such as described herein for the compounds of the Formulae above, or as described in particular examples herein, and as described in the classes of compounds encompassed by the present application.

[0126] "Pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio.

[0127] As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present application. Pharmaceutically acceptable salts are well known in the art, for examples, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts of the compounds of this application include those derived from pharmaceutically acceptable inorganic or organic acids and bases. Salts include, but are not limited to, those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, nitric, and organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, malonic, and other acids well known in the art. Further pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, sulfocyanate, tosylate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-C4alkyl)4salts. Also contemplated are quaternized salts of any cationic nitrogen-containing groups present in the compounds of the present application. Water or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed by the addition of inorganic or organic acids to a free amine group of the present compounds. Examples of such salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, nitrate, acetate, lactate, citrate, succinate, malate, maleate, fumarate, tartrate, mandelate, methanesulfonate, benzoate, salicylate, naphthoate, benzonate, pamoate, pantothenate, bitartrate, ascorbate, dodecylsulfate, glycerophosphate, gluconate, glutamate, glycolate, and the like.

[0128] Certain embodiments of the application are now described in greater detail by reference to the following examples and the accompanying structural and chemical formulas. The application intends to encompass all alternatives, modifications and equivalents as can be included within the scope of the application as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many methods and materials similar or equivalent to those described herein. The application is not intended to be limited to the methods and materials described herein. In the event that one or more of the incorporated references contradicts the disclosure contained herein, including but not limited to defined terms, term application, described techniques, etc., the disclosure herein controls.

[0129] It should be further recognized that certain of the described features of the application can be interchanged, or combined, with respect to one another, and / or with features of other embodiments, without departing from the scope of the application. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any appropriate subcombination.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The application resides in the field of pharmaceuticals.

[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The application resides in the field of pharmaceuticals.

[0132] Anti-CEACAM5 antibodies

[0133] The present disclosure provides an antibody or antigen-binding fragment thereof that binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0134] In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively.

[0135] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively.

[0136] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively.

[0137] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively.

[0138] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 38, 48, 50, 52, 54, 56, 58, 61, 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 43, 65, 67, 69, 71, 73, 75.

[0139] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.

[0140] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0141] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0142] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0143] In some embodiments, the VH comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5. In some embodiments, the VL comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5.

[0144] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0145] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0146] In some embodiments, the insertion, deletion, and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3, and / or FR4.

[0147] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within a group (a) to (e) below: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0148] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to lie; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie, or to Leu.

[0149] In preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0150] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 69.

[0151] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73.

[0152] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0153] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0154] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33.

[0155] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody. In preferred embodiments, the antibody is a humanized antibody.

[0156] Based on the amino acid sequences of the heavy chain constant region, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chain, the light chain of an antibody can be assigned to either a lambda (l) chain or a kappa (K) chain. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0157] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl antibody.

[0158] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; or (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0159] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 79 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 77; or (ii) the light chain comprises the amino acid sequence of SEQ ID NO: 83 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 81.

[0160] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to IL-13. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen binding activity.

[0161] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0162] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.

[0163] A number of tumor-associated antigens associated with particular cancers have been identified in the art. In some embodiments, a tumor-associated antigen is an antigen that can elicit a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. Numerous other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0164] Examples of tumor-associated antigens include, but are not limited to, 5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0165] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen binding region binds to any of the gamma, delta, epsilon, zeta, and eta chains of CD3.

[0166] An antibody disclosed herein can comprise an Fc region. The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgGl Fc.

[0167] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, FcyRI, FcyRII, or FcyRIIIA binding. For example, the Fc region can be of the IgGl isotype, or of a non-IgGl type, e.g., IgG2, IgG3, or IgG4, which has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall’Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, the Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred embodiment the Fc region comprises L234A and L235A (LA mutations).

[0168] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation receptor site or is otherwise manipulated to alter the glycosylation properties. For example, in an IgGl Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Thus, in a particular embodiment, the Fc region comprises an IgGl sequence with the N297Q mutation.

[0169] In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, e.g., by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, e.g., as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the method described in Shields et al. (1999) Nature Biotech 17:176 can be used to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, e.g., as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0170] ​In other embodiments, the Fc region has an extended serum half-life. For examples of altering (e.g., decreasing or increasing) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745, all of which are incorporated herein by reference in their entireties. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to decrease the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to increase the in vivo half-life of the antibody. In a particular embodiment, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and / or the third constant (CH3) domain (residues 341-447 of human IgGl) (numbered according to the EU numbering system). In a particular embodiment, the constant region of an antibody IgGl described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254; and a substitution of threonine (T) to glutamic acid (E) at position 256 (numbered according to the EU numbering system). See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. Such mutant IgGs, termed “YTE mutants,” have been demonstrated to exhibit a four-fold increase in half-life compared to the wild-type version of the same antibody (see Dall Acqua WF et al. (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the Fc region comprises M252Y, S254T, and T256E (YTE mutations).

[0171] Antibody drug conjugates (ADCs)

[0172] In another aspect, the present application provides an antibody drug conjugate comprising an antibody or antigen binding fragment thereof disclosed herein and a chemical moiety conjugated to the antibody or antigen binding fragment thereof.

[0173] In the context of the present disclosure, an "antibody drug conjugate," "antibody conjugate," or "antibody conjugate" is an antibody or antibody fragment (such as an antigen binding fragment) covalently linked to a chemical moiety. The chemical moiety can be selected from the group consisting of a cytotoxic drug, an immunostimulatory molecule, and a detectable label, e.g., a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. When the antibody conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody drug conjugate" or "ADC."

[0174] The term "conjugate," "conjugated," or "linked" can refer to making two polypeptides into one continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, an antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, where a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0175] Any number of ways known to those skilled in the art can be used to link a chemical moiety to an antibody of the present invention. In this context, the number of chemical moieties linked on a single antibody of the present invention can be represented by "DAR." For example, in some embodiments, "DAR" represents the number of linker-payloads linked on a single antibody of the present invention.

[0176] Covalent and non-covalent modes of attachment can be used. The procedure for attaching a chemical moiety to an antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with a suitable functional group on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of a number of known linker molecules. A linker can be any molecule used to link an antibody to a chemical moiety. The linker is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0177] In certain cases, it is desirable to release the chemical moiety from the antibody when the antibody conjugate reaches its target site. Thus, in these cases, the antibody conjugate will comprise a cleavable linkage near the target site.

[0178] The conditions experienced by the enzymatic activity or antibody conjugate within the target cell or near the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0179] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, the skilled artisan will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0180] In one embodiment, the present application provides an antibody drug conjugate of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof: y (I)

[0181] wherein,

[0182] T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CEACAM5;

[0183] L is a linker unit;

[0184] D is a biologically active fragment;

[0185] y is selected from an integer or decimal number from 0.1 to 20.

[0186] T

[0187] In one embodiment, T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds CEACAM5.

[0188] In one embodiment, the antibody or antigen-binding fragment thereof that binds CEACAM5 is as defined herein.

[0189] In a particular embodiment, T is mAb1; in another particular embodiment, T is mAb1-H6K3; in another particular embodiment, T is mAb1-H6K5; in another particular embodiment, T is mAb2; in another particular embodiment, T is mAb3; in another particular embodiment, T is mAb4.

[0190] L

[0191] In a particular embodiment, L comprises a cleavable linker or a non-cleavable linker.

[0192] In a particular embodiment, L comprises a cleavable linker comprising an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photo-labile linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

[0193] In a particular embodiment, the L comprises or is derived from a fragment of mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxycarbonyl (PABC), SPDB (N-succinimidyl-4-(2-pyridyldithio)-butyrate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate), beta-glucuronide, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butyric acid), dimethyl hydrazide (3-methyl-) 3-mercaptobutane hydrazide), AcBut-dimethyl hydrazide, or SMCC (N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-carboxylate).

[0194] In a particular embodiment, L is -L1-L2-L3-.

[0195] L is selected from the following structures: (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-val-cit-PABC), (SPDB), (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM),

[0196] L1

[0197] In a particular embodiment, L1 is -L 1a -L 1b -L 1c -L 1d -L 1e -.

[0198] In a particular embodiment, L 1a is In another specific implementation scheme, L 1a for In another specific implementation scheme, L 1a for In another specific implementation scheme, L 1a for

[0199] In a more specific implementation scheme, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody; in another, more specific embodiment, L 1a for L represents 1a The binding site with the antibody.

[0200] In one specific implementation plan, L 1b C 1-10 Alkylene; in another embodiment, L 1b C 1-6 Alkylene; in another embodiment, L 1b C 2-10 alkenyl; in another embodiment, L 1b C 2-6 alkenyl; in another embodiment, L 1b C 2-10 etymynyl; in another embodiment, L 1b C 2-6 alkenyl; in another embodiment, L 1b C 2-6 etyne group; in another embodiment, the aforementioned C 1-10 Alkylene, C 1-6 Alkylene, C 2-10 imidene group, C 2-6 imidene group, C 2-10 etyne and C 2-6 Each CH2 group in the ethynyl group is not substituted; in another embodiment, the aforementioned C 1-10 Alkylene, C 2-10 imide and C 2-10 Each CH2 group in the ethynyl group is optionally surrounded by one, two, or three R groups. x replace.

[0201] In a particular embodiment, each R x and R x are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, two R x on any identical or different carbon atom can be joined to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene, preferably a C 3-7 cycloalkylene or 3-7 membered heterocyclylene, more preferably a C 3-5 cycloalkylene, such as cyclopropylene.

[0202] In a particular embodiment, L 1c is a chemical bond; in another embodiment, L 1c is -C(O)-; in another embodiment, L 1c is -C(O)NH-; in another embodiment, L 1c is -NHC(O)-.

[0203] In a particular embodiment, L 1d is a chemical bond; in another embodiment, L 1d is C 1-8 alkylene; in another embodiment, L 1d is C 1-6 alkylene; in another embodiment, L 1d is -(CH2CH2O) n -C 1-4 alkylene-; in another embodiment, L 1d is C 1-8 alkylene or C 1-6 alkylene, said C 1-8 alkylene and C 1-6 alkylene being optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl; in another embodiment, L 1d is C 1-8 alkylene or C 1-6 alkylene, said C 1-8 alkylene and C 1-6 alkylene being optionally substituted with -C(O)NH-(CH2CH2O) w -C 1-4 alkyl.

[0204] In a particular embodiment, n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0205] In a particular embodiment, w is selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0206] In a particular embodiment, L 1e is -C(O)-; in another embodiment, L 1e is -NHC(O)-; in another embodiment, L 1e is -C(O)-NHC(O)-.

[0207] L2

[0208] In one embodiment, L2is a bond; in another embodiment, L2is selected from a divalent peptidyl group comprising 2 to 8 optionally substituted natural amino acid residues or non-natural amino acid residues.

[0209] In a particular embodiment, L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues; in another particular embodiment, L2is selected from gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-β-ala.

[0210] In a particular embodiment, L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, the amino acids being selected from glycine, phenylalanine, alanine, valine, citrulline, or lysine.

[0211] In a more particular embodiment, L2is -gly-gly-phe-gly-; in another more particular embodiment, L2is -val-cit-; in another more particular embodiment, L2is -val-lys-gly-.

[0212] In one embodiment, each amino acid residue in L2is unsubstituted; in another embodiment, each amino acid residue in L2is optionally substituted with 1, 2, 3, 4, or 5 R y substituents.

[0213] In one embodiment, each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy.

[0214] L3

[0215] In one embodiment, L3 is an optional spacer, which can or can not be substituted; in one embodiment, L3 is -NH-CH2-(AM); in one embodiment, L3 is (PABC); in one embodiment, L3 is (PAB).

[0216] In one embodiment, L3 is not substituted; in another embodiment, L3 is optionally substituted with 1, 2, or 3 R z .

[0217] In one embodiment, each R z is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy.

[0218] D

[0219] In one embodiment, D is selected from the group consisting of: metal complexes; antibiotics; DNA topoisomerase inhibitors; microtubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active agents that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis.

[0220] In one embodiment, D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors, and RNA splicesome inhibitors.

[0221] In one embodiment, D is selected from a tubulin inhibitor and a microtubule polymerization inhibitor, for example an auristatin, a maytansinoid, a tubulysin, a cryptophycin, or a rhizoxin.

[0222] In one embodiment, D is an antibiotic, for example a calicheamicin, an anthracycline, and an anthracycline antibiotic;

[0223] In one embodiment, D is selected from a DNA synthesis inhibitor, for example a duocarmycin, a PBD (pyrroloridine), or an IGN (indolinobenzodiazepine).

[0224] In one embodiment, D is selected from a DNA topoisomerase I inhibitor, for example a camptothecin or a camptothecin derivative.

[0225] In one embodiment, D is selected from an RNA polymerase II inhibitor, for example an alpha-amanitin.

[0226] In one embodiment, D is selected from an RNA splicesome inhibitor drug, for example a splicetastatin and a telatinastatin;

[0227] In one embodiment, D is selected from a camptothecin or a camptothecin derivative, for example a hydroxyl camptothecin, 9-amino camptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan, or other derivatives.

[0228] In one embodiment, D is selected from an auristatin drug, for example MMAE or MMAF.

[0229] In one embodiment, D is selected from a maytansinoid drug, for example DM1, DM2, DM3, or DM4.

[0230] In one embodiment, D is selected from a compound of Formula (D-I), Formula (D-II), Formula (D-III), or Formula (D-IV), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof:

[0231] In one embodiment, each of the compounds of Formula (D-I), Formula (D-II), Formula (D-III), and Formula (D-IV) is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from:

[0232] deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl.

[0233] In one embodiment, D is (MMAE); in another embodiment, D is (DM4); in another embodiment, D is (Dxd); in another embodiment, D is In another embodiment, D is In another embodiment, D is In another embodiment, D is In another embodiment, D is

[0234] *1, *2 and *3 and q

[0235] In one embodiment, *1 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof.

[0236] In one embodiment, *2 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof.

[0237] In one embodiment, *3 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in a preferred embodiment, *3 is in the (S) absolute configuration.

[0238] In one embodiment, q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3;

[0239] L D1 , L D2 , L D3 and L D4

[0240] In one embodiment, L D1 is a chemical bond; in another embodiment, L D1 is -NH-; in another embodiment, L D1 is -O-; in another embodiment, L D1 is -C(O)-; in another embodiment, L D1 is -NHC(O)-; in another embodiment, L D1 is -C(O)NH-.

[0241] In one embodiment, L D2 is -NH-; in another embodiment, LD2 is -O-; in another embodiment, L D2 is -C(O)-; in another embodiment, L D2 is -NHC(O)-; in another embodiment, L D2 is -C(O)NH-.

[0242] In one embodiment, L D3 is a bond; in another embodiment, L D3 is C 1-10 alkylene, for example C 1-6 alkylene; in another embodiment, L D3 is C 1-10 haloalkylene, for example C 1-6 haloalkylene; in another embodiment, L D3 is C 2-10 alkenylene, for example C 2-6 alkenylene; in another embodiment, L D3 is C 2-10 alkynylene, for example C 2-6 alkynylene.

[0243] In one embodiment, L D3 is unsubstituted; in another embodiment, L D3 is optionally substituted with 1, 2, or 3 R D3 .

[0244] In one embodiment, L D4 is -NH-; in another embodiment, L D4 is -O-; in another embodiment, L D4 is -C(O)-; in another embodiment, L D4 is -NHC(O)-; in another embodiment, L D4 is -C(O)NH-.

[0245] R D1 , R D2 , R D3 , R D4 , R D5 , and R D6

[0246] In one embodiment, R D1 is H; in another embodiment, R D1 is halogen; in another embodiment, R D1 is C 1-6 alkyl, for example Me; in another embodiment, R D1 is C 1-6Halogenated alkyl; in another embodiment, R D1 C 1-6 Alkyl group.

[0247] In one implementation, R D2 For H; in another implementation, R D2 For example, halogen, such as F; in another embodiment, R D2 C 1-6 Alkyl; in another embodiment, R D2 C 1-6 Halogenated alkyl; in another embodiment, R D2 C 1-6 Alkyl group.

[0248] In one implementation, R D1 R D2 Together with the carbon atoms they are attached to, they form C 3-7 cycloalkyl or 3-7 membered heterocyclic group; in another embodiment, R D1 R D2 Together with the carbon atoms they are attached to, they form 5-6 membered heterocyclic groups.

[0249] In one implementation, R D6 For H; in another implementation, R D6 For halogen; in another embodiment, R D6 C 1-6 Alkyl; in another embodiment, R D6 C 1-6 Halogenated alkyl; in another embodiment, R D6 C 1-6 Alkyl group.

[0250] In one implementation, R D1 R D6 Together with the carbon atoms they are attached to, they form C 3-7 cycloalkyl or 3-7 membered heterocyclic group; in another embodiment, R D1 R D6 Together with the carbon atoms they are attached to, they form C 5-6 Cycloalkyl.

[0251] In one implementation, R D3 For H; in another implementation, R D3 For halogen; in another embodiment, R D3 C 1-6 Alkyl; in another embodiment, R D3 C 1-6 Halogenated alkyl; in another embodiment, R D3 C1-6 alkyl; in another embodiment, R D3 is C 1-6 haloalkyl; in another embodiment, R D3 is C 3-7 cycloalkyl; in another embodiment, R D3 is 3-7 membered heterocyclyl.

[0252] in one embodiment, R D4 is H; in another embodiment, R D4 is halogen; in another embodiment, R D4 is C 1-6 alkyl; in another embodiment, R D4 is C 1-6 haloalkyl; in another embodiment, R D4 is C 1-6 alkoxy; in another embodiment, R D4 is C 1-6 haloalkoxy; in another embodiment, R D4 is C 3-7 cycloalkyl; in another embodiment, R D4 is 3-7 membered heterocyclyl.

[0253] in one embodiment, R D3 and R D4 are not simultaneously H.

[0254] in one embodiment, R D3 , R D4 and the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl; in another embodiment, R D3 , R D4 and the carbon atom to which they are attached form C 3-5 cycloalkyl.

[0255] in one embodiment, R D5 is halogen; in another embodiment, R D5 is C 1-6 alkyl; in another embodiment, R D5 is C 1-6 haloalkyl; in another embodiment, R D5 is C 3-10 cycloalkyl; in another embodiment, R D5 is 3-10 membered heterocyclyl; in another embodiment, R D5 is C 6-10 aryl; in another embodiment, R D5R is -C5-10aryl; in another embodiment, R D5 is -C 1-6 alkylene-C 3-10 cycloalkyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; in another embodiment, R D5 is -C 1-6 alkylene-3-10 membered heterocyclyl, preferably -C 1-4 alkylene-3-5 membered heterocyclyl; in another embodiment, R D5 is -C 1-6 alkylene-C 6-10 aryl; in another embodiment, R D5 is -C 1-6 alkylene-5-10 membered heteroaryl.

[0256] y

[0257] In one embodiment, y is selected from an integer or decimal number from about 0.1 to about 20; in another embodiment, y is selected from an integer or decimal number from about 1 to about 10; in another embodiment, y is selected from an integer or decimal number from about 2 to about 8.

[0258] In one specific embodiment, y is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; in another specific embodiment, y is about 5.5, 6.5, 7.5, 8.5, 9.5; in another specific embodiment, y is about 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, or 6.0; in another specific embodiment, y is about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0259] Any of the above specific embodiments, or any combination thereof, can be combined with any of the other specific embodiments, or any combination thereof. For example, any of the embodiments of T, or any combination thereof, can be combined with any of the embodiments of y, L1, L2, L3, L 1a , L 1b , L 1c , L 1d , L 1e , D, LD1 , L D2 , L D3 , L D4 , R D1 , R D2 , R D3 , R D4 , R D5 and R D6 Any of the technical solutions or any combination thereof can be combined. The present application is intended to include all combinations of the technical solutions, which are not listed one by one due to the limited space.

[0260] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0261] (1) the VH comprises HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively; or

[0262] (2) the VH comprises HCDR1, HCDR2, HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively; or

[0263] (3) the VH comprises HCDR1, HCDR2, HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively; or

[0264] (4) the VH comprises HCDR1, HCDR2, HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively.

[0265] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein:

[0266] (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; or

[0267] (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or

[0268] (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73; or

[0269] (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; or

[0270] (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; or

[0271] (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0272] In a more specific embodiment, the present application relates to the above antibody drug conjugate of Formula (I), wherein:

[0273] (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 43; or

[0274] (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 69; or

[0275] (3) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73; or

[0276] (4) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13; or

[0277] (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0278] (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33.

[0279] In a more specific embodiment, the present application relates to the above antibody drug conjugate of Formula (I), wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0280] In a more specific embodiment, the present application relates to the above antibody drug conjugate of Formula (I), wherein the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0281] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody belongs to a subtype selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.

[0282] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0283] (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 79 and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 77; or

[0284] (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 83 and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 81 ;

[0285] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:

[0286] (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 79 and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 77; or

[0287] (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 83 and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 81 ;

[0288] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv and ds-scFv.

[0289] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0290] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen.

[0291] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein,

[0292] said L comprises a cleavable linker or a non-cleavable linker;

[0293] Preferably, said cleavable linker comprises an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photo-labile linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

[0294] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein,

[0295] said L comprises the following structure or a fragment derived from the following structure: mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxycarbonyl (PABC), SPDB (N-succinimidyl-4-(2-pyridyldithio)-butyrate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate), beta-glucuronide, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG- aminomethyl, AcBut (4-(4-acetylphenoxy)-butyric acid), dimethyl hydrazide (3-methyl-) 3- mercaptobutane hydrazide), AcBut-dimethyl hydrazide, or SMCC (N-succinimidyl-4-(N- maleimidomethyl) cyclohexane-carboxylate).

[0296] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein,

[0297] L is -L1-L2-L3-;

[0298] L1is -L 1a -L 1b -L 1c -L 1d -L 1e -;

[0299] L 1a is selected from

[0300] L1b selected from C 1-10 alkylene, C 2-10 alkenylene, or C 2-10 alkynylene, wherein, C 1-10 alkylene, C 2-10 alkenylene, and C 2-10 alkynylene, each CH2is optionally substituted with 1, 2, or 3 R x ;

[0301] each R x is independently selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl; or, two R x on any identical or different carbon atom can be linked to form a C 3-10 cycloalkylene, or 3-10 membered heterocyclyl ene;

[0302] L 1c is selected from a bond, -C(O)-, -C(O)NH-, or -NHC(O)-;

[0303] L 1d is selected from a bond, C 1-8 alkylene, or -(CH2CH2O) n -C 1-4 alkylene-; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl, or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0304] n and w are independently selected from 1, 2, 3, 4, 5, 6, 7, or 8;

[0305] L 1e is selected from -C(O)-, -NHC(O)-, or -C(O)-NHC(O)-;

[0306] L2is a bond or is selected from a divalent peptidyl group comprising 2 to 8 optionally substituted natural amino acid residues or non-natural amino acid residues,

[0307] each amino acid residue in said L2is optionally substituted with 1, 2, 3, 4, or 5 R y ;

[0308] each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6haloalkoxy;

[0309] L3is an optional spacer that is optionally substituted or unsubstituted, e.g., L3is selected from -NH-CH2-(AM), (PABC), (PAB), or

[0310] L3is optionally substituted with 1, 2, or 3 R z each R z is independently selected from H, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1- 6haloalkyl, or C 1-6 haloalkoxy;

[0311] Preferably,

[0312] L is -L1-L2-L3-;

[0313] L1is -L 1a -L 1b -L 1c -L 1d -L 1e -;

[0314] L 1a is selected from represents L 1a and the attachment site to the antibody;

[0315] L 1b is selected from C 1-6 alkylene, C 2-6 alkenylene, or C 2-6 alkynylene, wherein each CH2in C 1-6 alkylene, C 2-6 alkenylene, and C 2-6 alkynylene is optionally substituted with 1 or 2 R x ;

[0316] each R x is independently selected from H, halo, or C 1-6 alkyl; or, two R x on any identical or different carbon atom can be joined to form a C 3-7 cycloalkylene, or 3-7 membered heterocyclyliden, preferably C 3-7 cycloalkyl, more preferably C 3-5 cycloalkylene, e.g., cyclopropylidene;

[0317] L 1c is selected from a bond, -C(O)-, -C(O)NH-, or -NHC(O)-, preferably a bond or -C(O)NH-;

[0318] L 1d is selected from a chemical bond or C 1-6 alkylene; said C 1-6 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0319] w is selected from 2, 3 or 4, preferably 3;

[0320] L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably a chemical bond or -C(O)-;

[0321] L2is selected from a divalent peptidic moiety comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, each amino acid residue in said L2is optionally substituted with 1, 2 or 3 R y ;

[0322] each R y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0323] L3is an optionally substituted or unsubstituted spacer, for example L3is selected from -NH-CH2-(AM) or (PABC);

[0324] L3is optionally substituted with 1, 2 or 3 R z , each R z is independently selected from H, halogen or C 1-6 alkyl.

[0325] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein,

[0326] L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, selected from gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit or val-lys-beta-ala;

[0327] each amino acid residue in said L2 is optionally substituted with 1, 2 or 3 R y ;

[0328] each R y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0329] Preferably,

[0330] L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, said amino acids being selected from glycine, phenylalanine, alanine, valine, citrulline or lysine, preferably -gly-gly-phe-gly- or -val-cit- or -val-lys-gly-;

[0331] each amino acid residue in said L2 is optionally substituted with 1, 2 or 3 R y ;

[0332] each R y is independently selected from H, halogen or C 1-6 alkyl.

[0333] In a more specific embodiment, the application relates to the antibody drug conjugate of formula (I) above, wherein,

[0334] L is selected from the following structures: (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-val-cit-PABC), (SPDB), (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM),

[0335] Preferably, L is selected from the following structures: (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM),

[0336] In a more specific embodiment, the present application relates to the antibody drug conjugate of formula (I) above, wherein

[0337] D is selected from the group consisting of metal complexes; antibiotics; DNA topoisomerase inhibitors; microtubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis;

[0338] Preferably,

[0339] D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors and RNA splicesome inhibitors;

[0340] More preferably,

[0341] said D is selected from the group consisting of microtubulin inhibitors and microtubule polymerization inhibitors, such as auristatins, maytansinoids, tubulysins, cryptophycins or rhizoxin;

[0342] Alternatively, said D is an antibiotic, such as a calicheamicin, an anthracycline and an anthracycline antibiotic;

[0343] Alternatively, said D is selected from the group consisting of DNA synthesis inhibitors, such as duocarmycins, PBDs (pyrroloridine dioxanes) or IGNs (indolinobenzodiazepines);

[0344] or said D is selected from a DNA topoisomerase I inhibitor, such as camptothecin or a camptothecin derivative;

[0345] or said D is selected from an RNA polymerase II inhibitor, such as an α-amanitin;

[0346] or said D is selected from an RNA splicesome inhibitor drug, such as a splicetastatin or a talanstatin;

[0347] Preferably, D is selected from a camptothecin or a camptothecin derivative, such as hydroxycamptothecin, 9-aminocamptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or other derivatives;

[0348] Preferably, D is selected from an auristatin drug, such as MMAE or MMAF;

[0349] Preferably, D is selected from a maytansinoid drug, such as DM1, DM2, DM3 or DM4.

[0350] In a more specific embodiment, the present application relates to an antibody drug conjugate of formula (I) as described above, wherein,

[0351] D is selected from an auristatin drug, such as MMAE or MMAF;

[0352] or D is selected from a compound of formula (D-I) or formula (D-II), or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, racemic, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0353] *1, *2 and *3 are chiral centers independently selected from (S) or (R) absolute configuration, or mixtures thereof, preferably *3 is in (S) absolute configuration;

[0354] L D1 is selected from a chemical bond, -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0355] q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3;

[0356] L D2 is selected from -NH-, -O-, -C(O)-, -NHC(O)- or -C(O)NH-;

[0357] L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene or C 2-10alkynylene, said L D3 optionally substituted by 1, 2, or 3 R D3 substituents;

[0358] L D4 is selected from -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0359] R D1 , R D2 , and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D1 , R D2 , and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a 5-6 membered heterocyclyl; or, R D1 , R D6 , and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 5-6 cycloalkyl;

[0360] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl, or 3-7 membered heterocyclyl; or, R D3 , R D4 , and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 3-5 cycloalkyl;

[0361] R D5 is selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl, or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl;

[0362] each of the compounds of formula (D-I) and formula (D-II) is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from:

[0363] deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl.

[0364] preferably,

[0365] D is selected from an auristatin or a maytansinoid, such as MMAE, MMAF, DM1, DM2, DM3, or DM4, preferably MMAE or DM4;

[0366] Alternatively, D is selected from a compound of formula (D-III) or formula (D-IV), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, stereoisomer, racemate, polymorph, hydrate, or solvate thereof, or mixtures thereof:

[0367] *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0368] L D1 selected from a bond, -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0369] q is 0, 1, 2, 3, or 4, preferably 0 or 1;

[0370] L D2 selected from -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0371] L D3 selected from a bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene, or C 2-10 alkynylene;

[0372] L D4 selected from -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0373] R D1 , R D2 , and R D6 are independently selected from H, halogen, C1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 alkoxy; or, R D1 R D2 Together with the carbon atoms they are attached to, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; or, R D1 R D6 Together with the carbon atoms they are attached to, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic groups, preferably C 5-6 cycloalkyl;

[0374] Each R D3 and R D4 Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 alkoxy; or, R D3 R D4 Together with the carbon atoms they are attached to, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic groups, preferably C 3-5 cycloalkyl;

[0375] R D5 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-10 heteroaryl, -C 1-6 Alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10-membered heterocyclic group, -C 1-6 Alkylene-C 6-10 Aryl or -C 1-6 Alkyl-5-10 heteroaryl groups, preferably -C 1-6 Alkylene-C 3-10 Cycloalkyl.

[0376] In a more specific embodiment, the present invention relates to antibody-drug conjugates of formula (I) above, wherein,

[0377] D is selected from aurestatin or maytansine drugs, such as MMAE, MMAF, DM1, DM2, DM3 or DM4, preferably MMAE or DM4;

[0378] Alternatively, D is selected from compounds of formula (D-III) or (D-IV), or pharmaceutically acceptable salts, isotopic variants, tautomers, stereoisomers, racemates, polymorphs, hydrates or solvates, or mixtures thereof:

[0379] *1 and *2 are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0380] L D1 is selected from a chemical bond, -NHC(O)- or -C(O)NH-;

[0381] q is 0, 1, 2, 3 or 4, preferably 0 or 1;

[0382] L D2 is selected from -NH- or -O-;

[0383] L D3 is selected from a chemical bond, C 1-6 alkylene or C 1-6 haloalkylene, more preferably C 1-6 alkylene;

[0384] L D4 is selected from -NH- or -O-;

[0385] R D1 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me;

[0386] R D2 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, for example F;

[0387] or, R D1 , R D2 and the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0388] each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0389] R D5 is selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C1-4 alkylene-C 3-5 cycloalkyl;

[0390] R D6 is H;

[0391] Alternatively, D is selected from a compound of Formula (D-V), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof:

[0392] *2 is a chiral center independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0393] q is 1, 2, or 3, preferably 1;

[0394] R D1 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, such as F; 1-6 alkyl, such as Me;

[0395] R D2 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, such as F;

[0396] Alternatively, R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0397] each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably R D3 and R D4 are not simultaneously H; or R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl;

[0398] R D5 is selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; more preferably RD5 is selected from -methylene-cyclopropyl or -methylene-cyclobutyl.

[0399] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0400] Preferably, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemate, polymorph, hydrate or solvate thereof, or mixtures thereof:

[0401] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I), wherein y is an integer or decimal number selected from 0 to 20, preferably an integer or decimal number selected from 0 to 10, more preferably an integer or decimal number selected from 3 to 9.

[0402] In a more particular embodiment, the present application relates to the above antibody drug conjugate of formula (I) having the structure of formula (I-1), (I-2) or (I-3):

[0403] wherein,

[0404] T, D and y are as defined herein;

[0405] each m is selected from 1, 2, 3, 4 or 5;

[0406] each w is selected from 1, 2, 3, 4, 5, 6, 7 or 8;

[0407] each R x and R x are independently selected from H, halogen or C 1-6 alkyl; or, two R x on any identical or different carbon atom can be linked to form a C 3-7 cycloalkylene or 3-7 membered heterocyclylene, preferably C 3-7 cycloalkylene, more preferably C 3-5 cycloalkylene;

[0408] each R y and R y are independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy.

[0409] In a more particular embodiment, the present application relates to an antibody drug conjugate of formula (I) as described above, wherein the antibody drug conjugate is selected from the following structures:

[0410] wherein,

[0411] T, D and y are as defined herein;

[0412] Preferably,

[0413] D is selected from an auristatin, a maytansinoid, a camptothecin or a camptothecin derivative, such as MMAE, MMAF, DM1, DM2, DM3, DM4, hydroxycamptothecin, 9-aminocamptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or a compound of formula (D-I), formula (D-II), formula (D-III) or formula (D-IV), wherein the variables are as defined herein;

[0414] y is selected from an integer or a decimal number from 1 to 10.

[0415] In a more particular embodiment, the present application relates to an antibody drug conjugate of formula (I) as described above, wherein the antibody drug conjugate is selected from the following structures:

[0416] T and y are as defined herein;

[0417] Preferably,

[0418] y is selected from an integer or a decimal number from 1 to 10.

[0419] In a more particular embodiment, the present application relates to an antibody drug conjugate of formula (I) as described above, wherein the antibody drug conjugate is selected from the following structures:

[0420] wherein,

[0421] the mAb1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 38 and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 43; or

[0422] the mAb 1 H6K3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 69; or

[0423] the mAb 1 H6K5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 73; or

[0424] the mAb 2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 13; or

[0425] the mAb 3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 23; or

[0426] the mAb 4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprising an amino acid sequence as set forth in SEQ ID NO: 33.

[0427] Preferably,

[0428] the mAb 1 H6K3 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 77, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 79;

[0429] the mAb 1 H6K5 comprises a heavy chain (HC) and a light chain (LC), the HC comprising an amino acid sequence as set forth in SEQ ID NO: 81, and the LC comprising an amino acid sequence as set forth in SEQ ID NO: 83;

[0430] More preferably,

[0431] the -L-D structure is attached to a cysteine in the antibody or antigen-binding fragment thereof that binds to CEACAM5.

[0432] In a more particular embodiment, the present application relates to a pharmaceutical composition comprising an antibody drug conjugate according to the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0433] In a more particular embodiment, the present application relates to the above pharmaceutical composition, wherein the composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, monoclonal antibody drugs, bi- / multi-specific antibody drugs, recombinant protein drugs, nucleotide drugs (including siRNA and antisense oligonucleotides), small molecule drugs, immunomodulatory drugs, and cell therapy drugs.

[0434] In a more particular embodiment, the present application relates to a method for preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject an antibody drug conjugate according to the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof.

[0435] In a more particular embodiment, the present application relates to the above method, further comprising administering to the subject a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, monoclonal antibody drugs, bi- / multi-specific antibody drugs, recombinant protein drugs, nucleotide drugs (including siRNA and antisense oligonucleotides), small molecule drugs, immunomodulatory drugs, and cell therapy drugs.

[0436] In a more particular embodiment, the present application relates to an antibody drug conjugate according to the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof, for use in the prevention and / or treatment of a disease.

[0437] In a more particular embodiment, the present application relates to the use of an antibody drug conjugate according to the present application, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof, in the manufacture of a medicament for the prevention and / or treatment of a disease.

[0438] In a preferred embodiment, the disease is cancer, for example a cancer associated with CEACAM5 expression;

[0439] Preferably, the disease is selected from the group consisting of gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder carcinoma, or epithelial carcinoma;

[0440] More preferably, the disease is selected from the group consisting of pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, and breast cancer.

[0441] Pharmaceutical compositions

[0442] The present application provides pharmaceutical compositions comprising the antibody drug conjugates disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0443] The antibody drug conjugates of the present application (also referred to herein as "active compounds") can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise an antibody drug conjugate, together with a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such media and agents for use in

[0444] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0445] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0446] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0447] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0448] Oral composition generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash in which the compound is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0449] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide.

[0450] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and the like. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into

[0451] The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or in the form of retention enemas for rectal delivery.

[0452] In one embodiment, the active compounds are prepared with an agent that protects the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0453] The present application provides therapeutic compositions comprising the antibody drug conjugates of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary of all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing TM DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions with

[0454] Methods of treatment and uses

[0455] The present disclosure provides methods for treating a disease in a subject comprising administering to the subject an effective amount of an antibody drug conjugate disclosed herein or a pharmaceutical composition disclosed herein.

[0456] The present disclosure also provides use of an antibody drug conjugate disclosed herein, or a pharmaceutical composition disclosed herein, in the manufacture of a medicament for treating a disease in a subject.

[0457] The present disclosure also provides an antibody drug conjugate disclosed herein, or a pharmaceutical composition disclosed herein, for use in treating a disease in a subject.

[0458] The present disclosure also provides a method for preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject an antibody drug conjugate disclosed herein, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof.

[0459] In some embodiments, the method further comprises administering to the subject a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody drug, a bi- / multi-specific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

[0460] The present disclosure also provides an antibody drug conjugate disclosed herein, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or a mixture thereof, for use in preventing and / or treating a disease.

[0461] In some embodiments, the disease is a cancer, for example a cancer associated with CEACAM5 expression. In more preferred embodiments, the disease is selected from the group consisting of gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, thyroid cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial carcinoma. In preferred embodiments, the disease is selected from the group consisting of pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, and breast cancer.

[0462] In some embodiments, the dose administered to a subject can vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dose should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of the drug to be administered to a human or other subject to treat a medical condition. The precise amount required can depend on many factors, such as the activity of the drug conjugate and the route of administration.

[0463] The antibody drug conjugates or compositions described herein can be administered to a mammal in a single dose or in a series of sub-doses over a period of time, e.g., daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annually, or annually, as desired. Dose units comprising an effective amount of the antibody drug conjugates or compositions can be administered as a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily, as desired.

[0464] The appropriate mode of administration can be chosen by the physician. The route of administration can be parenteral administration, e.g., administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody drug conjugates or compositions are selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, e.g., orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0465] In some embodiments, the methods further comprise administering a second therapeutic agent to the subject. In some embodiments, the antibody drug conjugates or pharmaceutical compositions disclosed herein are used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, the antibody drug conjugates or compositions disclosed herein are administered prior to, substantially simultaneously with, or following administration of the second therapeutic agent.

[0466] Kits / Dosing Devices

[0467] The present disclosure provides kits or dosing devices comprising the antibody drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein.

[0468] In some embodiments, the kits or dosing devices comprise one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, e.g., the antibody drug conjugates disclosed herein.

[0469] In specific embodiments, the kits comprise a first container that contains a pharmaceutical conjugate disclosed herein. In specific embodiments, the kits comprise a first container that is a vial containing a pharmaceutical conjugate as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.

[0470] In specific embodiments, injection devices containing a pharmaceutical conjugate are provided herein. In specific embodiments, the injection devices comprise a pharmaceutical conjugate in a sterile solution. In specific embodiments, the injection devices are syringes.

[0471] In one embodiment, the kit includes instructional materials that disclose the manner in which the pharmaceutical conjugate of the disclosure is used. The instructional materials can be written, in electronic format (such as a computer diskette or CD-ROM), or visual (such as a video cassette). The kit can also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit can additionally contain tools for detection of the label (e.g., enzyme substrates for enzymatic labels, filter sets for detection of fluorescent labels, appropriate secondary labels such as secondary antibodies, etc.). The kit can also include buffers and other reagents commonly used in the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0472] Examples

[0473] For the purpose of illustrating the present embodiments, the examples that follow are presented in the following order. However, it is not intended to limit the present invention in any way. The present embodiments, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary and are not intended as limitations on the scope of the present invention. Changes therein and other uses will occur to those skilled in the art upon reading the description and drawings herein.

[0474] The control targeting CEACAM5 antibody is Tusamitamab, prepared according to the patent WO2014079886A1, hereinafter ref1. Another control targeting CEACAM5 antibody is hu8G4 (M9140 antibody moiety), prepared according to WO2022048883A, hereinafter ref8.

[0475] The control CEACAM5 ADCs include M9140 and Tusamitamab ravtansine, prepared according to the patents WO2022048883A and WO2014079886A1, hereinafter ADC-15 (M9140) and ADC-17 (Tusamitamab ravtansine), respectively.

[0476] Example 1, Preparation of antigens and stable cell lines

[0477] 1.1 Expression of recombinant CEACAM5 antigen protein and CEACAM family antigen proteins (CEACAM1, 6, 7, 8)

[0478] The nucleic acid sequence encoding the antigen protein with his, Fc tag was integrated into a mammalian cell expression vector, after bacterial infection, lysis, plasmid extraction, and washing, the plasmid expressing the antigen protein was obtained. P11 was subcultured to a suitable number of HEK293F cells in the inner generation, and the complex formed by PEI and plasmid was added dropwise to the HEK293F cells in the inner generation in the clean bench the day before. The cells were cultured for 48-72 hours, and the supernatant was collected and stored at 4°C for use. 6HEK293F cells with a cell density of 1.5x106cells / ml were transfected with the complex by adding the complex to the flask while shaking the flask, or by adding part of the complex to the flask, mixing the cells, and then repeating the addition. The HEK293F cells were cultured in a cell incubator shaker at 37°C, 120 rpm, and 5% CO2, and the day of transfection was designated as day 0. On days 1 and 3 after transfection, 5% OPM-293Profeed (Opmay, P82019) was added to the cells. On day 4 after transfection, the cell viability was measured using a Vi-cell XR cell viability analyzer (Beckman). When the cell viability decreased to about 70%, the fermentation broth was collected and filtered to obtain the antigen protein.

[0479] The amino acid sequences of the related antigen proteins are as follows:

[0480] Human CEACAM5 (SEQ ID NO. 1)

[0481] Cynomolgus monkey CEACAM5 (SEQ ID NO. 2)

[0482] Rat CEACAM5 (SEQ ID NO. 3)

[0483] Human CEACAM1 (SEQ ID NO. 4)

[0484] Human CEACAM6 (SEQ ID NO. 5)

[0485] Human CEACAM7 (SEQ ID NO. 6)

[0486] Human CEACAM8 (SEQ ID NO. 7)

[0487] 1.2 Construction of HEK293 stable cell lines expressing membrane CEACAM5 antigen protein

[0488] The nucleic acid sequence encoding the membrane protein is integrated into a lentivirus expression vector, and after bacterial infection, lysis, plasmid extraction, and washing, a plasmid expressing the antigen protein is obtained. After the HEK293 cells (ATCC, Cat No. CRL-3216) are recovered for 1-2 days, when the confluence reaches 70-80%, the cells are passaged, the complete culture medium, trypsin solution, and PBS are preheated at 37°C, the cells are gently washed with PBS, an appropriate amount of trypsin solution is added (to ensure complete coverage of the cells by gently shaking the culture bottle, about 2-3 mL for a 10 cm culture dish, and about 3-4 mL for a 15 cm culture dish), and incubated at 37°C or room temperature for 2-4 minutes. Under a microscope, the cells become rounded, and the cells are detached from the culture surface by gently tapping the side of the culture dish. The volume of trypsin is 2-3 times that of the complete culture medium, and the cells are resuspended and transferred to a centrifuge tube, centrifuged at 300g for 3-5 minutes, resuspended with complete culture medium, counted, and inoculated into new culture bottles at a 1:3-1:10 passage ratio. The cells are removed, the supernatant is discarded, and 9 mL of Opti-MEM medium (Gibco, Cat No. 31985070) is slowly added, trying not to blow up the cells. The pre-prepared PEI / DNA complex is evenly dropped into the cells, the culture bottle is gently shaken left and right and front and back, and placed in a 37°C, 5% CO2 incubator for culture. After 16-24 hours of transfection, the supernatant is discarded, 30 mL of complete culture medium is added, and the culture is continued in a 37°C, 5% CO2 incubator. Transfection detection: after 24-72 hours of transfection, the expression of the relevant receptor is detected by flow cytometry. After pressurization, sorting, and retesting, monoclonal HEK293 cells expressing human / monkey CEACAM5 membrane protein (hereinafter referred to as HEK293-hCEACAM5 / HEK293-cyCEACAM5) are obtained.

[0489] Example 2, Production of Anti-CEACAM5 Chimeric Antibody

[0490] 2.1 Mouse Immunization and Hybridoma Cell Preparation

[0491] Take 6-8 weeks female Balb / c mice (Vantianlihua, strain code: 211), after the mice are weighed and anesthetized, immunize with a plasmid encoding CEACAM5 protein or CEACAM5 antigen protein, or alternately immunize. When DNA is used for immunization, 20 μg of plasmid is mixed with 1 μg of CpG, and then directly injected into the abdomen of the mouse at 40 psi using a gene gun (Biorad). Immunization is performed once a week. Three days before fusion preparation, CEACAM5 antigen protein is used for tail vein injection impact immunization. When protein is used for immunization, 79 μl of antigen protein is mixed with 401 μL of PBS, 480 μL of the prepared antigen mixture is taken up with a 3 mL syringe, and another syringe is used to take up 480 μL of CFA. A T-shaped three-way joint is used for mixing until a white emulsion of water-in-oil type is formed. The prepared antigen is subcutaneously injected at a dose of 50 μg per mouse at the abdomen near the thigh root. Three days before fusion preparation, CEACAM5 antigen protein 10 μg per mouse is injected into the foot pad for immunization.

[0492] After immunization, the mice are sacrificed by cervical dislocation, and then soaked in 75% alcohol solution for 5 min for disinfection. The spleen, popliteal lymph node, inguinal lymph node and iliac lymph node of the mouse are collected, and a lymphocyte-rich suspension is obtained by grinding in DMEM medium. Sp2 / 0 cells and an equal proportion of lymphocytes are mixed, centrifuged and resuspended, and then cell fusion is performed by an electrofusion instrument to obtain hybridoma cells.

[0493] 2.2 Hybridoma clone screening

[0494] 384-well plate ELISA screening

[0495] Dilute the hCEACAM5 antigen protein with coating solution to a concentration of 1 pg / mL, 30 pL / well into the enzyme-labeled plate, 4°C overnight. Take the enzyme-labeled plate out of the refrigerator, wash three times with 0.05% PBST. Add 80 pL / well of blocking solution 2% milk into the plate, incubate at room temperature for 1 h. Wash once with 0.05% PBST. Add samples and controls according to 30 pL / well into the enzyme-labeled plate, incubate at room temperature for 1 h. Wash five times with 0.05% PBST. Dilute the secondary antibody with 0.5% milk goat anti-mouse IgG-Fc HRP (Jackson, Cat: 115-035-071) 4000 times, 30 pL / well into the enzyme-labeled plate, incubate at room temperature for 1 h. Wash five times with 0.05% PBST. Add color developing solution, 30 pL / well, in a 37°C constant temperature incubator, develop for 10 min. Add 2M concentrated sulfuric acid stop solution, 15 pL / well, avoid bubbles, read within 15 min with an enzyme-labeled instrument (wavelength: 450 nm). As shown in Table 1, mAb1, mAb2, mAb3 and mAb4 clones have strong binding activity to human CEACAM5 protein. At the same time, mAb1, mAb3 and mAb4 clones have stronger binding activity to cynomolgus monkey CEACAM5 protein than ref1 antibody. mAb1, mAb2, mAb3 and mAb4 clones have weaker binding to the same family protein mixture than ref1 antibody, proving that the hybridoma clones have better specificity for binding to CEACAM5 protein.

[0496] Table 1. ELISA binding activity of supernatant culture solution of different hybridoma clones to human monkey species CEACAM5 protein

[0497] FACS screening

[0498] Label HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells with CFSE (Thermo Fisher, Cat: C34554), final density 1 x 10 7 7 Incubate 1 x 10 6 ​Cells / 500 μL 3% BSA; after the end of blocking, HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells were mixed together, 0.5% BSA was added according to the number of plates, 100 μL / well; after centrifugation at 200g for 3 min, the aggregated cell mass was gently tapped; the hybridoma supernatant and control antibody were both incubated at 50 μL / well for 60 min; after incubation, 0.2 ml 0.5% BSA was added for washing twice, and after centrifugation at 200g for 3 min, the supernatant was discarded, and the cell mass was tapped; 30 μL / well of anti-human IgG or anti-mouse IgG was added for incubation. After 30 min of secondary antibody incubation, 0.2 ml 0.5% BSA was added for washing three times, and after centrifugation at 200g for 3 min, the supernatant was discarded, and the cells were tapped; finally, 50 μL / well of PBS was added for resuspension, and flow cytometry was performed.

[0499] As shown in Table 2, mAb1, mAb2, mAb3 and mAb4 clones have strong binding activity to HEK293 cells expressing human or cynomolgus monkey.

[0500] Table 2. FACS binding activity of supernatant culture of different hybridoma clones to HEK293 surface human monkey species CEACAM5 target

[0501] 2.3 Hybridoma mouse anti-sequence determination and chimeric antibody construction

[0502] cDNA synthesis

[0503] The hybridoma clone that binds CEACAM5 in the selection screening was amplified, and the RNA was extracted, and the RNA (mRNA) was reverse transcribed into cDNA under the action of reverse transcriptase. The first strand of cDNA was used as a template for PCR amplification. The gene-specific upstream and downstream primers for PCR amplification were designed according to the target gene. The gene-specific upstream primer was annealed to the first strand of cDNA, and the second strand of cDNA was synthesized under the action of Taq DNA polymerase. The first and second strands of cDNA were used as templates for PCR amplification with gene-specific upstream and downstream primers to obtain a large amount of cDNA.

[0504] Reverse transcription and amplification of cDNA, 65°C for 5 min, and incubation on ice for 2 min. The reaction system is as follows:

[0505] Prepare the cDNA synthesis reaction solution (5 μl reaction system), and add the following table:

[0506] Take 5 μl of cDNA synthesis reaction solution into 200 μl PCR tube respectively, mix well, spin, 50℃ reaction for 50 min. After 85℃ termination reaction for 5 min, insert into ice immediately. After spin, add 1 μl of RNase H to the PCR tube, 37℃ reaction for 20 min. Dilute the synthesized cDNA 5 times, and store at -20℃.

[0507] Chimeric antibody plasmid construction

[0508] The PCR amplification V region fragment reaction system is as follows:

[0509] Put the amplified fragments and the vector plasmid into the optimal enzyme cutting reaction temperature, add restriction endonuclease, and perform enzyme cutting reaction for 1 hour. After electrophoretic separation of the corresponding target fragments, purify and recover the required fragments according to the operation manual of NucleoSPIN Gel and PCR Clean-up recovery kit. Add 2xHiFi Assembly ligase to the recovered linearized gel recovery product and the target fragment recovery product, and place it in a 50℃ water bath for 15 min of ligation reaction to obtain a chimeric expression plasmid. After competent cell transfection, amplification, lysis, filtration collection, and washing purification, a monoclonal chimeric plasmid is obtained. The heavy chain and light chain variable region sequences of the chimeric antibody are shown in Table 3.

[0510] Table 3. Anti-CEACAM5 chimeric antibody light and heavy chain variable region sequences

[0511] The heavy chain / light chain variable region sequences of the anti-CEACAM5 antibody are as follows (wherein the CDR sequences are defined according to the Kabat definition rule):

[0512] mAb1

[0513] The amino acid sequence of the heavy chain VH of mAb1 is shown in SEQ ID NO. 38, the encoding nucleic acid is shown in SEQ ID NO. 39, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 40, 41 and 42 respectively.

[0514] Nucleotide sequence

[0515] The amino acid sequence of the light chain VL of mAb1 is shown in SEQ ID NO. 43, the encoding nucleic acid is shown in SEQ ID NO. 44, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 45, 46 and 47 respectively.

[0516] Nucleotide sequence

[0517] mAb2

[0518] The amino acid sequence of the heavy chain VH of mAb2 is shown as SEQ ID NO. 8, the encoding nucleic acid is shown as SEQ ID NO. 9, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 10, 11, 12 respectively.

[0519] Nucleotide sequence

[0520] The amino acid sequence of the light chain VL of mAb2 is shown as SEQ ID NO. 13, the encoding nucleic acid is shown as SEQ ID NO. 14, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 15, 16, 17 respectively.

[0521] Nucleotide sequence

[0522] mAb3

[0523] The amino acid sequence of the heavy chain VH of mAb3 is shown as SEQ ID NO. 18, the encoding nucleic acid is shown as SEQ ID NO. 19, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 20, 21, 22 respectively.

[0524] Nucleotide sequence

[0525] The amino acid sequence of the light chain VL of mAb3 is shown as SEQ ID NO. 23, the encoding nucleic acid is shown as SEQ ID NO. 24, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 25, 26, 27 respectively.

[0526] Nucleotide sequence

[0527] mAb4

[0528] The amino acid sequence of the heavy chain VH of mAb4 is shown as SEQ ID NO. 28, the encoding nucleic acid is shown as SEQ ID NO. 29, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 30, 31, 32 respectively.

[0529] Nucleotide sequence

[0530] The amino acid sequence of the VL of the light chain of mAb4 is shown as SEQ ID NO. 33, the encoding nucleic acid is shown as SEQ ID NO. 34, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 35, 36, 37, respectively.

[0531] Nucleotide sequence

[0532] Example 3, Characterization of Anti-CEACAM5 Chimeric Antibodies

[0533] 3.1 Binding assay of chimeric antibodies to tumor cells and stable transfected cells

[0534] The binding of chimeric antibodies to stable cell lines was detected by FACS. The CEACAM5 stable cell lines HEK293-hCEACAM5 and HEK293-cyCEACAM5 and CEACAM5 positive tumor cells MKN45 (National Experimental Cell Resource Sharing Platform, item number: 1101HUM-PUMC000229), BxPc-3 (ATCC, item number: CRL-1687), Caco-2 (ATCC, item number: HTB-37) and KatoIII (ATCC, item number: HTB-103) cells in logarithmic growth phase were adjusted to 5x10 5 cells / ml with PBS containing 1% BSA, 100 μl / well of cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, the supernatant was discarded, 100 μl of gradient-diluted chimeric antibody (initial concentration 200 nM, 4-fold dilution) was added to each well, and incubated at 4°C for 60 minutes. Secondary antibody was added with 50 μl / well of Alexa Fluro 647-labeled goat anti-human IgG Fc (1:300 dilution), and incubated on ice for 20 minutes. The binding activity of CEACAM5 chimeric antibody to cells was detected by flow cytometry. The results of cell binding are shown in Table 4, and mAb1 clone has strong binding activity to tumor cells expressing CEACAM5 and HEK293 cells expressing human or cynomolgus CEACAM5 antigen protein.

[0535] Table 4. EC50 of anti-CEACAM5 chimeric antibodies to tumor cells and stable transfected cells

[0536] 3.2 Endocytosis activity of anti-CEACAM5 chimeric antibodies

[0537] DT3C is a fusion protein composed of diphtheria toxin and protein G 3C fragment that binds IgG, which can bind to the Fc of antibodies, enter cells during antibody endocytosis, and release diphtheria toxin DT under the action of furin, leading to cell death. It was developed by researchers at Sapporo Medical University in 2014 (Yamaguchi, Miki, et al. Biochem Biophys Res Commun 454.4 (2014): 600-603.) and has been widely used as an evaluation method for ADC screening.

[0538] MKN45 cells expressing CEACAM5 were used as target cells, and the cell density was adjusted to 1 x 10 4 cells / well and plated one day in advance. The next day, the DT3C protein (homemade) was gradient diluted (133 nM as the starting concentration, 5-fold gradient, a total of 9 dilutions), and incubated with 10 ug / mL experimental antibody at 37°C for 30 min to form antibody-DT3C complexes, and then the antibody-DT3C complexes were added to the cell culture medium and incubated with the cells for 3 days. The number of viable cells in each experimental group was detected by the CCK-8 kit, the cell survival rate was calculated, and the antibody concentration-survival rate curve was fitted by the four-parameter model. The results of the endocytosis activity are shown in Table 5, and the mAb1 clone has good endocytosis activity.

[0539] Table 5. DT3C endocytosis activity IC50 of anti-CEACAM5 chimeric antibody

[0540] Example 4, Humanization of anti-CEACAM5 chimeric antibody

[0541] The germline gene with high homology to mAb1 was selected as the template, the humanized light chain template was selected as IGKV1-39*01 and IGKV3-15*01, and the J region was selected as IGKJ2*01; the humanized heavy chain template was selected as IGHV1-69*01 and IGHV3-30*01, and the J region was selected as IGHJ4*01. The CDR region of the murine anti-CEACAM5 antibody was transplanted into the corresponding humanized gene template, and part of the amino acids were mutated according to the in silico simulation results to improve the degree of humanization.

[0542] The sequences of the heavy chain variable region and the light chain variable region of the anti-CEACAM5 humanized antibody are shown in Tables 6 and 7, respectively.

[0543] Table 6. Heavy chain variable region sequence of anti-CEACAM5 humanized antibody

[0544] Table 7. Light chain variable region sequence of anti-CEACAM5 humanized antibody

[0545] The heavy chain / light chain variable region sequences of the anti-CEACAM5 humanized antibodies are as follows:

[0546] The amino acid sequence of mAb1 H1 is set forth in SEQ ID NO. 48, the encoding nucleic acid is set forth in SEQ ID NO. 49, and the CDR1, CDR2 and CDR3 are set forth in SEQ ID NO. 40, 41, 42, respectively.

[0547] Nucleotide sequence

[0548] The amino acid sequence of mAb1 H2 is set forth in SEQ ID NO. 50, the encoding nucleic acid is set forth in SEQ ID NO. 51, and the CDR1, CDR2 and CDR3 are set forth in SEQ ID NO. 40, 41, 42, respectively.

[0549] Nucleotide sequence

[0550] The amino acid sequence of mAb1 H3 is set forth in SEQ ID NO. 52, the encoding nucleic acid is set forth in SEQ ID NO. 53, and the CDR1, CDR2 and CDR3 are set forth in SEQ ID NO. 40, 41, 42, respectively.

[0551] Nucleotide sequence

[0552] The amino acid sequence of mAb1 H4 is set forth in SEQ ID NO. 54, the encoding nucleic acid is set forth in SEQ ID NO. 55, and the CDR1, CDR2 and CDR3 are set forth in SEQ ID NO. 40, 41, 42, respectively.

[0553] Nucleotide sequence

[0554] The amino acid sequence of mAb1 H5 is set forth in SEQ ID NO. 56, the encoding nucleic acid is set forth in SEQ ID NO. 57, and the CDR1, CDR2 and CDR3 are set forth in SEQ ID NO. 40, 41, 42, respectively.

[0555] Nucleotide sequence

[0556] The amino acid sequence of mAb1 H6 is shown as SEQ ID NO. 58, the encoding nucleic acid is shown as SEQ ID NO. 59, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, SEQ ID NO. 60 (YINPNTGYTEYSQKFKG), SEQ ID NO. 42, respectively.

[0557] Nucleotide sequence

[0558] The amino acid sequence of mAb1 H7 is shown as SEQ ID NO. 61, the encoding nucleic acid is shown as SEQ ID NO. 62, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 60, 42, respectively.

[0559] Nucleotide sequence

[0560] The amino acid sequence of mAb1 H8 is shown as SEQ ID NO. 63, the encoding nucleic acid is shown as SEQ ID NO. 64, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 60, 42, respectively.

[0561] Nucleotide sequence

[0562] The amino acid sequence of mAb1 K1 is shown as SEQ ID NO. 65, the encoding nucleic acid is shown as SEQ ID NO. 66, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47, respectively.

[0563] Nucleotide sequence

[0564] The amino acid sequence of mAb1 K2 is shown as SEQ ID NO. 67, the encoding nucleic acid is shown as SEQ ID NO. 68, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47, respectively.

[0565] Nucleotide sequence

[0566] The amino acid sequence of mAb1 K3 is shown as SEQ ID NO. 69, the encoding nucleic acid is shown as SEQ ID NO. 70, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47, respectively.

[0567] Nucleotide sequence

[0568] The amino acid sequence of mAb1 K4 is shown as SEQ ID NO. 71, its encoding nucleic acid is shown as SEQ ID NO. 72, and its CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0569] Nucleotide sequence

[0570] The amino acid sequence of mAb1 K5 is shown as SEQ ID NO. 73, its encoding nucleic acid is shown as SEQ ID NO. 74, and its CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0571] Nucleotide sequence

[0572] The amino acid sequence of mAb1 K6 is shown as SEQ ID NO. 75, its encoding nucleic acid is shown as SEQ ID NO. 76, and its CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0573] Nucleotide sequence

[0574] The heavy chain amino acid sequence of mAb1 H6K3 is shown as SEQ ID NO. 77, its encoding nucleic acid is shown as SEQ ID NO. 78, and the light chain amino acid sequence is shown as SEQ ID NO. 79, its encoding nucleic acid is shown as SEQ ID NO. 80.

[0575] Heavy chain amino acid sequence

[0576] Heavy chain nucleic acid sequence

[0577] Light chain amino acid sequence

[0578] Light chain nucleic acid sequence

[0579] The heavy chain amino acid sequence of mAb1 H6K5 is shown as SEQ ID NO. 81, and the encoding nucleic acid is shown as SEQ ID NO. 82; the light chain amino acid sequence is shown as SEQ ID NO. 83, and the encoding nucleic acid is shown as SEQ ID NO. 84.

[0580] Heavy chain amino acid sequence

[0581] Heavy chain nucleic acid sequence

[0582] Light chain amino acid sequence

[0583] Light chain nucleic acid sequence

[0584] Example 5, affinity characterization of anti-CEACAM5 humanized antibodies

[0585] 5.1 Affinity of anti-CEACAM5 humanized antibodies to hCEACAM5

[0586] After the light chain variable region and the heavy chain variable region are synthesized in full sequence, respectively, they are cloned into eukaryotic expression vectors containing the antibody kappa chain constant region Ckappa or human IgG1 constant region CH1-CH3. After the light chain and heavy chain plasmids are combined and paired, cells are transfected to express the antibody. The affinity between the antibody and the antigen is detected by Gator (BLI, Bio-Layer Interferometry). The commercial Protein A or HFC (anti-hIgG Fc) probe is used to fix the antibody, and then the antigen is combined to record the intermolecular binding and dissociation process in real time, so as to determine the affinity between the antigen and the antibody. One column of Protein A probes is placed on a MAX plate containing 260 μl of buffer, soaked for 10 minutes, and one column of regeneration solution and buffer is added to the MAX plate as a neutralizing liquid. The antibody is diluted with buffer to 5 μg / ml, and 200 μl / well is transferred to the 96-well sample. The antibody solidification height is 1.5 nm. The monovalent antigen is diluted with buffer to a maximum concentration of 10×KD, and is diluted in a 3-fold concentration gradient, a total of 3 concentration gradients and a blank buffer as a negative control. Two columns of buffer are set in the 96-well plate for balancing the probe. The sample plate and the MAX plate are placed in the corresponding positions of the instrument, and the sample plate is set to tilt mode. The program baseline1 is set to 30 seconds, loading is set to 100 seconds, baseline2 is set to 30 seconds, association is set to 150 seconds, dissociation is set to 150 seconds, regeneration / neutralization is set to 5 seconds each and repeated 3 times.

[0587] The affinity of the humanized VH and VL pairs is shown in Table 8. The H5, H6 heavy chain and K3, K4, K5, K6 light chain pairs have high affinity.

[0588] Table 8. Affinity of humanized light-heavy chain paired antibodies

[0589] 5.2 Binding of anti-CEACAM5 humanized antibodies to tumor cells

[0590] The binding of the candidate antibodies to CEACAM5 positive tumor cells was detected by FACS. MKN45 cells in the logarithmic growth phase were adjusted to 5 x 105cells / ml with PBS containing 1% BSA, 100 μl / well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, 100 μl of a gradient dilution of the chimera was added to each well, and incubated at 4°C for 60 minutes. The secondary antibody was added at 50 μl / well of Alexa Fluro 647 labeled goat anti-human IgG Fc (1:300 dilution), and incubated on ice for 20 minutes. The binding activity of the CEACAM5 candidate antibodies to the cells was detected by flow cytometry. 5

[0591] The results are shown in Table 9. The humanized antibodies have strong binding activity to CEACAM5 positive tumor cells.

[0592] Table 9. EC50 of humanized light-heavy chain paired antibodies binding to tumor cells

[0593] 5.3 Binding of anti-CEACAM5 humanized antibodies to various species of CECAM5 antigens and CEACAM family proteins

[0594] The monoclonal antibody was digested into Fab with papain to detect the affinity to various species of CECAM5 antigens and CEACAM family proteins. The antibody and activated papain were mixed at a mass ratio of 10:1, incubated at 37°C for 3 hours, then an appropriate amount of Protein A filler (40 μg of protein added to 1 μL of Protein A filler) was added to the digestion product and incubated for 20 min to remove the undigested IgG and digested Fc, and finally obtain the Fab fragment.

[0595] ​The surface plasmon resonance technology (Biacore T200 / SPR) was used to couple Anti-His monoclonal antibody to the chip surface by amino coupling on the surface of CM5 chip, and various genus antigen proteins were captured on the chip surface as ligands by specific binding of the 6*His tag of Anti-His monoclonal antibody to the antigen, then gradient-diluted Fab was flowed through the chip surface as analyte, the monovalent affinity of Fab to the antigen was detected, and finally the data was analyzed by Biacore analysis software 3.0 analysis software, the data was processed by langmiur 1:1 kinetics fitting, and the 1:1 affinity between antigen and antibody was obtained.

[0596] The affinity detection results are shown in FIG. 1, the affinity of mAb1 H6K3 and mAb1 H6K5 to human and cynomolgus CEACAM5 proteins is stronger than that of ref1, and they do not bind to mouse CEACAM5 and CEACAM5 family proteins. The affinity of CEACAM5 mAb1 H6K3 to human and cynomolgus CEACAM5 proteins is within 10 times.

[0597] Example 6, endocytosis activity of anti-CEACAM5 humanized antibody in CEACAM5 highly-expressed tumor cells

[0598] The density of CEACAM5-expressing cells MKN45 was adjusted to 1×10 4 cells / well, and plated one day in advance. The next day, gradient-diluted DT3C protein (133 nM as the starting concentration, 5-fold gradient, a total of 9 dilutions) was incubated with experimental group antibodies (10 ug / mL) at 37°C for 30 min to form antibody-DT3C complexes, and then the antibody-DT3C complexes were added to the cell culture medium and incubated with the cells for 3 days. The CCK-8 kit was used to detect the number of living cells in each experimental group, the cell survival rate was calculated, and the DT3C concentration-survival rate curve was obtained by fitting with a four-parameter model.

[0599] The results are shown in FIG. 2, mAb1 H6K3 and mAb1 H6K5 antibodies have good endocytosis activity, and the endocytosis activity is better than that of ref1.

[0600] Example 7, FACS binding of anti-CEACAM5 humanized antibody to CEACAM5 highly-expressed tumor cells

[0601] The candidate antibodies were detected by FACS for binding to tumor cells with high expression of CEACAM5. MKN45 (National Experimental Cell Resource Sharing Platform, item number: 1101HUM-PUMC000229), BxPc-3 (ATCC, item number: CRL-1687), Caco-2 (ATCC, item number: HTB-37), KatoIII (ATCC, item number: HTB-103), SNU16 (ATCC, item number: CRL-5974), HPAC (ATCC, item number: CRL-2119), NCI-H2110 (ATCC, item number: CRL-5924) cells in logarithmic growth phase were adjusted to 5x10 5 cells / ml, 100 μl / well of cell suspension was added to a 96-well U-shaped plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, 100 μl of gradient-diluted chimera (starting concentration 200 nM, 4-fold dilution) was added to each well, and incubated at 4°C for 60 minutes. Secondary antibody was added with 50 μl / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 minutes, and the binding activity of CEACAM5 candidate molecules to cells was detected by flow cytometry.

[0602] The results are shown in Figure 3. The mAb1 H6K3 antibody had good binding capacity to tumor cells expressing CEACAM5.

[0603] Example 8, Immunogenicity stimulation of anti-CEACAM5 humanized antibody to PBMC

[0604] Peripheral venous blood was collected from 24 healthy volunteers, PBMC was collected by Ficoll centrifugation, washed once with PBS, counted, and the cell density was adjusted to 2.5x10 6 cells / ml, 100 μl was added to a 96-well U-shaped plate. The diluted antibody sample 100 μl was added to the cells, and incubated at 37°C, 5% CO2 for 48 hours. Donanemab (homemade) was used as a positive control, mcKLH (Thermo Fisher, item number 77600) was used as a positive control 2, and the well without sample was used as a negative control.

[0605] 96-well plate discarded supernatant, 200 μl / well flow buffer cleaning once, add blocking fluid 50 μl, incubate on ice for 30 min, 1 μl / well add anti-human CD4, APC anti-human CD137 (OX40), PE anti-human CD134 (4-1BB) fluorescent antibody, incubate on ice for 45 min, add 5 μg / ml PI 50 μl staining 5 min, flow buffer cleaning 2 times, 100 μl PBS resuspended, flow cytometry analysis.

[0606] The results are shown in Figure 4, and the CEACAM5 humanized candidate antibody mAb1 H6K3 has no obvious immunogenicity reaction.

[0607] Example 9, PK parameters of anti-CEACAM5 humanized antibody in FcRn humanized mice

[0608] 6-8 week old female B-hFcRn mice (Bioss, Cat No: 110001) were raised in a sterile animal room at 25°C with 12h light-dark alternation, and free diet and water. The experimental group mice (4 mice per group) were injected with 10 mg / kg of antibody via the tail vein, and the antibody was detected for endotoxin and diluted in about 100 μL of sterile saline. 100-200 μL of orbital venous blood was collected before injection (0h) and 5min, 7h, D1, D3, D5, D7, D14, D21, D28, D35 after injection, and the serum was separated by centrifugation and stored at -80°C for detection.

[0609] hCEACAM5 (cHis) was diluted with coating solution to 0.5 pg / ml, 100 pL / well was added to the enzyme-labeled plate, and coated overnight at 2-8°C. Then wash the plate 3 times with 0.05% PBST (plate 1), then add 200 pL / well of 2% milk into the plate, room temperature blocking for 60 min, wash the plate 1 time with 0.05% PBST. The standard was diluted with blank C57 mouse serum from 10 ug / mL, 2-fold gradient dilution, then diluted with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. The standard was diluted with blank mouse serum to high concentration quality control (3750 ng / mL), medium concentration quality control (800 ng / mL), low concentration quality control (58.5 ng / mL), then diluted with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. The sample was diluted with 0.5% milk, then diluted with C57 mouse serum to the corresponding times, then washed the plate 5 times with 0.05% PBST. Anti-hIgG1 HRP (biolegend, Cat: 410603) was diluted 6000 times with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. Wash the plate 5 times, add color developing solution, 100 pL / well, avoid bubbles, paste the sealing film, and develop color in the 37°C constant temperature incubator for 10 min in the dark. Add 2M sulfuric acid termination solution, 50 pL / well, avoid bubbles, shake well, and read on the enzyme-labeled instrument within 5 min (wavelength: 450 nm).

[0610] The results are shown in Figure 5. The half-life of the CEACAM5 humanized clone mAb1 H6K3 hIgG1 in the FcRn humanized mice was 165.7 h, and the half-life of the ref1 antibody was 116.5 h. The blood clearance rate of the mAb1 H6K3 hIgG1 in the humanized mice was lower than that of the ref1 antibody.

[0611] Example 10, ADC preparation example

[0612] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The chemical shift d is given in units of 10 -6 (ppm). The NMR was measured by a Bruker nuclear magnetic instrument, and the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0613] The LCMS was measured by: Agilent 1260 Infinity II (ESI) mass spectrometer, Waters UPLC H Class plus (ESI), or Shimadzu LCMS-2020 (ESI).

[0614] High performance liquid chromatography (HPLC) analysis uses Agilent 1260 or Shimadzu LC-20AD.

[0615] Preparative high performance liquid chromatography (pre-HPLC) uses GILSON GX-281 or Agilent 1260 Infinity II preparative liquid.

[0616] Chiral preparation uses critical fluid chromatography (SFC), and the instrument uses Shimadzu LC-30Adsf or Shimadzu LC-20AD.

[0617] Thin layer chromatography silica gel plate uses Anhui Liangchen Silicon Source Material Co., Ltd. GF254 acrylic adhesive silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.2 mm silica gel plate, and the thin layer chromatography separation and purification product adopts a specification of 0.5 mm silica gel plate.

[0618] Column chromatography generally uses 200-300 mesh silica gel from Anhui Liangchen Silicon Source Material Co., Ltd. as a carrier.

[0619] Determination of average inhibition rate and IC 50 of kinases uses SpectraMax i3X microplate reader (MD, USA).

[0620] Known starting materials of the present disclosure can be synthesized or purchased from companies such as Bid pharmaceutical, Leyan, Shaoyuan Chemical Technology, and Anning Chemicals, etc. according to methods known in the art.

[0621] Unless otherwise specified, the reactions in the following examples were carried out under an argon or nitrogen atmosphere.

[0622] An argon or nitrogen atmosphere refers to a reaction bottle connected to an argon or nitrogen balloon with a volume of about 1 L.

[0623] A hydrogen atmosphere refers to a reaction bottle connected to a hydrogen balloon with a volume of about 1 L.

[0624] Hydrogenation reactions are usually vacuumed, filled with hydrogen, and repeated 3 times.

[0625] An oxygen atmosphere refers to a reaction bottle connected to an oxygen balloon with a volume of about 1 L.

[0626] Unless otherwise specified, the solution in the following examples refers to an aqueous solution, and the reaction temperature is room temperature, which is 20-30°C.

[0627] The monitoring of the reaction progress in the examples employs thin layer chromatography (TLC), the developing solvent used for the reaction, the eluent system of the column chromatography used for purifying the compounds, and the developing solvent system of the thin layer chromatography include: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system, the volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagent such as triethylamine and acetic acid can also be added for adjustment.

[0628] Example 10-1: Preparation of Compound 1 and Isomers 1-A, 1-B, 1-C and 1-D thereof

[0629] N-((1R,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxyacetamide 1-A

[0630] N-((1S,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxyacetamide 1-B

[0631] N-((1S,9R)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxyacetamide 1-C

[0632] N-((1R,9R)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxyacetamide 1-D

[0633] First step

[0634] 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolane]-7-yl)-3- cyclopropylpropanoic acid ethyl ester 1b

[0635] Dissolve 1a (1.01 g, 3.31 mmol, prepared using the method disclosed in Example 30, page 28 of the patent application “WO2019238046A1”) in 15 mL of acetonitrile, add bromomethylcyclopropane (894.93 mg, 6.63 mmol) and potassium carbonate (916.16 mg, 6.63 mmol), stir at 80°C for 13 hours. Add 10 mL of water, extract the diluted reaction solution with ethyl acetate (15 mL x 2), wash the organic phase with saturated sodium chloride solution (10 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with the developing system A to obtain the title product 1b (1.12 g, yield: 92%) in the form of a yellow solid.

[0636] MS m / z (ESI): 359.1 [M+1].

[0637] Second step

[0638] 3-cyclopropyl-2-(6-formyl-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7- yl)propanoic acid ethyl ester 1c

[0639] Dissolve 1b (1.12 g, 3.05 mmol) in a mixture solvent of 5 mL of water, 5 mL of acetonitrile and 5 mL of formic acid, protect under nitrogen, add Raney nickel (261.72 mg), replace with hydrogen three times, stir the reaction solution under hydrogen (15 Psi) at 60°C for 4 hours. Filter the reaction solution with celite, wash the filter cake with dichloromethane (50 mL x 3), wash the filtrate with hydrochloric acid aqueous solution (4 M, 20 mL) and then with sodium carbonate aqueous solution (12 M, 50 mL), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by reverse phase liquid chromatography (separation conditions: column: 120 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water: B-acetonitrile, gradient elution, flow rate: 85 mL / min, instrument: ISCO) to obtain the title product 1c (680 mg, yield: 60%) in the form of a yellow solid.

[0640] MS m / z (ESI): 362.1 [M+1].

[0641] Third step

[0642] 4-(cyclopropylmethyl)-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4-f]indolizine-6,2'- [1,3]dioxolan]-3,10-dione 1d

[0643] Dissolve 1c (680 mg, 1.85 mmol) in 10 mL of dichloromethane, protect under nitrogen, and cool to 0 °C in an ice water bath. Add sodium borohydride (108.02 mg, 2.86 mmol) in portions. Stir the reaction at 0 °C for 30 minutes. Add acetic acid (133.15 mg, 2.22 mmol) dropwise at 25 °C. Gas is generated. Continue stirring for 2 hours. Add 30 mL of water dropwise at 15 °C. Gas is generated. Stir at 15 °C for 1.5 hours. Wash the reaction with water (50 mL). Add p-toluenesulfonic acid monohydrate (35.15 mg, 184.78 μmol) to the washed organic phase at 15 °C. Stir at 15 °C for 12 hours. Add 50 mL of water. Extract the reaction with dichloromethane (45 mL x 3). Dry the organic phase over anhydrous sodium sulfate. Filter and concentrate the filtrate by distillation under reduced pressure. Purify the resulting residue by silica gel column chromatography with developing system B to obtain the title product 1d (200 mg, yield: 32%) as a yellow oil.

[0644] MS m / z (ESI): 318.1 [M+1].

[0645] Fourth Step

[0646] 4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e

[0647] Dissolve 1d (202.13 mg, 598.74 μmol) in 0.5 mL of methanol. Cool to 0 °C in an ice water bath. Add potassium carbonate (82.75 mg, 598.74 μmol) in portions. Stir at 0 °C for 5 hours under oxygen bubbling (15 psi). Pour the reaction into 10 mL of saturated aqueous ammonium chloride. Concentrate under reduced pressure to remove methanol. Extract the reaction with dichloromethane (20 mL x 3). Dry the organic phase over anhydrous sodium sulfate. Filter and concentrate the filtrate by distillation under reduced pressure to obtain the crude title product 1e (140 mg) as a yellow oil. The product is used directly in the next reaction without purification.

[0648] MS m / z (ESI): 334.1 [M+1].

[0649] Fifth Step

[0650] 4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)- trione 1f

[0651] Dissolve 1e (64.52 mg, 180.00 μmol) in a mixture solution of 0.5 mL trifluoroacetic acid and 0.125 mL water, stir at 25 °C for 1 h. Concentrate the reaction solution under reduced pressure, the obtained residue gives the crude product of title 1f (40 mg) as a yellow solid without purification, the product is used directly in the next step without purification.

[0652] MS m / z (ESI): 290.1 [M+1].

[0653] Sixth step

[0654] N-(9-(cyclopropylmethyl)-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]indolizino[1,2-b]quinolin-1-yl)acetamide 1h

[0655] Dissolve 1f (40 mg, 128.59 μmol) and 1g (38.62 mg, 154.31 μmol, prepared by the method disclosed in patent application "CN111065621A" page 61 example 5-1) with pyridine 4-methylbenzenesulfonic acid (6.46 mg, 25.72 μmol) in 0.5 mL toluene, protect under nitrogen, stir at 130 °C for 2 h. Filter the reaction solution with diatomite, add 10 mL water to the filtrate, extract with dichloromethane (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue by preparative thin layer chromatography with developing system A, to give the title product 1h (15 mg, yield: 23%) as a brown solid.

[0656] MS m / z (ESI): 504.2 [M+1].

[0657] Seventh step

[0658] 1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate 1i

[0659] Dissolve 1h (50 mg, 96.5 μmol) in 1 mL ethyleneglycol dimethyl ether, add 0.5 mL methylsulfonic acid and 0.5 mL water, stir at 85 °C for 18 h. Pour the reaction solution into 10 mL water, adjust the pH to 7-8 with saturated sodium bicarbonate solution, extract with dichloromethane (10 mL x 5), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, to give the crude product of title 1i (40 mg) as a brown solid without purification, the product is used directly in the next step without purification.

[0660] MS m / z (ESI): 462.3 [M+1].

[0661] Eighth step

[0662] N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxyacetamide 1-A

[0663] N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxyacetamide 1-B

[0664] N-((1S,9R)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxyacetamide 1-C

[0665] N-((1R,9R)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxyacetamide 1-D

[0666] The methanesulfonate salt of 1i (35 mg, 62.2 μmol) and glycolic acid (5.7 mg, 74.6 μmol) were dissolved in 1.5 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (12.6 mg, 93.2 μmol), N,N-diisopropylethylamine (16.1 mg, 124 μmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.9 mg, 93.2 μmol) were added successively, and stirred at 25 °C for 1 hour. 20 mL of water was added to the reaction solution, and the diluted reaction solution was extracted with dichloromethane (10 mL x 5), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by supercritical fluid chromatography to obtain four single configuration title products 1-A, 1-B, 1-C and 1-D:

[0667] Single configuration compound one of compound 1 (labeled as 1-B, 1.02 mg)

[0668] SFC analysis: Retention time 1.472 min, purity: 98%. (Chromatographic column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol and Acetonitrile (0.05% Diethylamine), Isocratic elution: B: 60%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0669] MS m / z (ESI): 520.5 [M+1].

[0670] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.61 (m, 2H), 5.73 - 5.66 (m, 1H), 5.63 - 5.56 (m, 1H), 5.40 - 5.32 (m, 2H), 5.12 - 5.06 (m, 1H), 4.28 - 4.22 (m, 1H), 4.18 - 4.12 (m, 1H), 3.32 - 3.32 (m, 1H), 3.20 - 3.11 (m, 1H), 2.42 (s, 3H), 2.39 - 2.30 (m, 1H), 2.29 - 2.19 (m, 1H), 1.97 - 1.89 (m, 1H), 1.88 - 1.80 (m, 1H), 0.94 - 0.86 (m, 1H), 0.51 - 0.38 (m, 2H), 0.14 - 0.07 (m, 1H), 0.04 - -0.01 (m, 1H).

[0671] Single configuration compound two of compound 1 (labeled as 1-A, 1.02 mg)

[0672] SFC analysis: Retention time 2.075 min, purity: 93%. (Chromatographic column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5%-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0673] MS m / z (ESI): 520.4 [M+1].

[0674] 1H NMR (400 MHz, CD3OD) δ 7.66 - 7.53 (m, 2H), 5.70 - 5.63 (m, 1H), 5.60 - 5.54 (m, 1H), 5.41 - 5.31 (m, 2H), 5.22 - 5.14 (m, 1H), 4.34 - 4.23 (m, 1H), 4.22 - 4.11 (m, 1H), 3.43 - 3.36 (m, 1H), 3.22 - 3.13 (m, 1H), 2.43 - 2.37 (m, 3H), 2.35 - 2.26 (m, 2H), 1.95 - 1.80 (m, 2H), 0.93 - 0.86 (m, 1H), 0.55 - 0.35 (m, 2H), 0.17 - 0.08 (m, 1H), 0.07 - -0.03 (m, 1H).

[0675] Single configuration compound three of compound 1 (1.21 mg)

[0676] SFC analysis: Retention time 0.468 min, purity: 98%. (Chromatographic column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), gradient elution: B%: 5-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0677] MS m / z (ESI): 520.1 [M+1].

[0678] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.64 (m, 2H), 5.73 - 5.67 (m, 1H), 5.62 - 5.56 (m, 1H), 5.43 - 5.40 (m, 1H), 5.38 - 5.35 (m, 1H), 5.18 - 5.16 (m, 1H), 4.28 - 4.21 (m, 1H), 4.18 - 4.12 (m, 1H), 3.50 - 3.45 (m, 1H), 3.20 - 3.12 (m, 1H), 2.49 - 2.40 (m, 3H), 2.36 - 2.23 (m, 2H), 1.96 - 1.82 (m, 2H), 0.93 - 0.88 (m, 1H), 0.52 - 0.39 (m, 2H), 0.15 - 0.08 (m, 1H), 0.06 - -0.02 (m, 1H).

[0679] Single configuration compound four of compound 1 (0.27 mg)

[0680] SFC analysis: Retention time 0.464 min, purity: 98%. (Column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0681] MS m / z (ESI): 520.1 [M+1].

[0682] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.62 (m, 2H), 5.73 - 5.67 (m, 1H), 5.62 - 5.56 (m, 1H), 5.41 - 5.35 (m, 2H), 5.23 - 5.20 (m, 1H), 4.33 - 4.23 (m, 1H), 4.21 - 4.12 (m, 1H), 3.38 - 3.36 (m, 1H), 3.22 - 3.13 (m, 1H), 2.44 (s, 3H), 2.37 - 2.28 (m, 2H), 1.96 - 1.83 (m, 2H), 0.91 - 0.86 (m, 1H), 0.50 - 0.38 (m, 2H), 0.14 - 0.09 (m, 1H), 0.06 - -0.01 (m, 1H).

[0683] Examples 10-2 to 10-6

[0684] The following compounds were prepared according to the procedure of Reference Example 10-1, selecting the corresponding starting materials:

[0685] Example 10-2: Preparation of compound 2 and its isomers 2-A and 2-B

[0686] (S)-N-((1S,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 2-A

[0687] 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 2-A

[0688] (S)-N-((1R,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 2-A

[0689] 1H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)-2- hydroxypropanamide 2-B

[0690] First step

[0691] (S)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-1

[0692] (R)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-2

[0693] 1e (1.30 g, 3.88 mmol) was separated by SFC (Separation condition: Column: DAICEL CHIRALPAK AS 250 mm x 50 mm, 10 pm; Mobile phase: A-Carbon dioxide: B-Methanol (0.1% NH3-H2O), Isocratic elution: B: 20%, Flow rate: 120 mL / min, Instrument: Shimadzu LC-30ADsf) to give the title product 1e-1 (301 mg, yield: 22.1%) as a yellow solid, and the title product 1e-2 (285 mg, yield: 19.4%) as a yellow solid.

[0694] Single configuration compound 1e-1

[0695] SFC analysis: Retention time 1.392 min. (Column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), Gradient elution: B%: 5%-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0696] MS m / z (ESI): 334.0 [M+1].

[0697] Single configuration compound 1e-2

[0698] SFC analysis: Retention time 1.762 min. (Column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), Gradient elution: B%: 5%-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0699] MS m / z (ESI): 333.9 [M+1].

[0700] Second Step

[0701] (S)-4-(Cyclopropylmethyl)-4-hydroxy-7,8-dihydro-lH-pyrano[3,4- f]indolizine-3,6,10(4H)-trione If-1

[0702] Dissolve le-1 (301 mg, 857 μmol) in 2 mL of a mixture of trifluoroacetic acid and 0.5 mL of water, stir at 25 °C for 4 hours. Concentrate the reaction solution under reduced pressure, the obtained residue gives the crude title product If-1 (209 mg) as a yellow solid without purification, the product is used directly in the next step reaction without purification.

[0703] MS m / z (ESI): 290.1 [M+1].

[0704] Third Step

[0705] N-((9S)-9-(Cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)acetamide Ih-1

[0706] Dissolve If-1 (50.0 mg, 159 μmol) and Ig (48.0 mg, 192 μmol), pyridine 4- methylbenzenesulfonic acid (8.0 mg, 31.8 μmol) in 2 mL of toluene, protect under nitrogen, stir at 120 °C for 16 hours. Filter the reaction solution with diatomite, add 20 mL of water to the filtrate, extract with dichloromethane (15 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue with preparative thin layer chromatography with developing system A, to give the title product Ih-1 (190 mg, yield: 56%) as a yellow solid.

[0707] MS m / z (ESI): 504.1 [M+1].

[0708] Fourth Step

[0709] (9S)-l-Amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 1,2,3,9,12,15-hexahydro-10H, 13H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinoline-10,13-dione Ii-1

[0710] Dissolve 1h-1 (60.0 mg, 111 μmol) in 4.5 mL ethyleneglycol dimethyl ether, add 1.5 mL methylsulfonic acid and 1.5 mL water, stir at 85 °C for 16 hours. Add the reaction solution dropwise into 10 mL water with stirring, extract with dichloromethane (10 mL x 5), wash the organic phase with 25 mL 0.05 M aqueous hydrochloric acid, filter the aqueous phase, combine all the aqueous extracts, adjust the aqueous phase to pH 7-8 with saturated potassium bicarbonate solution at 0 °C, extract with mixed solvent (dichloromethane / methanol: 20 / 3, 15 mL x 5), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to give the crude title product 1i-1 (22 mg) as a brown solid, use the product directly in the next step without purification. MS m / z (ESI): 462.1 [M+1].

[0711] Fifth step

[0712] (S)-N-((1R,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxypropanamide 2-B

[0713] (S)-N-((1R,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b] quinolin-1-yl)-2-hydroxypropanamide 2-B

[0714] The mesylate salt of 1i-1 (22.0 mg, 47.7 μmol) and (2S)-2-hydroxypropanoic acid (6.4 mg, 71.5 μmol) were dissolved in 1.5 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (9.7 mg, 71.5 μmol), N,N-diisopropylethylamine (18.5 mg, 143 μmol) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.7 mg, 71.5 μmol) were added successively, and stirred at 25 °C for 4 hours. To the reaction solution was added toluene (4 mL x 2), and concentrated under reduced pressure at 25 °C, the obtained residue was diluted with N,N-dimethylformamide to 1.5 mL, and the diluted solution was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 26%-56%), to obtain a mixture (10 mg), which was further purified by supercritical fluid chromatography to obtain two single configuration title products 2-A and 2-B:

[0715] Single configuration compound 2-A of compound 2 (5.55 mg, yield: 20%, off-white solid)

[0716] SFC analysis: retention time 1.381 minute, purity: 92%. (column: Chiralcel OJ-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0717] MS m / z (ESI): 534.1 [M+1].

[0718] 1H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.53 - 7.49 (m, 1H), 5.65 - 5.54 (m, 2H), 5.36 - 5.29 (m, 1H), 5.25 - 5.18 (m, 1H), 4.81 - 4.77 (m, 1H), 4.41 - 4.33 (m, 1H), 3.28 - 3.23 (m, 1H), 3.15 - 3.05 (m, 1H), 2.36 - 2.33 (m, 3H), 2.33 - 2.27 (m, 1H), 2.17 - 2.10 (m, 1H), 1.95 - 1.87 (m, 1H), 1.85 - 1.77 (m, 1H), 1.47 - 1.44 (m, 3H), 0.91 - 0.87 (m, 1H), 0.51 - 0.35 (m, 2H), 0.15 - 0.06 (m, 1H), 0.04 - -0.05 (m, 1H).

[0719] Single configuration compound 2-B of compound 2 (4.06 mg, yield: 16%, yellow solid)

[0720] SFC analysis: Retention time 1.299 min, purity: 97%. (Chromatographic column: Chiralcel OJ-3 50x4.6 mm I.D., 3 pm, mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), gradient elution: B%: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0721] MS m / z (ESI): 534.1 [M+1].

[0722] 1 H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.57 - 7.51 (m, 1H), 5.65 - 5.52 (m, 2H), 5.42 - 5.30 (m, 2H), 5.12 - 5.02 (m, 1H), 4.38 - 4.30 (m, 1H), 3.29 - 3.24 (m, 1H), 3.20 - 3.07 (m, 1H), 2.43 - 2.37 (m, 3H), 2.35 - 2.26 (m, 2H), 1.97 - 1.80 (m, 2H), 1.64 - 1.58 (m, 3H), 0.92 - 0.83 (m, 1H), 0.51 - 0.36 (m, 2H), 0.16 - 0.07 (m, 1H), 0.06 - -0.02 (m, 1H).

[0723] Example 10-3: Preparation of compound 3 and its isomers 3-A and 3-B

[0724] (R)-N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-A

[0725] (R)-N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-B

[0726] First step

[0727] (R)-N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-A

[0728] (R)-N-((1R,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b] quinolin-l-yl)-2-hydroxypropanamide 3-B

[0729] Dissolve li-1 (40.0 mg, 80.8 μmol) and (2R)-2-hydroxypropanoic acid (10.9 mg, 121 μmol) in 1.5 mL of N,N-dimethylformamide, then add 1-hydroxybenzotriazole (16.4 mg, 121 μmol), N,N-diisopropylethylamine (31.3 mg, 242 μmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.2 mg, 121 μmol) successively, stir at 25 °C for 4 hours. Add toluene (4 mL x 2) to the reaction solution, concentrate under reduced pressure, dilute the obtained residue with N,N-dimethylformamide to 1.5 mL, and purify the diluted solution by pre-HPLC (column: Xtimate C18 150 x 40 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 23%-53%), and purify the obtained mixture by supercritical fluid chromatography to give two single configuration title products 3-A and 3-B:

[0730] Single configuration compound 3-A of compound 3 (6.11 mg, yield: 14%, off-white solid)

[0731] SFC analysis: Retention time 1.413 min, purity: 97%. (Column: Chiralcel OJ-3 50x4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0732] MS m / z (ESI): 534.3 [M+1].

[0733] 1 H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.54 - 7.48 (m, 1H), 5.70 - 5.62 (m, 1H), 5.59 - 5.51 (m, 1H), 5.38 - 5.30 (m, 1H), 5.29 - 5.21 (m, 1H), 4.83 - 4.78 (m, 1H), 4.33 - 4.25 (m, 1H), 3.30 - 3.23 (m, 1H), 3.20 - 3.09 (m, 1H), 2.40 - 2.36 (m, 3H), 2.35 - 2.31 (m, 1H), 2.25 - 2.14 (m, 1H), 1.95 - 1.88 (m, 1H), 1.86 - 1.79 (m, 1H), 1.60 - 1.54 (m, 3H), 0.95 - 0.82 (m, 1H), 0.52 - 0.35 (m, 2H), 0.15 - 0.07 (m, 1H), 0.04 - -0.04 (m, 1H).

[0734] Single configuration compound 3-B of compound 3 (2.78 mg, yield: 6%, off-white solid)

[0735] SFC analysis: Retention time 2.013 min, purity: 97%. (Column: Chiralcel OD-3 50x4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol (0.05% Diethylamine), Gradient elution: B%: 5-40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0736] MS m / z (ESI): 534.1 [M+1].

[0737] 1H NMR (400 MHz, DMSO-d6) δ 8.42 - 8.36 (m, 1H), 7.81 - 7.75 (m, 1H), 7.37 (s, 1H), 6.57 (s, 1H), 5.58 - 5.49 (m, 2H), 5.41 (s, 2H), 5.26 - 5.12 (m, 2H), 4.17 - 4.09 (m, 1H), 3.19 - 3.14 (m, 1H), 2.42 - 2.36 (m, 3H), 2.23 - 2.10 (m, 2H), 1.90 - 1.81 (m, 1H), 1.79 - 1.71 (m, 1H), 1.32 - 1.27 (m, 3H), 0.85 - 0.77 (m, 1H), 0.39 - 0.27 (m, 2H), 0.10 - 0.02 (m, 1H), -0.03 - -0.11 (m, 1H).

[0738] Example 10-4: Preparation of Compound 4 and its isomers 4-A and 4-B

[0739] (S)-2-cyclopropyl-N-((1S,9S)-9-(cyclopropylmethyl)-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] indolizino[1, 2-b]quinolin-1 -yl)-2-hydroxyacetamide 4-A

[0740] (S)-2-cyclopropyl-N-((1R,9S)-9-(cyclopropylmethyl)-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] indolizino[1, 2-b]quinolin-1 -yl)-2-hydroxyacetamide 4-B

[0741] First Step

[0742] The mesylate salt of 1i-1 (37.0 mg, 78.5 μmol) and (2S)-2-cyclopropyl-2- hydroxyacetic acid (9.59 mg, 78.5 μmol) were dissolved in 1 mL of N,N- dimethylformamide, then 1-hydroxybenzotriazole (15.9 mg, 118 μmol), N,N- diisopropylethylamine (30.4 mg, 236 μmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (22.6 mg, 118 μmol) were added in turn, and stirred at 25 °C for 12 hours. To the reaction solution was added ethyl acetate (10 mL), washed with water (5 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 31%-61%), to obtain a mixture in the form of a yellow solid (15 mg, yield 31.2%), which was separated by preparative SFC (column: REGIS (s,s) WHELK-O1 250 x 30 mm, 5 μm; mobile phase: A-carbon dioxide (isopropanol), B-acetonitrile; isocratic elution: B: 65%), to obtain two single configuration title products 4-A and 4-B:

[0743] Single configuration compound 4-A of compound 4 (7.0 mg, yield: 50.2%, yellow solid)

[0744] SFC analysis: retention time 0.761 min, purity: 98%. (column: (S,S) WHELK-O1 50 x 4.6 mm I.D., 3.5 μm, mobile phase: A-carbon dioxide, B-isopropanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 65%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0745] MS m / z (ESI): 560.5 [M+1].

[0746] 1H NMR (400 MHz, CD3OD) δ 7.67 (s, 1H), 7.64-7.60 (m, 1H), 5.68-5.62 (m, 1H), 5.61-5.55 (m, 1H), 5.41-5.37 (m, 1H), 5.36-5.33 (m, 1H), 5.06-5.00 (m, 1H), 3.87-3.84 (m, 1H), 3.21-3.11 (m, 2H), 2.43-2.40 (m, 3H), 2.37-2.31 (m, 1H), 2.25-2.18 (m, 1H), 1.96-1.89 (m, 1H), 1.87-1.80 (m, 1H), 1.32-1.28 (m, 1H), 0.92-0.87 (m, 1H), 0.61-0.55 (m, 2H), 0.53-0.45 (m, 3H), 0.44-0.38 (m, 1H), 0.14-0.08 (m, 1H), 0.04- -0.03 (m, 1H).

[0747] Single configuration compound 4-B of compound 4 (6.0 mg, yield: 41.9%, yellow solid)

[0748] SFC analysis: Retention time 1.486 min, purity: 96%. (Chromatographic column: (S, S) WHELK-01 50 x 4.6 mm I.D., 3.5 pm, Mobile phase: A-Carbon dioxide, B-Isopropyl alcohol and Acetonitrile (0.05% Diethylamine), Isocratic elution: B: 65%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0749] MS m / z (ESI): 560.3 [M+1].

[0750] 1H NMR (400 MHz, CD3OD) δ 7.64 (s, 1H), 7.61 - 7.57 (m, 1H), 5.67 - 5.62 (m, 1H), 5.59 - 5.52 (m, 1H), 5.46 - 5.39 (m, 1H), 5.38 - 5.32 (m, 1H), 5.21 - 5.14 (m, 1H), 3.72 - 3.68 (m, 1H), 3.22 - 3.11 (m, 2H), 2.42 - 2.39 (m, 3H), 2.34 - 2.28 (m, 2H), 1.96 - 1.89 (m, 1H), 1.87 - 1.81 (m, 1H), 1.42 - 1.35 (m, 1H), 0.91 - 0.85 (m, 1H), 0.69 - 0.58 (m, 3H), 0.56 - 0.51 (m, 1H), 0.48 - 0.40 (m, 2H), 0.15 - 0.07 (m, 1H), 0.06 - -0.02 (m, 1H).

[0751] Example 10-5: Preparation of Compound 5 and its isomers 5-A and 5-B

[0752] (R)-2-cyclopropyl-N-((1S,9S)-9-(cyclopropylmethyl)-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] indolizino[1,2-b]quinolin-1 -yl)-2-hydroxyacetamide 5-A

[0753] (R)-2-cyclopropyl-N-((1R,9S)-9-(cyclopropylmethyl)-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] indolizino[1,2-b]quinolin-1 -yl)-2-hydroxyacetamide 5-B

[0754] First Step

[0755] The mesylate salt of 1i-1 (25.0 mg, 42.3 pmol) and (2R)-2-cyclopropyl-2- hydroxyacetic acid (5.89 mg, 50.7 pmol) were dissolved in 1.5 mL of N,N- dimethylformamide, then 1-hydroxybenzotriazole (8.57 mg, 63.4 pmol), N,N- diisopropylethylamine (16.4 mg, 127 pmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (12.2 mg, 63.4 pmol) were added successively, stirred at 20 °C for 4 hours. Toluene (4 mL x 2) was added to the reaction solution, concentrated under reduced pressure at 25 °C, the obtained residue was diluted with N,N- dimethylformamide to 1.5 mL, the diluted solution was purified by pre-HPLC (column: Phenomenex luna C18 150 x 25 mm x 10 pm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 28%-58%), to obtain two single configuration title products 5-A and 5-B:

[0756] Single configuration compound 5-A of compound 5 (4.95 mg, yield: 20%, off-white solid)

[0757] SFC analysis: retention time 2.115 minutes, purity: 97%. (column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-carbon dioxide, B-methanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 50%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0758] MS m / z (ESI): 560.1 [M+1].

[0759] 1 H NMR (400 MHz, CD3OD) d 7.70 - 7.64 (m, 2H), 5.70 - 5.65 (m, 1H), 5.60 - 5.54 (m, 1H), 5.46 - 5.38 (m, 1H), 5.37 - 5.32 (m, 1H), 5.23 - 5.17 (m, 1H), 3.72 - 3.67 (m, 1H), 3.36 - 3.34 (m, 1H), 3.23 - 3.17 (m, 1H), 2.48 - 2.42 (m, 3H), 2.37 - 2.31 (m, 1H), 2.31 - 2.25 (m, 1H), 1.96 - 1.89 (m, 1H), 1.88 - 1.80 (m, 1H), 1.34 - 1.31 (m, 1H), 0.92 - 0.89 (m, 1H), 0.68 - 0.42 (m, 6H), 0.15 - 0.07 (m, 1H), 0.05 - -0.02 (m, 1H).

[0760] Single configuration compound 5-B of compound 5 (1.95 mg, yield: 8%, white solid)

[0761] SFC analysis: Retention time 1.179 min, purity: 97%. (Chromatographic column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A-Carbon dioxide, B-Methanol and Acetonitrile (0.05% Diethylamine), Isocratic elution: B: 50%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30AD sf).

[0762] MS m / z (ESI): 560.1 [M+1].

[0763] 1 H NMR (400 MHz, CD3OD) δ 7.69 (s, 1H), 7.69 - 7.62 (m, 1H), 5.69 - 5.63 (m, 1H), 5.61 - 5.55 (m, 1H), 5.49 - 5.42 (m, 1H), 5.40 - 5.34 (m, 1H), 5.24 - 5.16 (m, 1H), 3.91 - 3.84 (m, 1H), 3.38 - 3.36 (m, 1H), 3.24 - 3.16 (m, 1H), 2.44 (s, 3H), 2.40 - 2.23 (m, 2H), 1.97 - 1.80 (m, 2H), 1.29 - 1.21 (m, 1H), 0.95 - 0.85 (m, 1H), 0.64 - 0.36 (m, 6H), 0.17 - 0.07 (m, 1H), 0.07 - -0.02 (m, 1H).

[0764] Example 10-6: Preparation of compound 6

[0765] First step

[0766] (1S,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl 1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 1i-1-1

[0767] (1R,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl 1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 1i-1-2

[0768] The 1i-1 (550 mg, 1.10 mmol) was separated by SFC (Separation condition: Column: DAICEL CHIRALPAK IC 250 mm x 50 mm, 10 pm; Mobile phase: A - n- hexane: B - Ethanol, Isocratic elution: B: 45%,) to get the title product 1i-1-1 (260 mg, yield: 41%) as yellow solid, the title product 1i-1-2 (290 mg, yield: 46%) as yellow solid.

[0769] Single configuration compound 1i-1-1

[0770] SFC analysis: Retention time 3.039 min. (Column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - n-hexane (0.05% isopropylamine), B - Ethanol and Acetonitrile (0.05% isopropylamine), Isocratic elution: B: 45%, Flow rate: 1 mL / min, Instrument: Shimadzu LC-20AD).

[0771] MS m / z (ESI): 462.3 [M+1].

[0772] Single configuration compound 1i-1-2

[0773] SFC analysis: Retention time 4.951 min. (Column: Chiralpak IC-3 50 x 4.6 mm I.D., 3 pm, Mobile phase: A - n-hexane (0.05% isopropylamine), B - Ethanol and Acetonitrile (0.05% isopropylamine), Isocratic elution: B: 45%, Flow rate: 1 mL / min, Instrument: Shimadzu LC-20AD).

[0774] MS m / z (ESI): 462.3 [M+1].

[0775] Second step

[0776] Dissolve 1i-1-1 (13 mg, 25.35 µmol), 1-hydroxycyclopropanecarboxylic acid (4 mg, 38.03 µmol) and 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12 mg, 30.42 µmol) in 1 mL of N,N-dimethylformamide, add N,N-diisopropylethylamine (10 mg, 76.06 µmol), stir at 25 °C for 1 hour. Purify the reaction solution by pre-HPLC (column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 µm; mobile phase: A-water (0.1% formic acid), B-acetonitrile; gradient elution: B%: 20%-80%), to obtain the title product 6 (2.25 mg, yield: 16%) in the form of a white solid.

[0777] MS m / z (ESI): 546.3 [M+1].

[0778] 1 H NMR (400 MHz, DMSO-d6) δ 8.66 - 8.58 (m, 1H), 7.80 - 7.74 (m, 1H), 7.41 - 7.34 (m, 1H), 6.62 - 6.58 (m, 1H), 6.37 - 6.29 (m, 1H), 5.61 - 5.53 (m, 1H), 5.48 - 5.37 (m, 2H), 5.33 - 5.24 (m, 1H), 5.12 - 5.04 (m, 1H), 3.28 - 3.22 (m, 1H), 3.17 - 3.06 (m, 1H), 2.44 - 2.33 (m, 3H), 2.28 - 2.15 (m, 2H), 1.86 - 1.73 (m, 2H), 1.27 - 1.15 (m, 2H), 1.00 - 0.89 (m, 2H), 0.87 - 0.75 (m, 1H), 0.40 - 0.25 (m, 2H), 0.08 - 0.03 (m, 1H), -0.06 - -0.14 (m, 1H).

[0779] Examples 10-7 to 10-13

[0780] The following compounds were prepared according to the method of Reference Example 10-1, selecting the corresponding substrates:

[0781] Example 10-24: Preparation of compound 24

[0782] (S)-N-((8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide 24

[0783] First step

[0784] 4-bromo-7-fluoro-5-nitro-2,3-dihydro-1H-indene 24b

[0785] Dissolve 24a (17 g, 79.05 mmol, prepared by the method disclosed in patent application “WO2021093820 A1” page 31 example 9) in 200 mL trifluoroacetic acid, protect with nitrogen, cool to 0 °C with ice water bath, slowly add nitric acid (20.45 g, 292.08 mmol, content 90%) dropwise to the reaction solution, stir for 10 minutes at 0 °C, remove the ice water bath, stir for 30 minutes at 25 °C. Add 100 mL water, extract the diluted reaction solution with ethyl acetate (500 mL x 3), wash the organic phase with water (500 mL x 3), dry over anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system B, to obtain the title product 24b (8.96 g, yield: 44%) in the form of colorless oil.

[0786] 1 H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.0 Hz, 1H), 3.05-3.16 (m, 4H), 2.18-2.27 (m, 2H).

[0787] Second step

[0788] 4-bromo-7-fluoro-2,3-dihydro-1H-inden-5-amine 24c

[0789] Dissolve 24b (8.96 g, 34.61 mmol) in a mixture solution of 100 mL ethanol and 20 mL water, add iron powder (5.80 g, 103.82 mmol) and ammonium chloride (5.55 g, 103.82 mmol) successively, protect with nitrogen, stir the reaction solution at 80 °C for 1 hour. Filter the reaction solution, concentrate the filtrate by reduced pressure distillation, to obtain the crude title product 24c (7.2 g) in the form of white solid. The product is directly used in the next step reaction without purification.

[0790] MS m / z (ESI): 229.9 [M+1].

[0791] Third step

[0792] tert-Butyl (4-bromo-7-fluoro-2,3-dihydro-1H-inden-5-yl)(tert-butoxycarbonyl)carbamate 24d

[0793] Dissolve 24c (7.2 g, 31.29 mmol) in 50 mL of tetrahydrofuran, add di-tert-butyl dicarbonate (27.32 g, 125.18 mmol), 4-dimethylaminopyridine (38.23 mg, 312.94 μmol) and triethylamine (12.67 g, 125.18 mmol), stir the reaction solution at 25 °C for 16 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 24d (9.2 g, yield: 68%) as a white solid.

[0794] MS m / z (ESI): 317.9 [M+1-56-56].

[0795] Fourth step

[0796] tert-Butyl (tert-butoxycarbonyl)(7-fluoro-4-vinyl-2,3-dihydro-1H-inden-5-yl)carbamate 24e

[0797] Dissolve 24d (1.5 g, 4.38 mmol) and vinylboronic acid pinacol ester (810.20 mg, 5.26 mmol) in a mixture solution of 6 mL of 1,4-dioxane and 2 mL of water, protect under nitrogen, add potassium phosphate tribasic (2.79 g, 13.15 mmol) and methane sulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'- biphenyl) (2'-amino-1,1'-biphenyl-2-yl)palladium(II) (371.07 mg, 438.38 μmol) in sequence, stir the reaction solution at 95 °C for 10 hours under nitrogen protection. Add 20 mL of water, dilute the reaction solution with ethyl acetate (10 mL x 3), wash the organic phase with saturated sodium chloride solution (5 mL x 2), dry over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 24e (350 mg, yield: 26%) as a colorless oil.

[0798] MS m / z (ESI): 222.1 [M+1-100-56].

[0799] Fifth step

[0800] tert-Butyl (tert-butoxycarbonyl)(7-fluoro-4-formyl-2,3-dihydro-1H-inden-5-yl)carbamate 24f

[0801] Dissolve 24e (755 mg, 2.00 mmol) in a mixture of 1.5 mL of dioxane and 1.5 mL of water, add sodium periodate (1.29 g, 6.01 mmol) and potassium osmate dihydrate (73.8 mg, 200 µmol), stir at 25 °C for 5 hours. Filter the reaction solution, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 24f (538 mg, yield: 71%) as a yellow solid.

[0802] MS m / z (ESI): 180.2 [M+1-100-100].

[0803] Sixth step

[0804] (S)-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24h

[0805] Dissolve 24f (538 mg, 1.42 mmol) and 24g (i.e. compound 22c as above) (375 mg, 1.42 mmol, prepared by the method disclosed in patent application “WO2019238046 A1” page 28, example 30) in 10 mL of ethanol, add concentrated hydrochloric acid (12 M, 1.58 mL), stir at 80 °C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure to remove ethanol, add 10 mL of water, filter to obtain the filter cake, to obtain the crude title product 24h (500 mg) as a yellow solid. The product is used directly in the next step without purification.

[0806] MS m / z (ESI): 407.2 [M+1].

[0807] Seventh step

[0808] (S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline 6-oxide 24i

[0809] Dissolve 24h (500 mg, 1.21 mmol) in 70 mL acetic acid, under nitrogen protection, add hydrogen peroxide (16.9 g, 149 mmol, content 30%), stir at 75 °C for 3 hours. LCMS shows the raw material 24h remaining, add hydrogen peroxide (16.5 g, 145 mmol, content 30%) again, stir at 75 °C for 4 hours. Pour the reaction liquid into 100 mL water, filter to get the filter cake, get the crude title product 24i (400 mg) in the form of yellow solid. The product is directly used in the next step reaction without purification.

[0810] MS m / z (ESI): 423.2 [M+1].

[0811] Eighth step

[0812] (S)-15-chloro-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24j

[0813] Dissolve 24i (100 mg, 142 μmol) in 2 mL N,N-dimethylformamide, under nitrogen protection, cool to 0 °C in ice water bath, add oxalyl chloride (45.0 mg, 355 μmol), stir at 25 °C for 1 hour. Pour the reaction liquid into 10 mL water, filter to get the filter cake, get the crude title product 24j (70 mg) in the form of brown solid. The product is directly used in the next step reaction without purification.

[0814] MS m / z (ESI): 441.2 [M+1].

[0815] Ninth step

[0816] (S)-((8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)carbamic acid tert-butyl ester 24k

[0817] Dissolve 24j (20.0 mg, 26.3 μmol) and potassium ((tert-butoxycarbonylamino)methyl)trifluoroborate (14.0 mg, 59.2 μmol) in 1 mL of a mixture of dioxane and 0.2 mL of water, protect with nitrogen, add 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (2.89 mg, 3.95 μmol) and potassium carbonate (25.1 mg, 118 μmol), stir at 95 °C under nitrogen for 1 hour. Pour the reaction into aqueous hydrochloric acid (1 M, 2 mL), concentrate the filtrate by distillation under reduced pressure to obtain the crude title product 24k (20 mg) as a brown oil. Use the product directly in the next reaction without purification.

[0818] MS m / z (ESI): 436.2 [M + 1 - 100].

[0819] Tenth step

[0820] (S)-15-(Aminomethyl)-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione 24l

[0821] Dissolve 24k (1.00 mg, 1.87 μmol) in 0.6 mL of dichloromethane, add hydrochloric acid in dioxane (4 M, 0.2 mL), stir at 25 °C for 1 hour. Add 1 mL of water, extract the diluted reaction with ethyl acetate (1 mL x 3), filter the aqueous phase, concentrate the filtrate by distillation under reduced pressure to obtain the crude title product 24l (0.5 mg) as a yellow solid. Use the product directly in the next reaction without purification.

[0822] MS m / z (ESI): 436.1 [M + 1].

[0823] Eleventh step

[0824] (S)-N-((8-Ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H- cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide 24

[0825] Dissolve 24l (13.3 mg, 22.9 μmol) and 2-hydroxyacetic acid (2.10 mg, 27.5 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (8.90 mg, 68.9 μmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.60 mg, 34.4 μmol) and 1-hydroxybenzotriazole (4.65 mg, 34.4 μmol), stir at 25 °C for 30 minutes. Concentrate the reaction solution by distillation under reduced pressure, add 2 mL of water, extract with ethyl acetate (1 mL x 3), wash the organic phase with saturated sodium chloride solution (1 mL), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by preparative HPLC (separation conditions: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A-water (containing 0.225% formic acid), B-acetonitrile, gradient elution, flow rate: 25 mL / min, instrument: GILSON GX-281) to obtain the title product 24 (0.98 mg, yield: 8%) as a yellow solid.

[0826] MS m / z (ESI): 494.1 [M+1].

[0827] 1 H NMR (400 MHz, CD3OD) δ 7.71 - 7.66 (m, 1H), 7.63 (s, 1H), 5.61 - 5.56 (m, 1H), 5.50 - 5.48 (m, 1H), 5.43 - 5.35 (m, 3H), 5.18 - 5.09 (m, 3H), 3.73 - 3.65 (m, 2H), 3.20 - 3.12 (m, 2H), 2.39 - 2.29 (m, 2H), 2.02 - 1.93 (m, 2H), 1.05 - 0.94 (m, 3H).

[0828] Example 10-25 to 12-33: Preparation of compounds 25 to 33

[0829] Referring to Example 10-24, the corresponding substrates were selected to prepare the following compounds 25 to 33:

[0830] Example 10-25: Preparation of compound 25

[0831] (S)-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro- 1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2- hydroxypropanamide 25

[0832] Dissolve 24l (22.3 mg, 45.1 μmol) and (2R)-2-hydroxypropanoic acid (4.07 mg, 45.2 μmol) in 0.5 mL of dichloromethane, add N,N-diisopropylethylamine (17.5 mg, 135 μmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (10.4 mg, 54.2 μmol) and 1-hydroxybenzotriazole (7.33 mg, 54.2 μmol), stir at 25 °C for 3 hours. Concentrate the reaction by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Phenomenex luna C18 250 x 50 mm x 15 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, B%: 23% - 53%), to obtain the title product 25 (0.49 mg, yield: 2%) as a yellow gum. SFC analysis: retention time 0.931 min, purity: 97%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A - carbon dioxide, B - isopropanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0833] MS m / z (ESI): 508.1 [M+1].

[0834] 1 H NMR (400 MHz, CD3OD) δ 7.70 (d, J = 9.6 Hz, 1H), 7.63 (s, 1H), 5.63 - 5.56 (m, 1H), 5.43 - 5.36 (m, 3H), 5.15 - 5.10 (m, 2H), 4.25 - 4.18 (m, 1H), 3.71 - 3.66 (m, 2H), 3.19 - 3.13 (m, 2H), 2.39 - 2.31 (m, 2H), 2.01 - 1.93 (m, 2H), 1.43 - 1.38 (m, 3H), 1.04 - 0.98 (m, 3H).

[0835] Example 10-26: Preparation of compound 26

[0836] (R)-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro- 1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-15-yl)methyl)-2- hydroxypropanamide 26

[0837] Dissolve 24l (22.3 mg, 45.2 μmol) and (2S)-2-hydroxypropanoic acid (4.07 mg, 45.1 μmol) in 0.5 mL of dichloromethane, add N,N-diisopropylethylamine (17.5 mg, 135 μmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (10.4 mg, 54.2 μmol) and 1-hydroxybenzotriazole (7.33 mg, 54.2 μmol), stir at 25 °C for 3 hours. Concentrate the reaction by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, B%: 20% - 50%), to obtain the title product 26 (1.09 mg, yield: 5%) as a yellow gum. SFC analysis: retention time 0.745 min, purity: 98%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A - carbon dioxide, B - isopropanol and acetonitrile (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0838] MS m / z (ESI): 508.1 [M+1].

[0839] 1 H NMR (400 MHz, CD3OD) δ 7.72 (d, J = 9.4 Hz, 1H), 7.66 (s, 1H), 5.63 - 5.59 (m, 1H), 5.45 - 5.39 (m, 3H), 5.17 - 5.14 (m, 2H), 4.26 - 4.22 (m, 1H), 3.74 - 3.68 (m, 2H), 3.21 - 3.16 (m, 2H), 2.40 - 2.35 (m, 2H), 2.01 - 1.95 (m, 2H), 1.44 - 1.39 (m, 3H), 1.06 - 1.00 (m, 3H).

[0840] Example 10-27: Preparation of compound 27

[0841] (S)-2-cyclopropyl-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15-yl)methyl)- 2-hydroxyacetamide 27

[0842] Dissolve 24l (25.0 mg, 50.5 μmol) and (2S)-2-cyclopropyl-2-hydroxyacetic acid (8.80 mg, 75.8 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (26.1 mg, 202 μmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (14.5 mg, 75.8 μmol) and 1-hydroxybenzotriazole (10.2 mg, 75.8 μmol), stir at 25 °C for 30 min. Concentrate the reaction by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Welch ultimate C18 150 x 25 mm x 7 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, flow rate: 25 mL / min) to give the title product 27 (2.15 mg, yield: 8%) as a brown oil. SFC analysis: retention time 1.131 min, purity: 97%. (Chromatography column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm; mobile phase: A - carbon dioxide: B - ethanol (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0843] MS m / z (ESI): 534.3 [M+1].

[0844] 1 H NMR (400 MHz, CD3OD) δ 7.72-7.66 (m, 1H), 7.63 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.34 (m, 3H), 5.12 (s, 2H), 3.75-3.64 (m, 3H), 3.19-3.10 (m, 2H), 2.40-2.30 (m, 2H), 2.02-1.91 (m, 2H), 1.21-1.14 (m, 1H), 1.06-0.93 (m, 3H), 0.57-0.40 (m, 4H).

[0845] Example 10-28: Preparation of compound 28

[0846] (R)-2-cyclopropyl-N-(((S)-8-ethyl-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15-yl)methyl)- 2-hydroxyacetamide 28

[0847] Dissolve 24l (25.0 mg, 50.5 μmol) and (2R)-2-cyclopropyl-2-hydroxyacetic acid (8.80 mg, 75.8 μmol) in 1 mL of dichloromethane, add N,N-diisopropylethylamine (26.1 mg, 202 μmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (14.5 mg, 75.8 μmol) and 1-hydroxybenzotriazole (10.2 mg, 75.8 μmol), stir at 25 °C for 30 min. Concentrate the reaction by distillation under reduced pressure, purify the resulting residue by pre-HPLC (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 10 μm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution) to give the title product 28 (2.77 mg, yield: 10%) as a brown oil. SFC analysis: retention time 0.898 min, purity: 99%. (column: Chiralcel OD-3 50 x 4.6 mm I.D., 3 μm; mobile phase: A - carbon dioxide: B - ethanol (0.05% diethylamine), isocratic elution: B: 40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0848] MS m / z (ESI): 534.4 [M+1].

[0849] 1 H NMR (400 MHz, CD3OD) δ 7.75 - 7.71 (m, 1H), 7.66 (s, 1H), 5.63 - 5.59 (m, 1H), 5.47 - 5.43 (m, 2H), 5.40 - 5.37 (m, 1H), 5.16 - 5.14 (m, 2H), 3.75 - 3.66 (m, 3H), 3.21 - 3.15 (m, 2H), 2.41 - 2.32 (m, 2H), 2.04 - 1.95 (m, 2H), 1.21 - 1.15 (m, 1H), 1.07 - 1.00 (m, 3H), 0.56 - 0.42 (m, 4H).

[0850] Example 10-34: Preparation of compound 34

[0851] (S)-N-((8-(cyclopropylmethyl)-4-fluoro-8-hydroxy-9,12-dioxo-2,3,8,9,12,14- hexahydro-lH,l lH-cyclopenta[f]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-15- yl)methyl)-2-hydroxyacetamide 34

[0852] First step

[0853] Dissolve 24f (500 mg, 1.32 mmol) and 1e-1 (578 mg, 1.73 mmol) in 20 mL of ethanol, add concentrated hydrochloric acid (12 M, 5.02 mL), stir at 80 °C for 16 hours. Concentrate the reaction solution by distillation under reduced pressure, add 10 mL of water, precipitate the solid, and collect the solid by filtration to obtain the title product 34a (496 mg) as a yellow solid. The product is used directly in the next step without purification.

[0854] MS m / z (ESI): 433.1 [M+1].

[0855] Second step

[0856] Dissolve 34a (596 mg, 1.38 mmol) in 36 mL of acetic acid, add hydrogen peroxide (3.86 g, 34.0 mmol, content 30%), stir at 75 °C for 3 hours. Add 100 mL of water to the reaction solution, precipitate the solid, and collect the solid by filtration to obtain the title product 34b (500 mg) as a yellow solid. The product is used directly in the next step without purification.

[0857] MS m / z (ESI): 449.0 [M+1].

[0858] Third step

[0859] Dissolve 34b (490 mg, 915 μmol) in 30 mL of N,N-dimethylformamide, protect under nitrogen, and cool to 0 °C in an ice water bath. Add oxalyl chloride (465 mg, 3.66 mmol), and stir at 0 °C for 1 hour. Pour the reaction solution into 100 mL of water, precipitate the solid, and collect the solid by filtration to obtain the title product 34c (500 mg) as a yellow solid. The product is used directly in the next step without purification.

[0860] MS m / z (ESI): 467.1 [M+1].

[0861] Fourth step

[0862] Dissolve 34c (200 mg, 428 μmol) in 5 mL of a mixed solvent of dioxane and 0.5 mL of water, add potassium N-tert-butyloxo-methyltrifluoroborate (203 mg, 856 μmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (31.3 mg, 42.8 μmol), and potassium phosphate tribasic (272 mg, 1.29 mmol), protect under nitrogen, and stir at 95 °C for 5 hours. Concentrate the reaction solution by distillation under reduced pressure to obtain the title product 34d (250 mg) as a brown solid. The product is used directly in the next step without purification.

[0863] MS m / z (ESI): 562.3 [M+1].

[0864] Fifth step

[0865] Dissolve 34d (240 mg, 427 pmol) in 5 mL of aqueous hydrochloric acid (6 M) and stir at 40 °C for 10 h. Add 5 mL of water to the reaction mixture and extract with dichloromethane (10 mL x 3). Lyophilize the aqueous phase to obtain the hydrochloride salt of the title product 34e as a yellow solid (200 mg). The product is used directly in the next step without purification.

[0866] MS m / z (ESI): 462.3 [M+1].

[0867] Sixth step

[0868] Dissolve 34e hydrochloride salt (20.0 mg, 40.1 pmol) and glycolic acid (4.6 mg, 60.2 pmol) in 2 mL of N,N-dimethylformamide. Add N,N-diisopropylethylamine (15.5 mg, 120 pmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.5 mg, 60.2 pmol) and 1-hydroxybenzotriazole (8.1 mg, 60.2 pmol) and stir at 25 °C for 2 h. Add 1 mL of water to the reaction mixture and extract with dichloromethane (3 mL x 3). Dry the organic phase over anhydrous sodium sulfate, filter and concentrate the filtrate by distillation under reduced pressure. Purify the obtained residue by pre-HPLC (separation conditions: column: Phenomenex Luna C18 150 x 25 mm x 10 pm, mobile phase: A - water (0.225% formic acid), B - acetonitrile, gradient elution, B%: 26% - 56%) to obtain the title product 34 as a yellow solid (2.51 mg, yield: 12%).

[0869] MS m / z (ESI): 520.3 [M+1]. 1 H NMR (400 MHz, CD3OD) d 7.78 - 7.62 (m, 2H), 5.64 - 5.55 (m, 1H), 5.47 - 5.32 (m, 3H), 5.20 - 5.10 (m, 2H), 4.15 - 4.04 (m, 2H), 3.74 - 3.63 (m, 2H), 3.21 - 3.10 (m, 2H), 2.40 - 2.29 (m, 2H), 1.96 - 1.82 (m, 2H), 0.94 - 0.86 (m, 1H), 0.51 - 0.38 (m, 2H), 0.15 - -0.02 (m, 2H).

[0870] Example 10-35: Preparation of compound 35

[0871] (S)-N-((4-(cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo- 3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin- 11-yl)methyl)-2-hydroxyacetamide 35

[0872] First Step

[0873] Dissolve 2-amino-4-fluoro-5-methylbenzaldehyde hydrochloride (138 mg, 686 μmol) and If-1 (200 mg, 686 μmol) in 35 mL of toluene, add 4-methylbenzenesulfonic acid pyridine (103 mg, 412 μmol) and o-methylphenol (596 mg, 5.52 mmol), stir at 135 °C for 3 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 35a (213 mg, yield: 73%) in the form of yellow solid.

[0874] MS m / z (ESI): 407.2 [M+1].

[0875] Second Step

[0876] Dissolve 35a (250 mg, 479 μmol) in 12 mL of acetic acid, add hydrogen peroxide (4.41 g, 38.9 mmol, content 30%), stir at 70 °C for 1.5 hours. Quench the reaction solution with 20 mL of sodium thiosulfate at 0 °C, extract with ethyl acetate (25 mL x 2), wash the organic phase with saturated sodium chloride solution (25 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 35b (120 mg, yield: 55%) in the form of yellow solid.

[0877] MS m / z (ESI): 423.2 [M+1].

[0878] Third Step

[0879] Dissolve 35b (120 mg, 263 μmol) in 2 mL of N,N-dimethylformamide, protect with nitrogen, cool to 0 °C with ice water bath, add oxalyl chloride (167 mg, 1.32 mmol), stir at 0 °C for 1 hour. Pour the reaction solution into 10 mL of water, precipitate the solid, collect the solid by filtration, dry to obtain the title product 35c (115 mg) in the form of yellow solid. The product is used directly in the next step without purification.

[0880] MS m / z (ESI): 441.1 [M+1].

[0881] Fourth Step

[0882] Dissolve 35c (90.0 mg, 167 μmol) in 3 mL of a mixture solvent of dioxane and 0.2 mL of water, add potassium N-tert-butyloxy carbonylmethyl trifluoroborate (198 mg, 838 μmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (24.5 mg, 33.5 μmol) and cesium carbonate (163 mg, 503 μmol), stir for 5 hours at 105 °C under nitrogen protection. Add 5 mL of water to the reaction solution, extract with ethyl acetate (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, and obtain the title product 35d (110 mg) in the form of a yellow solid. The product is directly used in the next step without purification.

[0883] MS m / z (ESI): 536.1 [M+1].

[0884] Fifth step

[0885] Dissolve 35d (110 mg, 205 μmol) in 3 mL of aqueous hydrochloric acid (6 M), stir for 10 hours at 40 °C. Add 5 mL of water to the reaction solution, extract with dichloromethane (10 mL x 3), and freeze dry the aqueous phase to obtain the hydrochloride salt of the title product 35e (70 mg) in the form of a yellow solid. The product is directly used in the next step without purification.

[0886] MS m / z (ESI): 436.0 [M+1].

[0887] Sixth step

[0888] Dissolve the hydrochloride salt of 35e (30.0 mg, 68.8 μmol) and glycolic acid (5.76 mg, 75.7 μmol) in 2 mL of N,N-dimethylformamide, add N,N-diisopropylethylamine (26.7 mg, 206 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.8 mg, 103 μmol) and 1-hydroxybenzotriazole (13.9 mg, 103 μmol), and stir for 1.5 hours at 25 °C. Add 2 mL of water to the reaction solution, extract with dichloromethane (5 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, and purify the obtained residue by pre-HPLC (separation condition: column: Phenomenex Luna C18 150 x 25 mm x 10 μm, mobile phase: A-water (0.225% formic acid), B-acetonitrile, gradient elution, B%: 19%-49%), to obtain the title product 35 (1.63 mg, yield: 5%) in the form of a yellow solid.

[0889] MS m / z (ESI): 494.3 [M+1].

[0890] 1 H NMR (400 MHz, CD3OD) δ 8.34 - 8.29 (m, 1H), 7.83 - 7.77 (m, 1H), 7.73 - 7.70 (m, 1H), 5.64 - 5.58 (m, 1H), 5.56 - 5.49 (m, 2H), 5.44 - 5.37 (m, 1H), 5.07 - 5.01 (m, 2H), 4.06 - 3.99 (m, 2H), 2.62 - 2.51 (m, 3H), 1.95 - 1.83 (m, 2H), 0.93 - 0.88 (m, 1H), 0.50 - 0.38 (m, 2H), 0.15 - 0.08 (m, 1H), 0.07 - -0.01 (m, 1H).

[0891] Example 10-36: Preparation of compound 36

[0892] (S)-N-(((S)-4-(Cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll-yl)methyl)-2- hydroxypropanamide 36

[0893] (S)-2-Hydroxypropanoic acid (2.1 mg, 23.3 μmol) was dissolved in 0.5 mL of N,N- dimethylformamide, followed by the addition of 1H-benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (13.1 mg, 25.2 μmol), N,N-diisopropylethylamine (14.9 mg, 155 μmol) and 35e (10.2 mg, 23.4 μmol) in sequence, stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by pre-HPLC (separation condition: column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 10%-70%), to give the title product 36 (5.3 mg, yield: 44.56%) as a white solid.

[0894] MS m / z (ESI): 508.4 [M+l].

[0895] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (t, J = 6.0 Hz, 1H), 8.42 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H), 7.38 (s, 1H), 6.58 (s, 1H), 5.61 (d, J = 4.9 Hz, 1H), 5.49 (d, J = 2.1 Hz, 2H), 5.43 (d, J = 2.6 Hz, 2H), 4.92 - 4.75 (m, 2H), 4.05 - 3.94 (m, 1H), 3.09 - 2.95 (m, 4H), 1.89 - 1.75 (m, 2H), 1.75 - 1.68 (m, 4H), 1.20 (d, J = 6.8 Hz, 3H), 0.86 - 0.76 (m, 1H), 0.41 - 0.25 (m, 2H), 0.11 - 0.02 (m, 1H), -0.04 - -0.13 (m, 1H).

[0896] Example 10-37: Preparation of compound 37

[0897] (R)-N-(((S)-4-(cyclopropylmethyl)-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll-yl)methyl)-2-hydroxypropanamide 37

[0898] (R)-2-hydroxypropanoic acid (2.1 mg, 23.3 μmol) was dissolved in 0.5 mL of N,N- dimethylformamide, followed by the addition of 1H-benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (13.1 mg, 25.2 μmol), N,N-diisopropylethylamine (14.9 mg, 155 μmol) and 35e (10.2 mg, 23.4 μmol) in sequence, stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by pre-HPLC (separation condition: column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 10%-70%), to give the title product 37 (3.6 mg, yield: 30.27%) as a white solid.

[0899] MS m / z (ESI): 508.4 [M+l].

[0900] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.42 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H), 7.38 (s, 1H), 6.59 (s, 1H), 5.61 (d, J = 4.9 Hz, 1H), 5.49 (s, 2H), 5.43 (d, J = 3.0 Hz, 2H), 4.91 - 4.75 (m, 2H), 4.04 - 3.96 (m, 1H), 3.03 - 3.00 (m, 4H), 1.89 - 1.77 (m, 2H), 1.76 - 1.73 (m, 4H), 1.19 (d, J = 6.8 Hz, 3H), 0.85 - 0.76 (m, 1H), 0.40 - 0.25 (m, 2H), 0.12 - 0.03 (m, 1H), -0.04 - -0.13 (m, 1H).

[0901] Example 10-38: Preparation of compound 38

[0902] First step

[0903] Dissolve 38g (800 mg, 5.83 mmol) in 20 mL of 1,2-dichloroethane, protect with nitrogen, cool to 0°C with ice water bath, drop in boron trichloride dichloromethane solution (1M, 4.08 mL) and 4-pentenenitrile (709 mg, 8.75 mmol), stir for 2 hours at 80°C under nitrogen protection. Cool to room temperature, add hydrochloric acid aqueous solution (2M, 50 mL), stir for 0.5 hours at 80°C. Extract with dichloromethane (30 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, reduce pressure to concentrate the filtrate, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 38h (220 mg, yield: 17%) in the form of yellow oil.

[0904] MS m / z (ESI): 220.2 [M+1].

[0905] Second step

[0906] Dissolve 1f-1 (288 mg, 991 μmol) and 38h (220 mg, 991 μmol) in 10 mL of toluene, add 4-methylbenzenesulfonic acid pyridine (124 mg, 495 μmol) and o-cresol (214 mg, 1.98 mmol), stir for 2 hours at 120°C under nitrogen protection. Add 50 mL of water to the reaction solution, extract with dichloromethane (45 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, reduce pressure to concentrate the filtrate, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 38i (202 mg, yield: 41%) in the form of yellow solid.

[0907] MS m / z (ESI): 473.0 [M+1].

[0908] Third Step

[0909] Dissolve 38i (200 mg, 406 μmol) in 10 mL of dichloromethane, cool to -78 °C, bubble ozone for 0.5 hour (15 Psi), clean up the excess ozone with nitrogen, add triphenylphosphine (213 mg, 812 μmol) at -78 °C, stir for 0.5 hour at -78 °C. Add 50 mL of water to the reaction solution, extract with dichloromethane (45 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue by silica gel column chromatography with dichloromethane / tetrahydrofuran developing system to obtain the title product 38j (70 mg, yield: 36%) in the form of yellow solid.

[0910] MS m / z (ESI): 475.3 [M+1].

[0911] Fourth Step

[0912] Dissolve 38j (70.0 mg, 147 μmol) in 5 mL of tetrahydrofuran, protect with nitrogen, add sodium borohydride (2.79 mg, 73.7 μmol), stir for 1 hour at -78 °C, add 10 mL of ammonium chloride solution to the reaction solution, extract with ethyl acetate (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid):B-acetonitrile, gradient elution: B%: 22%-42%), to obtain the title product 38 (8.95 mg, yield: 21%) in the form of yellow solid.

[0913] MS m / z (ESI): 477.0 [M+1].

[0914] 1H NMR (400 MHz, CD3OD) δ 7.64-7.61 (m, 1H), 7.55-7.51 (m, 1H), 7.41-7.37 (m, 1H), 6.24-6.19 (m, 2H), 5.62-5.55 (m, 1H), 5.40-5.34 (m, 1H), 5.26-5.21 (m, 2H), 3.72-3.64 (m, 2H), 3.25-3.19 (m, 2H), 1.99-1.90 (m, 3H), 1.89-1.81 (m, 1H), 0.97-0.88 (m, 1H), 0.52-0.38 (m, 2H), 0.15-0.09 (m, 1H), 0.07-0.00 (m, 1H).

[0915] Example 10-39: Preparation of compound 39

[0916] First step

[0917] Dissolve 39a (1.00 g, 7.99 mmol) in 20 mL of 1,2-dichloroethane, protect with nitrogen, cool to 0°C with ice water bath, drop in boron trichloride solution in dichloromethane (1M, 10.4 mL) and 4-pentenenitrile (777 mg, 9.59 mmol), stir at 80°C for 5 hours under nitrogen protection. Cool to room temperature, add hydrochloric acid aqueous solution (2M, 9 mL), stir at 80°C for 0.5 hours. Extract with dichloromethane (30 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, reduce pressure to concentrate the filtrate, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 39b (315 mg, yield: 19%) in the form of yellow solid.

[0918] MS m / z (ESI): 208.1 [M+1].

[0919] Second step

[0920] Dissolve 1f-1 (400 mg, 1.37 mmol) and 39b (313 mg, 1.51 mmol) in 50 mL of toluene, add 4-methylbenzenesulfonic acid pyridine (207 mg, 823 µmol) and o-cresol (1.19 g, 11.0 mmol), stir at 135°C for 3 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 39c (420 mg, yield: 60%) in the form of yellow solid.

[0921] MS m / z (ESI): 461.3 [M+1].

[0922] Third step

[0923] Dissolve 39c (100 mg, 197 pmol) in 2 mL of dichloromethane, cool to -78 °C, bubble ozone for 0.5 hour (15 Psi), clean up the excess ozone with nitrogen, add triphenylphosphine (51.7 mg, 197 pmol) at -78 °C, stir for 0.5 hour at -78 °C. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 39d (53.0 mg, yield: 54%) in the form of yellow solid.

[0924] MS m / z (ESI): 463.2 [M+1].

[0925] Fourth step

[0926] Dissolve 39d (40.0 mg, 80.1 pmol) in 2 mL of tetrahydrofuran and 0.1 mL of N,N-dimethylacetamide mixed solvent, protect with nitrogen, add sodium borohydride (1.52 mg, 40.1 pmol), stir for 1 hour at -78 °C, add 1 mL of ammonium chloride solution to the reaction solution, extract with ethyl acetate (2 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 10 pm; mobile phase: A-water (0.225% formic acid):B-acetonitrile, gradient elution: B%: 27%-57%), then purify by supercritical fluid chromatography, column: Phenomenex-Cellulose-2 (250 mm x 30 mm, 10 pm), mobile phase: A-carbon dioxide, B-methanol and acetonitrile, isocratic elution: B%: 55%, to obtain the title product 39 (8.66 mg, yield: 45%) in the form of light yellow solid.

[0927] MS m / z (ESI): 464.9 [M+1].

[0928] 1 H NMR (400 MHz, CD3OD) d 8.24-8.17 (m, 1H), 7.80-7.73 (m, 1H), 7.73-7.67 (m, 1H), 5.64-5.57 (m, 1H), 5.44-5.33 (m, 3H), 3.75-3.66 (m, 2H), 3.40-3.34 (m, 2H), 2.60-2.52 (m, 3H), 2.04-1.96 (m, 2H), 1.94-1.82 (m, 2H), 0.93-0.89 (m, 1H), 0.52-0.38 (m, 2H), 0.17-0.08 (m, 1H), 0.07--0.04 (m, 1H).

[0929] Example 10-40: Preparation of compound 40

[0930] First step

[0931] Dissolve 38 g (1.5 g, 10.94 mmol) in 20 mL of 1,2-dichloroethane, protect with nitrogen, and cool to 0 °C with an ice water bath. Add a solution of boron trichloride in dichloromethane (1 M, 8.75 mL) and 5-hexenenitrile (1.56 g, 16.4 mmol) dropwise. Stir at 80 °C for 2 hours under nitrogen protection. Cool to room temperature, add aqueous hydrochloric acid (2 M, 50 mL), and stir at 80 °C for 0.5 hours. Extract with dichloromethane (50 mL x 3), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by silica gel column chromatography with developing system B to obtain the title product 40a (502 mg, yield: 19%) in the form of yellow oil.

[0932] MS m / z (ESI): 234.0 [M+1].

[0933] Second step

[0934] Dissolve 1f-1 (249 mg, 857 μmol) and 40a (200 mg, 780 μmol) in 5 mL of toluene, add 4-methylbenzenesulfonic acid pyridine (150 mg, 600 μmol) and o-cresol (185 mg, 1.71 mmol), and stir at 120 °C for 2 hours under nitrogen protection. Add 50 mL of water to the reaction solution, extract with dichloromethane (45 mL x 3), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by silica gel column chromatography with developing system A to obtain the title product 40b (310 mg, yield: 53%) in the form of yellow solid.

[0935] MS m / z (ESI): 487.3 [M+1].

[0936] Third step

[0937] Dissolve 40b (250 mg, 513 μmol) in 5 mL of dichloromethane, cool to -78 °C, and bubble ozone for 0.5 hours (15 Psi). Remove excess ozone with nitrogen, add triphenylphosphine (674 mg, 2.57 mmol) at -78 °C, and stir at -78 °C for 0.5 hours. Concentrate the reaction solution under reduced pressure, and purify the resulting residue by silica gel column chromatography with dichloromethane / tetrahydrofuran developing system to obtain the title product 40c (65.0 mg, yield: 26%) in the form of yellow solid.

[0938] MS m / z (ESI): 489.2 [M+1].

[0939] Step 4

[0940] Dissolve 40c (65.0 mg, 133 μmol) in 15 mL of tetrahydrofuran, protect with nitrogen, add sodium borohydride (2.5 mg, 66 μmol), stir at -78 °C for 1 hour, add 20 mL of ammonium chloride solution to the reaction solution, extract with ethyl acetate (20 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 10 μm; mobile phase: A-water (0.225% formic acid):B-acetonitrile, gradient elution: B%: 25%-45%) to obtain the title product 40 (5.69 mg, yield: 9%) as a yellow solid.

[0941] MS m / z (ESI): 491.0 [M+1].

[0942] 1 H NMR (400 MHz, CD3OD) δ 7.62 - 7.58 (m, 1H), 7.50 - 7.46 (m, 1H), 7.37 - 7.34 (m, 1H), 6.24 - 6.18 (m, 2H), 5.62 - 5.54 (m, 1H), 5.42 - 5.32 (m, 1H), 5.21 - 5.16 (m, 2H), 3.69 - 3.62 (m, 2H), 3.17 - 3.10 (m, 2H), 1.98 - 1.90 (m, 1H), 1.88 - 1.79 (m, 3H), 1.78 - 1.70 (m, 2H), 0.96 - 0.85 (m, 1H), 0.52 - 0.40 (m, 2H), 0.18 - 0.08 (m, 1H), 0.07 - -0.00 (m, 1H).

[0943] Example 11. Preparation of conjugate intermediate linker-drug

[0944] Example 11-1: Preparation of LD-1

[0945] 2-(((S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-l,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazahenicosan-21-yl)oxy)-5- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N 1 , N 3Di(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl) isophthalamide LD-1

[0946] First step

[0947] 2-(4-(tert-butoxy)-4-oxobutoxy)-5-nitroisophthalic acid dimethyl ester LD-1b

[0948] LD-1a (5 g, 19.55 mmol, prepared by the method disclosed in the literature “Journal of Physical Organic Chemistry, 2010, vol. 16, #10, p. 682-690”), tert-butyl 4-hydroxybutanoate (3.76 g, 23.47 mmol) and triphenylphosphine (7.69 g, 29.33 mmol) were dissolved in 40 mL of a mixture solvent of N,N-dimethylformamide and 120 mL of tetrahydrofuran, protected by nitrogen, and cooled to 0 °C with an ice water bath. Diisopropyl azodicarboxylate (5.93 g, 29.33 mmol) was slowly added dropwise to the reaction solution, and stirred at 25 °C for 20 hours. 150 mL of water was added, and the diluted reaction solution was extracted with ethyl acetate (200 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The obtained residue was purified by reverse phase high performance liquid chromatography (separation conditions: column: I.D. 100 mm x H 300 mm Welch Ultimate XB_C18 20-40 μm, mobile phase: A-water, B-acetonitrile, gradient elution, flow rate: 200 mL / min, instrument: Agela Astra), to obtain the title product LD-1b (4.8 g, yield: 61%) as a yellow solid.

[0949] MS m / z (ESI): 420.2 [M+23].

[0950] Second step

[0951] 2-(4-(tert-butoxy)-4-oxobutoxy)-5-nitroisophthalic acid LD-1c

[0952] LD-1b (1.5 g, 3.71 mmol) was dissolved in a mixture solvent of 15 mL of water and 15 mL of tetrahydrofuran, lithium hydroxide monohydrate (311.10 mg, 7.41 mmol) was added, and stirred at 25°C for 2 hours. 1N hydrochloric acid solution was added to the reaction solution to adjust the pH value to 2-3, and extracted with ethyl acetate (40 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation to obtain the crude product of the title compound LD-1c (1.49 g) as a yellow solid, which was used directly in the next step without purification.

[0953] MS m / z (ESI): 392.1 [M+23].

[0954] Third step

[0955] 4-(2,6-bis((2,5,8,11,14,17,20,23-octaoxa-25-yl)carbamoyl)-4-nitrophenoxy)butyric acid tert-butyl ester LD-1d

[0956] butyl ester LD-1d

[0957] LD-1c (400 mg, 933.60 µmol) was dissolved in 5 mL of N,N-dimethylformamide, 1-hydroxybenzotriazole (378.45 mg, 2.80 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (536.92 mg, 2.80 mmol), N,N-diisopropylethylamine (723.97 mg, 5.60 mmol) and 3,6,9,12,15,18,21,24-octaoxa-25-ylamine (895.03 mg, 2.33 mmol) were added, and stirred at 25°C for 2 hours. 10 mL of water was added, extracted with dichloromethane (10 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by silica gel column chromatography with the developing system B to obtain the title product LD-1d (515 mg, yield: 50%) as a yellow oil.

[0958] MS m / z (ESI): 1117.7 [M+18].

[0959] Fourth step

[0960] 4-(2,6-bis((2,5,8,11,14,17,20,23-octaoxa-25-yl)carbamoyl)-4-nitrophenoxy)butyric acid tert-butyl ester LD-1d

[0961] LD-1d (515 mg, 466.74 μmol) was dissolved in a mixed solvent of 20 mL of ethanol and 10 mL of water, iron powder (130.32 mg, 2.33 mmol) and ammonium chloride (249.66 mg, 4.67 mmol) were added, and stirring was performed at 80°C for 16 hours. The reaction solution was filtered with celite, 10 mL of water was added to the filtrate, the diluted filtrate was extracted with dichloromethane (10 mL x 3), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing system A to obtain the title product LD-1e (435 mg, yield: 87%) as a yellow oil.

[0962] MS m / z (ESI): 1070.8 [M+1].

[0963] Fifth step

[0964] 4-(2,6-bis((2,5,8,11,14,17,20,23-oxa-25-yl)carbamoyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenoxy)butyric acid tert-butyl ester LD-1f

[0965] LD-1e (380 mg, 352.47 μmol) and 2,5-furandione (38.02 mg, 387.71 μmol) were dissolved in 6 mL of dioxane, and stirring was performed at 25°C for 1 hour. After the completion of the reaction of the starting material LD-1e was confirmed by TLC, ammonium persulfate (160.87 mg, 704.93 μmol) and dimethyl sulfoxide (55.08 mg, 704.93 μmol) were added to the reaction solution, and stirring was performed at 100°C for 1 hour. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative HPLC (separation conditions: column: Welch Xtimate C18 150 x 25 mm x 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution, B%: 25%-55%, flow rate 25 mL / min) to obtain the title product LD-1f (91 mg, yield: 21%) as a yellow oil.

[0966] MS m / z (ESI): 1167.8 [M+18].

[0967] Sixth step

[0968] 4-(2,6-bis((2,5,8,11,14,17,20,23-oxa-25-yl)carbamoyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenoxy)butyric acid LD-1g

[0969] LD-1f (2 mg, 1.74 μmol) was dissolved in 0.5 mL of dichloromethane, and trifluoroacetic acid (0.1 mL) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was concentrated by vacuum distillation to obtain crude product LD-1g (1.5 mg) as a colorless oil. The product was used directly in the next reaction without purification.

[0970] MS m / z(ESI):1111.8[M+18].

[0971] Step 7

[0972] (9H-fluorene-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-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-tetraazahexadecane-16-yl)carbamate LD-1i

[0973] LD-1h (68.7 mg, 94.7 μmol, prepared using the method disclosed in Example LND1067-L1 on page 9 of patent application “CN113402584 A”) and 1i-1-1 (50.0 mg, 78.9 μmol, trifluoroacetate) were dissolved in 2 mL of N,N-dimethylformamide, and 1-hydroxybenzotriazole (16.0 mg, 118 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.7 mg, 118 μmol), and N,N-diisopropylethylamine (30.6 mg, 236 μmol) were added. The mixture was stirred at 25 °C for 3 hours. Add 1 mL of water and extract with dichloromethane (3 mL × 3). Dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by silica gel column chromatography with system A as the developing system. The title product LD-1i (60 mg, yield: 57%) was obtained as a yellow solid.

[0974] MS m / z(ESI):1089.6[M+1].

[0975] Step 8

[0976] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-(cyclopropylmethyl)-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)

[0977] [1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3- phenylpropanamide LD-1j

[0978] LD-1i (55.0 mg, 41.5 μmol) was dissolved in 1.5 mL of acetonitrile, diethylamine (213 mg, 2.91 mmol) was added, and stirring was carried out at 25 °C for 1 hour. The reaction solution was concentrated by distillation under reduced pressure to obtain the crude title product LD-1j (35 mg) as a yellow solid. The product was used directly in the next reaction without purification.

[0979] MS m / z (ESI): 867.3 [M+1].

[0980] Ninth step

[0981] 2-(((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-

[0982] 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazahenicosan-21-yl)oxy)-5- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N 1 , N 3 bis(2,5,8,11,14,17,20,23-o xoahenicosan-25-yl)isophthalamide LD-1

[0983] The title compound LD-1 was obtained by reaction of LD-1j with LD-1g in the presence of 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine.

[0984] MS m / z (ESI): 972.5 [1 / 2 (M+2)].

[0985] Example 11-11: Preparation of LD-11

[0986] N-((S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3 ',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9, 12, 15-pentaoxo-3-oxa-5,8, 11, 14- tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide LD-11

[0987] MS m / z (ESI): 559.8 [1 / 2 (M+2)].

[0988] Example 11-12: Preparation of LD-12

[0989] (S)-N 5 -((S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3 ',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9, 12, 15-pentaoxo-3-oxa-5,8, 11, 14- tetraazahexadec-16-yl)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-N 1 -(2,5,8,11,14,17,20,23-octaoxa-25-yl)pentanediamide LD-12

[0990] MS m / z (ESI): 806.9 [1 / 2 (M+2)].

[0991] Example 11-13: Preparation of LD-13

[0992] (S)-N 5 -((2S,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3 ',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9, 12, 15-pentaoxo-3-oxa-5,8, 11, 14- tetraazahexadec-16-yl)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-N 1(2,5,8,11,14,17,20,23-octaoxapentacosyl) glutaramide LD-13

[0993] First step

[0994] (5S, 13S)-5-benzyl-1-(9H-fluor-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10- triazatetradecan-14-oic acid benzyl ester LD-13b

[0995] LD-13a (1.70 g, 3.17 mmol, prepared by the method disclosed in patent application “WO2022056696 A1” page 8, example 3) was dissolved in 20 mL of N,N-dimethylformamide, 1,8-diazabicyclo[5.4.0]undec-7-ene (241 mg, 1.59 mmol) was added, and stirring was carried out at 20 °C for 1 hour. Then (( (9H-fluoren-9-yl)methoxy) carbonyl)-L-phenylalanine (1.16 g, 3.00 mmol), 1- hydroxybenzotriazole (608 mg, 4.51 mmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (863 mg, 4.51 mmol) were sequentially added to the reaction solution, and stirring was carried out at 25 °C for 1 hour. 50 mL of water was added, extraction was carried out with ethyl acetate (45 mL x 3), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing system A to obtain the title product LD-13b (1.35 g, yield: 65%) in the form of a white solid.

[0996] MS m / z (ESI): 658.1 [M+23].

[0997] Second step

[0998] (11S, 19S)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa- 4,7,10,13,16-pentazadocosan-20-oic acid benzyl ester LD-13c

[0999] LD-13b (1.25 g, 1.81 mmol) was dissolved in 20 mL of N,N-dimethylformamide, 1,8-diazabicyclo[5.4.0]undec-7-ene (165 mg, 1.09 mmol) was added, and stirring was performed at 25 °C for 1 hour. Then ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (750 mg, 2.12 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.01 g, 2.65 mmol) were added to the reaction solution in sequence, and stirring was performed at 25 °C for 1 hour. 50 mL of water was added, extraction was performed with ethyl acetate (45 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing system A to obtain the title product LD-13c (850 mg, yield: 53%) in the form of a white solid.

[1000] MS m / z (ESI): 767.4 [M+18].

[1001] Third step

[1002] (11S,19S)-11-benzyl-1-(9H-fluoren-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazadocosan-20-oic acid LD-13d

[1003] LD-13c (750 mg, 817 µmol) and palladium on carbon (434 mg, 10%, wetted with about 55% water) were dissolved in 4 mL of ethyl acetate and 8 mL of ethanol under nitrogen protection, hydrogen gas was replaced three times, and the reaction solution was stirred at 25 °C under hydrogen gas (15 Psi) for 1 hour. The reaction solution was filtered with diatomite, and the filtrate was concentrated by distillation under reduced pressure to obtain the crude title product LD-13d (450 mg, yield: 67%) in the form of a white solid. The product was directly used in the next step reaction without purification.

[1004] MS m / z (ESI): 677.2 [M+18].

[1005] Fourth step

[1006] (9H-fluoren-9-yl)methyl ((2S,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15- pentoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate LD-13e

[1007] LD-13d (30.0 mg, 36.8 μmol) was dissolved in 1 mL of N,N-dimethylformamide, 1- hydroxybenzotriazole (7.47 mg, 55.2 μmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (10.6 mg, 55.3 μmol) and N,N-diisopropylethylamine (14.2 mg, 110 μmol) were added in turn, and stirred at 25 °C for 1 hour. 2 mL of water was added, extracted with dichloromethane (5 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system A to obtain the title product LD-13e (32.8 mg, yield: 65%) in the form of a yellow solid.

[1008] MS m / z (ESI): 1103.2 [M+1].

[1009] Fifth step

[1010] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((((S)-l-(((lS,9S)-9-(cyclopropylmethyl)-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l-oxoprop-2- yl)oxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide LD-13f

[1011] LD-13e (32.0 mg, 23.5 μmol) was dissolved in 1 mL of acetonitrile, diethylamine (142 mg, 1.94 mmol) was added, and stirred at 25 °C for 1 hour. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing system A to obtain the title product LD-13f (18 mg, yield: 87%) in the form of a white solid.

[1012] MS m / z (ESI): 881.2 [M+1].

[1013] Step 6

[1014] LD-13g (1.0 g, 2.97 mmol) was dissolved in 10 mL of N,N-dimethylformamide, N,N- diisopropylethylamine (1.15 g, 8.90 mmol), 1-hydroxybenzotriazole (400 mg, 2.97 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (570 mg, 2.97 mmol) were added successively, stirred at 0 °C for 15 min. 3,6,9,12,15,18,21,24- octaoxapentacosan-1-amine (1.14 g, 2.97 mmol) was added, and the reaction was gradually warmed to 25 °C and stirred for 2 h. 20 mL of water was added, extracted with ethyl acetate (30 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The obtained residue was purified by silica gel column chromatography with developing system A to give the title product LD-13h (1.28 g, yield: 62%) as a colorless oil.

[1015] MS m / z (ESI): 703.4 [M+1].

[1016] Step 7

[1017] LD-13h (1.28 g, 1.82 mmol) was dissolved in 13 mL of methanol, and palladium on carbon (10%, wetted with about 55% water, 300 mg) was added under nitrogen protection, and stirred at 20 °C for 1 h. Hydrogen was replaced for three times, and the reaction was stirred at 25 °C for 2 h under hydrogen (15 Psi). The reaction was filtered with celite, and the filtrate was concentrated by reduced pressure distillation to give the crude title product LD-13i (900 mg, yield: 87%) as a colorless oil. The product was used directly in the next step without purification.

[1018] MS m / z (ESI): 569.3 [M+1].

[1019] Step 8

[1020] LD-13i (300 mg, 0.53 mmol) was dissolved in 5 mL of N,N-dimethylformamide, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (200 mg, 0.53 mmol) and N,N-diisopropylethylamine (204 mg, 1.58 mmol) were added, then LD-13j (142 mg, 0.53 mmol, prepared by the method disclosed in patent application "WO2021228141 A1" page 30, example 2) was added, and stirred at 25 °C for 2 hours. 10 mL of water was added, extracted with ethyl acetate (15 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by silica gel column chromatography with developing system A to obtain the title product LD-13k (107.85 mg, yield: 25%) in the form of colorless oil.

[1021] MS m / z (ESI): 819.4 [M+1].

[1022] Ninth step

[1023] LD-13k (260 mg, 0.32 mmol) was dissolved in 5 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, and stirred at 25 °C for 2 hours. The reaction solution was concentrated by reduced pressure distillation, and the obtained residue was purified by pre-HPLC (separation conditions: column: Sunfire Prep C18 OBD 10 µm, 19 x 250 mm, mobile phase: A-acetonitrile, B-water, gradient elution, A%: 35%-95%), to obtain the title product LD-13l (82.84 mg, yield: 34%) in the form of white solid.

[1024] MS m / z (ESI): 763.3 [M+1].

[1025] Tenth step

[1026] LD-13i (17.9 mg, 23.5 μmol) and LD-13f (18.0 mg, 20.4 μmol) were dissolved in 1 mL of N,N-dimethylformamide, 1-hydroxybenzotriazole (4.14 mg, 30.6 μmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.88 mg, 30.6 μmol) and N,N- diisopropylethylamine (7.92 mg, 61.3 μmol) were added successively, stirred at 20 °C for 1 h. The reaction solution was concentrated by distillation under reduced pressure, the residue was purified by pre-HPLC (separation condition: column: Welch Xtimate C18 150x25mmx5μm, mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution, B%: 30%-60%), to get the title product LD-13 (2.09 mg, yield: 6%) as a purple solid.

[1027] MS m / z (ESI): 813.9 [1 / 2 (M+2)].

[1028] MS m / z (ESI): 1625.5 [M+1].

[1029] 1H NMR(400MHz,CD3OD)δ8.99–8.88(m,2H),8.64–8.57(m,1H),8.54–8.49(m,1H),8.22–8 .14(m,1H),8.08–8.01(m,1H),7.70–7.59(m,2H),7.27–7.20(m,2H),7.20–7.07(m,3H) ,5.72–5.64(m,1H),5.61–5.54(m,1H),5.49–5.41(m,1H),5.40–5.25(m,2H),5.20–5. 13(m,1H),4.78–4.71(m,2H),4.39–4.25(m,3H),3.94–3.86(m,3H),3.80–3.75(m,1H), 3.73–3.55(m,26H),3.55–3.49(m,4H),3.38–3.33(m,6H),3.20–3.12(m,1H),2.97–2. 84(m,2H),2.60–2.53(m,2H),2.50–2.38(m,5H),2.38–2.27(m,3H),2.26–2.14(m,2H), 2.11–2.02(m,2H),1.98–1.83(m,4H),1.78–1.71(m,1H),1.65–1.56(m,1H),1.49–1.4 1(m,3H),0.94–0.80(m,2H),0.46–0.33(m,2H),0.09–0.03(m,1H),0.01–-0.07(m,1H).

[1030] Examples 11-17: Preparation of LD-17

[1031] (S)-N 5 -((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-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-tetraazahexadecane-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-N 1 -(2,5,8,11,14,17,20,23-octaoxaenocarbamo-25-yl)glutaramide LD-17

[1032] first step

[1033] (S)-28-amino-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azahentriacontan-31-oic acid tert-butyl ester LD-17b

[1034] LD-17a (75.0 mg, 92.0 μmol, prepared by the method disclosed in patent application “WO2022262516 A1” page 210, example 2) was dissolved in 1.5 mL of acetonitrile, 1,2-dichloroethane (375 mg, 3.80 mmol) was added, and stirred at 25 °C for 1 hour. The reaction solution was concentrated by distillation under reduced pressure to obtain the crude title product LD-17b (50 mg) in the form of a yellow solid. The product was used directly in the next step without purification.

[1035] MS m / z (ESI): 569.4 [M+1].

[1036] Second step

[1037] (S)-28-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azahentriacontan-31-oic acid tert-butyl ester LD-17c

[1038] LD-17b (80.0 mg, 140 μmol) and 6-(2,5-dioxopyrrol-1-yl)hexanoic acid (44.5 mg, 211 μmol) were dissolved in 2 mL of dichloromethane, 1-hydroxybenzotriazole (28.5 mg, 211 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (40.4 mg, 211 μmol) and N,N-diisopropylethylamine (54.5 mg, 422 μmol) were added in turn, and stirred at 25 °C for 8 hours. 10 mL of water was added to the reaction solution, extracted with dichloromethane (15 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by pre-HPLC (separation conditions: column: Welch Xtimate C18 150 x 25 mm x 5 μm, mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution, B%: 23%-53%), to obtain the title product LD-17c (60 mg, yield: 90%) in the form of a yellow gum.

[1039] MS m / z (ESI): 762.4 [M+1].

[1040] Third step

[1041] (S)-28-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-27-oxo- 2,5,8,11,14,17,20,23 octaoxa-26-azahentriacontane-31-oic acid LD-17d

[1042] LD-17c (20.0 mg, 26.2 μmol) was dissolved in 1 mL of dichloromethane, trifluoroacetic acid (307 mg, 2.69 mmol) was added, stirred at 25 °C for 1 hour. The reaction solution was filtered, the filtrate was concentrated by distillation under reduced pressure, the obtained residue was purified by pre-HPLC (separation condition: column: Welch Xtimate C18 150 x 25 mm x 5 μm, mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution, B%: 10%-40%), to get the title product LD-17d (12 mg, yield: 64%) as yellow gum.

[1043] MS m / z (ESI): 706.4 [M+1].

[1044] Fourth step

[1045] The title product LD-17 was prepared by a similar method to LD-13, using LD-17d and LD-1j as starting materials.

[1046] The reference examples 11-13, the corresponding substrates were selected to prepare LD-18 to LD-96:

[1047] Example 11-97: Preparation of LD-97

[1048] N-((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadecan-16-yl)-1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1- yl)cyclopropane-1-carboxamide LD-97

[1049] First step

[1050] LD-97a (2.20 g, 12.1 mmol, prepared by the method disclosed in patent application “WO2020077038 A1” page 62, example 4), silver nitrite (111 mg, 724 µmol), bis(benzonitrile)palladium(II) chloride (555 mg, 1.45 mmol), nitromethane (4.90 g, 80.3 mmol) and copper(II) chloride dihydrate (247 mg, 1.45 mmol) were dissolved in 44 mL of tert-butanol, oxygen was replaced three times, and stirred at 25 °C for 12 hours under oxygen. 150 mL of water was added to the reaction solution at 25 °C, extracted with dichloromethane (50 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-97b (800 mg, yield: 33%) as a white solid.

[1051] Second step

[1052] LD-97b (800 mg, 4.04 mmol) was dissolved in 24 mL of methanol, then potassium carbonate (1.12 g, 8.07 mmol) and (1-diazo-2-oxopropyl) dimethyl phosphonate (930 mg, 4.84 mmol) were added, and stirred at 25 °C for 12 hours. 50 mL of water was added to the reaction solution, extracted with ethyl acetate (20 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-97c (650 mg, yield: 83%) as a white solid.

[1053] Third step

[1054] LD-97c (650 mg, 3.35 mmol), 5-bromo-2-methylmercaptopyrimidine (686 mg, 3.35 mmol), triethylamine (3.39 g, 33.4 mmol), cuprous iodide (63.7 mg, 334 µmol) and bis(triphenylphosphine)palladium(II) chloride (244 mg, 349 µmol) were dissolved in 10 mL of tetrahydrofuran, replaced with nitrogen three times, and stirred at 60 °C for 2 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-97d (770 mg, yield: 68%) as a white solid.

[1055] MS m / z (ESI): 319.6 [M+1].

[1056] Fourth step

[1057] LD-97d (1.80 g, 5.65 mmol) was dissolved in 20 mL of dichloromethane, then m-chloroperoxybenzoic acid (2.87 g, 14.1 mmol, purity 85.0%) was added, stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-97e (1.70 g, yield: 85%) as a white solid.

[1058] MS m / z (ESI): 351.1 [M+1].

[1059] Fifth step

[1060] LD-97e (500 mg, 1.43 mmol) was dissolved in 6 mL of dichloromethane, then trifluoroacetic acid (2.30 g, 20.2 mmol) was added, stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the crude title product LD-97f (418 mg) as a white solid. The product was used directly in the next step without purification.

[1061] MS m / z (ESI): 295.2 [M+1].

[1062] Sixth step

[1063] LD-97f (14.2 mg, 48.4 μmol) and LD-1j (35.0 mg, 40.3 μmol) were dissolved in 2 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (8.2 mg, 60.5 μmol), N,N-diisopropylethylamine (15.6 mg, 121 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.6 mg, 60.6 μmol) were added in turn, stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, 1 mL of water was added to the obtained residue, extracted with dichloromethane (3 mL x 2), the organic phase was washed with saturated sodium chloride solution (2 mL), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by pre-HPLC (column: Phenomenex Kinetex EVO C18 150 x 30 mm x 5 μm; mobile phase: A-water (0.225% formic acid), B-acetonitrile; gradient elution: B%: 35%-55%) to obtain the title product LD-97 (8.00 mg, yield: 17%) as a yellow solid.

[1064] MS m / z (ESI): 1143.5 [M+1].

[1065] 1H NMR (400 MHz, DMSO-d6) δ 9.11 - 9.00 (m, 2H), 8.67 - 8.59 (m, 1H), 8.55 - 8.48 (m, 1H), 8.34 - 8.28 (m, 1H), 8.17 - 8.11 (m, 1H), 8.00 - 7.94 (m, 1H), 7.90 - 7.83 (m, 1H), 7.81 - 7.75 (m, 1H), 7.41 - 7.35 (m, 1H), 7.28 - 7.11 (m, 5H), 6.60 - 6.55 (m, 1H), 5.63 - 5.56 (m, 1H), 5.47 - 5.35 (m, 2H), 5.25 - 5.17 (m, 2H), 4.70 - 4.59 (m, 2H), 4.51 - 4.43 (m, 1H), 4.07 - 3.98 (m, 2H), 3.79 - 3.53 (m, 6H), 3.44 - 3.36 (m, 3H), 3.20 - 3.13 (m, 2H), 3.05 - 2.99 (m, 1H), 2.81 - 2.73 (m, 1H), 2.71 - 2.62 (m, 2H), 2.40 - 2.36 (m, 3H), 2.22 - 2.13 (m, 2H), 1.92 - 1.85 (m, 2H), 1.85 - 1.79 (m, 1H), 1.78 - 1.71 (m, 1H), 1.04 - 0.95 (m, 2H), 0.83 - 0.76 (m, 1H), 0.73 - 0.65 (m, 2H), 0.37 - 0.25 (m, 2H), 0.09 - 0.01 (m, 1H), -0.05 - -0.12 (m, 1H).

[1066] Example 11-98: Preparation of LD-98

[1067] N-((2S,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadec-16-yl)-l-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-l-yl)cyclopropane- 1-carboxamide LD-98

[1068] LD-97f (10.8 mg, 36.8 μmol) and LD-13f (27.0 mg, 30.6 μmol) were dissolved in 1 mL of N,N-dimethylformamide, then 1-hydroxybenzotriazole (6.21 mg, 45.9 μmol), N,N-diisopropyl ethylamine (11.9 mg, 91.9 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.81 mg, 45.9 μmol) were added successively, stirred at 25 °C for 1 hour. The reaction solution was concentrated under reduced pressure to remove the solvent, 3 mL of water was added to the obtained residue, extracted with dichloromethane (3 mL x 3), the organic phase was washed with saturated sodium chloride solution (2 mL), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing system A to obtain the crude product, which was further purified by pre-HPLC (column: Phenomenex C18 150 mm x 30 mm x 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile; gradient elution: B%: 25%-55%), to obtain the title product LD-98 (2.11 mg, yield: 6%) in the form of a white solid.

[1069] MS m / z (ESI): 1157.8 [M+1].

[1070] 1 H NMR (400 MHz, CD3OD) δ 8.89 - 8.80 (m, 2H), 7.66 - 7.58 (m, 2H), 7.26 - 7.19 (m, 2H), 7.19 - 7.08 (m, 3H), 5.71 - 5.65 (m, 1H), 5.62 - 5.54 (m, 1H), 5.50 - 5.42 (m, 1H), 5.33 - 5.25 (m, 1H), 5.22 - 5.15 (m, 1H), 4.78 - 4.71 (m, 2H), 4.33 - 4.25 (m, 2H), 3.91 - 3.72 (m, 4H), 3.61 - 3.45 (m, 3H), 3.36 - 3.33 (m, 3H), 3.22 - 3.12 (m, 1H), 2.94 - 2.84 (m, 2H), 2.73 - 2.64 (m, 2H), 2.46 - 2.37 (m, 3H), 2.37 - 2.28 (m, 1H), 2.26 - 2.18 (m, 1H), 2.01 - 1.90 (m, 2H), 1.89 - 1.81 (m, 1H), 1.76 - 1.69 (m, 1H), 1.51 - 1.42 (m, 3H), 1.17 - 1.12 (m, 2H), 0.94 - 0.83 (m, 3H), 0.45 - 0.31 (m, 2H), 0.06 - -0.08 (m, 2H).

[1071] Example 11-99: Preparation of LD-99

[1072] N-((2R,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinoline-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadec-16-yl)-l-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-l-yl)cyclopropane- 1-carboxamide LD-99

[1073] First step

[1074] (R)-l-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazoundecan-11-oic acid benzyl ester LD-99b

[1075] LD-99a (3.00 g, 7.87 mmol, prepared by the method disclosed in Patent Application “WO2022135332 A1” page 226, Example 1.15) was dissolved in 50 mL of dichloromethane, 4-methylbenzenesulfonic acid pyridine (1.98 g, 7.87 mmol) and (2R)-2-hydroxypropionic acid benzyl ester (2.84 g, 15.7 mmol) were added in turn, and stirred at 50°C for 10 hours. 40 mL of water was added to the reaction solution, extracted with dichloromethane (50 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product LD-99b (1.55 g, yield: 40%) in the form of yellow gum.

[1076] Second step

[1077] (5S,13R)-5-benzyl-l-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10- triazatetradecan-14-oic acid benzyl ester LD-99c

[1078] LD-99b (2.63 g, 5.38 mmol) was dissolved in 30 mL of dichloromethane, 1,8-diazabicyclo[5.4.0]undec-7-ene (491 mg, 3.23 mmol) was added, and stirring was performed at 25°C for 1 hour. Then, (( (9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (2.29 g, 5.91 mmol), 1-hydroxybenzotriazole (1.09 g, 8.06 mmol), and 1-(3-dimethylaminop...

Claims

1. An antibody drug conjugate of Formula (I), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, polymorph, hydrate or solvate thereof, or mixtures thereof: T— [L— D] (I) y (I) wherein, T is a targeting moiety, and is an antibody or antigen-binding fragment thereof that binds to CEACAM5; L is a linker unit; D is a biologically active fragment; y is selected from an integer or decimal number from 0.1 to 20.

2. The antibody drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 45, 46, 47, respectively; or (2) the VH comprises HCDR1, HCDR2, HCDR3 having amino acid sequences as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 15, 16, 17, respectively; or (3) the VH comprises HCDR1, HCDR2, HCDR3 having amino acid sequences as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, 26, 27, respectively; or (4) the VH comprises HCDR1, HCDR2, HCDR3 having amino acid sequences as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 35, 36, 37, respectively.

3. The antibody drug conjugate of claim 2, wherein: (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73; or (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; or (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; or (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

33.

4. The antibody drug conjugate of claim 3, wherein: (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 38, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 43; or (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 58, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 69; or (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 58, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 73; or (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 8, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 13; or (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 18, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 23; or (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 28, and the VL comprises the amino acid sequence set forth in SEQ ID NO:

33.

5. The antibody drug conjugate of any one of claims 1-4, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

6. The antibody drug conjugate of any one of claims 1-5, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

7. The antibody drug conjugate of any one of claims 1-6, wherein the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

8. The antibody drug conjugate of any one of claims 1-7, wherein the antibody comprises a heavy chain and a light chain, wherein: (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; or (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81; Preferably, (i) the light chain comprises the amino acid sequence set forth in SEQ ID NO: 79, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 77; (ii) the light chain comprises the amino acid sequence set forth in SEQ ID NO: 83, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

81.

9. The antibody drug conjugate according to any one of claims 1-8, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

10. The antibody drug conjugate according to any one of claims 1-9, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody; Preferably, wherein the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds to a second antigen.

11. The antibody drug conjugate of any one of claims 1-10, wherein, the L comprises a cleavable linker or a non-cleavable linker; Preferably, the cleavable linker comprises an acid-labile linker, a hydrophilic linker, a protease-sensitive linker, a photo-labile linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

12. The antibody drug conjugate of any one of claims 1-11, wherein, L comprises or is derived from a fragment of mc(6-maleimidocaproyl), Val-Cit(valine-citrulline), p-amino-benzyloxycarbonyl (PABC), SPDB (N-succinimidyl-4-(2- pyridyldithio)-butyrate), Sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo- butyrate), beta-glucuronic acid, dimethyl ethyl amine (DMEA), Val-Cit-PABC, mc- Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, mc-VC-PABC-DMEA, GGFG (glycine- glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)- butyric acid), dimethyl hydrazide (3-methyl-) 3-mercaptobutane hydrazide), AcBut- dimethyl hydrazide, or SMCC (N-succinimidyl-4-(N-maleimidomethyl)cyclohexane- carboxylate).

13. The antibody drug conjugate of any one of claims 1-12, wherein, L is -L1-L2-L3-; L1is -L 1a -L 1b -L 1c -L 1d -L 1e -; L 1a selected from L 1b alkylene, C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein each CH2in C 1-10 alkylene, C 2-10 alkenylene and C 2-10 alkynylene is optionally substituted by 1, 2 or 3 R x ; each R x is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, two R x may be joined to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-; L 1d selected from a chemical bond, C 1-8 alkylene or -(CH2CH20) n -C 1-4 alkylene; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH20) w -C 1-4 alkyl or -C(O)NH-(CH2CH20) w -C 1-4 alkyl; n and w are independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-; L2 is a chemical bond or a divalent peptidic moiety comprising 2 to 8 optionally substituted natural or unnatural amino acid residues, each amino acid residue in said L2 is optionally substituted with 1, 2, 3, 4, or 5 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; L3 is an optional substituted or unsubstituted spacer, for example L3 is selected from -NH-CH2-(AM), (PABC), (PAB) or L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1- 6haloalkyl or C 1-6 haloalkoxy; Preferably, L is -L1-L2-L3-; L1is -L 1a -L 1b -L 1c -L 1d -L 1e -; L 1a selected from represents L 1a linkage site to an antibody; L 1b selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, wherein C 1-6 alkylene, C 2-6 alkenylene and C 2-6 alkynylene each CH2is optionally substituted with 1 or 2 R x ; each R is independently selected from H, halogen, or C x is independently selected from H, halogen, or C 1-6 alkyl; or, two R groups on any identical or different carbon atom can be joined to form a C x may be joined to form a C 3-7 cycloalkylene or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkyl, more preferably C 3-5 cycloalkylene, for example cyclopropylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-, preferably a chemical bond or -C(O)NH-; L 1d is selected from a chemical bond or C 1-6 alkylene; said C 1-6 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl; w is selected from 2, 3, or 4, preferably 3; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably a chemical bond or -C(O)-; L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, each amino acid residue in L2is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; L3 is an optional substituted or unsubstituted spacer, for example L3 is selected from -NH-CH2-(AM) or (PABC); L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, or C 1-6 alkyl.

14. The antibody drug conjugate of any one of claims 1-13, wherein, L2 is a divalent peptidic moiety comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, selected from gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-cit, or val-lys-beta-ala; each amino acid residue in said L2 is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Preferably, L2 is a divalent peptidic moiety comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, the amino acids being selected from glycine, phenylalanine, alanine, valine, citrulline, or lysine, preferably -gly-gly-phe-gly- or -val-cit- or -val-lys-gly-; each amino acid residue in said L2 is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, or C1-C6alkyl. y is independently selected from H, halogen, or C1-C6alkyl. 1-6 alkyl.

15. The antibody drug conjugate of any one of claims 1-14, wherein, L is selected from the following structures: (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-val-cit-PABC), (SPDB), (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM), Preferably, L is selected from the following structures: (mc-val-cit-pabc), (mc-val-cit-PABC), (mc-val-cit-PABC), (mc-GGFG-AM), (mc-GGFG-AM), (mc-GGFG-AM), 16. The antibody drug conjugate of any one of claims 1-15, wherein, D is selected from the group consisting of metal complexes; antibiotics; DNA topoisomerase inhibitors; tubulin inhibitors and microtubule polymerization inhibitors; DNA synthesis inhibitors; RNA polymerase II inhibitors; RNA splicesome inhibitors; agents acting on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor neovascularization inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit the growth of tumor cells, promote apoptosis or necrosis of tumor cells; Preferably, D is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors and RNA splicesome inhibitors; More preferably, D is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, such as auristatins, maytansinoids, tubulysins, cryptophycins or rhizoxin; Alternatively, D is an antibiotic, such as a calicheamicin, an anthracycline and an anthracycline antibiotic; Alternatively, D is selected from the group consisting of DNA synthesis inhibitors, such as duocarmycins, PBDs (pyrrolodinoindoles) or IGNs (indolinobenzodiazepines); Alternatively, D is selected from the group consisting of DNA topoisomerase I inhibitors, such as camptothecin or a camptothecin derivative; Alternatively, D is selected from the group consisting of RNA polymerase II inhibitors, such as an α-amanitin; Alternatively, D is selected from the group consisting of RNA splicesome inhibitors, such as splicetastatins and telatinastatins; Preferably, D is selected from the group consisting of camptothecin or a camptothecin derivative, such as hydroxy camptothecin, 9-amino camptothecin, SN-38, exatecan, Dxd, irinotecan, topotecan or other derivatives; Preferably, D is selected from the group consisting of auristatin drugs, such as MMAE or MMAF; Preferably, D is selected from the group consisting of maytansinoid drugs, such as DM1, DM2, DM3 or DM4.

17. The antibody drug conjugate of any one of claims 1 to 16, wherein D is selected from the group consisting of auristatin or maytansinoid drugs, such as MMAE, MMAF, DM1, DM2, DM3 or DM4, preferably MMAE or DM4; Alternatively, D is selected from a compound of Formula (D-I) or Formula (D-II), or a pharmaceutically acceptable salt, isotopologues, tautomer, stereoisomer, racemate, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1, *2 and *3 are chiral centers independently selected from the group consisting of (S) or (R) absolute configuration, or mixtures thereof, preferably *3 is (S) absolute configuration; L D1 is selected from a chemical bond, -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3; L D2 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; L D3 is selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene, or C 2-10 alkynylene, said L D3 is optionally substituted with 1, 2, or 3 R D3 groups; L D4 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; R D1 , R D2 and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a 5-6 membered heterocyclyl; or, R D1 , R D6 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 5-6 cycloalkyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl; each of the compounds of formula (D-I) and formula (D-II) is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl; Preferably, D is selected from the group consisting of auristatin or maytansinoid drugs, such as MMAE, MMAF, DM1, DM2, DM3 or DM4, preferably MMAE or DM4; Alternatively, D is selected from a compound of Formula (D-III) or Formula (D-IV), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1 and *2 are chiral centers independently selected from the group consisting of (S) or (R) absolute configuration, or mixtures thereof; L D1 is selected from a chemical bond, -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; q is 0, 1, 2, 3 or 4, preferably 0 or 1 ; L D2 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; L D3 selected from a chemical bond, C 1-10 alkylene, C 1-10 haloalkylene, C 2-10 alkenylene, or C 2-10 alkynylene; L D4 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; R D1 , R D2 , and R D6 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D1 , R D2 , and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a 5-6 membered heterocyclyl; or, R D1 , R D6 , and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably a C 5-6 cycloalkyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl.

18. The antibody drug conjugate of any one of claims 1 to 17, wherein D is selected from aurestatin or maytansine drugs, such as MMAE, MMAF, DM1, DM2, DM3 or DM4, preferably MMAE or DM4; Alternatively, D is selected from a compound of Formula (D-III) or Formula (D-IV), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: *1 and *2 are chiral centers, independently selected from the (S) or (R) absolute configuration, or a mixture thereof; L D1 is selected from a chemical bond, -NHC(O)-, or -C(O)NH-; q can be 0, 1, 2, 3 or 4, preferably 0 or 1; L D2 is selected from -NH- or -O-; L D3 selected from a chemical bond, C 1-6 alkylene or C 1-6 haloalkylene, more preferably C 1-6 alkylene; L D4 is selected from -NH- or -O-; R D1 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me; R D2 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F; or R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3- to 5-membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; R D6 is H; Alternatively, D is selected from a compound of Formula (D-V), or a pharmaceutically acceptable salt, isotopologue, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate, or mixtures thereof: *2 is a chiral center, independently selected from the (S) or (R) absolute configuration, or a mixture thereof; q can be 1, 2, or 3, preferably 1; R D1 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me; R D2 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F; or R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably R D3 and R D4 are not simultaneously H; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-5 cycloalkyl; R D5 selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl; more preferably, R D5 is selected from -methylene-cyclopropyl or -methylene-cyclobutyl.

19. The antibody drug conjugate of any one of claims 1-18, wherein, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, stereoisomer, racemic mixture, polymorph, hydrate, or solvate thereof, or mixtures thereof: (MMAE), (DM4), (Dxd), Preferably, D is selected from the following compounds, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, racemic, polymorph, hydrate, or solvate thereof, or mixtures thereof: (MMAE), (DM4), (Dxd), 20. The antibody-drug conjugate of any one of claims 1-19, wherein y is an integer or decimal selected from 0 to 20, preferably an integer or decimal selected from 0 to 10, and more preferably an integer or decimal selected from 3 to 9.

21. The antibody drug conjugate of any one of claims 1-20, having the structure of formula (I-1), (I-2), or (I-3): in, T, D, and y are as defined in any one of claims 1-20; Each m is selected from 1, 2, 3, 4, or 5; Each w is selected from 1, 2, 3, 4, 5, 6, 7, or 8; each R x and R x ' is independently selected from H, halogen or C 1-6 alkyl; or, two R x groups on any identical or different carbon atom can be joined to form a C 3-7 cycloalkylene or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkylene, more preferably C 3-5 cycloalkylene; each R y and R y ' is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy.

22. The antibody drug conjugate of any one of claims 1-21, wherein, The antibody drug conjugate is selected from the following formula: in, T, D, and y are as defined in any one of claims 1-20; Preferably, D is selected from aurestatins, maytansines, camptothecin or camptothecin derivatives, such as MMAE, MMAF, DM1, DM2, DM3, DM4, hydroxycamptothecin, 9-aminocamptothecin, SN-38, eczetidine, Dxd, irinotecan, topotecan or compounds of formula (DI), (D-II), (D-III) or (D-IV), wherein each variable is defined as in any one of claims 17-18; y is selected from integers or decimals from 1 to 10.

23. The antibody drug conjugate of any one of claims 1-22, wherein, The antibody drug conjugate is selected from the following structures: T and y are as defined in any one of claims 1-20; Preferably, y is selected from integers or decimals from 1 to 10.

24. The antibody drug conjugate of claims 1-23, wherein, The antibody drug conjugate is selected from the group consisting of the following structures: in, The mAb1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:38, and the VL comprises the amino acid sequence shown in SEQ ID NO:43; or The mAb1 H6K3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), (2) the VH comprises an amino acid sequence as shown in SEQ ID NO:58, and the VL comprises an amino acid sequence as shown in SEQ ID NO:69; or The mAb1 H6K5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:58, and the VL comprises the amino acid sequence shown in SEQ ID NO:73; or The mAb2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains the amino acid sequence shown in SEQ ID NO:8, and the VL contains the amino acid sequence shown in SEQ ID NO:13; or The mAb3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:18, and the VL comprises the amino acid sequence shown in SEQ ID NO:23; or the mAb4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33; Preferably, the mAb1 H6K3 comprises a heavy chain (HC) and a light chain (LC), the HC comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 79; the mAb1 H6K5 comprises a heavy chain (HC) and a light chain (LC), the HC comprises an amino acid sequence as set forth in SEQ ID NO: 81, and the LC comprises an amino acid sequence as set forth in SEQ ID NO: 83; More preferably, the -L-D structure is linked to a cysteine in the antibody or antigen-binding fragment thereof that binds to CEACAM5.

25. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-24, and optionally a pharmaceutically acceptable carrier or excipient.

26. The pharmaceutical composition of claim 25, wherein the composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody drug, a bi- / multi-specific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

27. Use of the antibody drug conjugate of any one of claims 1-24, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, polymorph, hydrate, or solvate thereof, or a mixture thereof, in the manufacture of a medicament for preventing and / or treating a disease.

28. The use of claim 27, wherein, the disease is cancer, for example a cancer associated with CEACAM5 expression; Preferably, the disease is selected from the group consisting of gastric cancer, colon cancer, rectal cancer, small intestine cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, thyroid cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, esophageal cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial carcinoma; More preferably, the disease is selected from the group consisting of pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, and breast cancer.

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